WO2012153153A1

WO2012153153A1 - Procedure for rapid determination of viruses using nucleic acid-based molecular diagnostics, and a kit for this purpose

Info

Publication number: WO2012153153A1
Application number: PCT/HU2011/000045
Authority: WO
Inventors: Gábor KISS; János KISS; Timea Kiss; Dániel Péter PAPP
Original assignee: Diagon Kft.
Priority date: 2011-05-11
Filing date: 2011-05-11
Publication date: 2012-11-15
Also published as: EP2707496A1

Abstract

Procedure for rapid determination of viruses using nucleic acid-based molecular diagnostics, for the qualitative and quantitative determination of DNA viruses or RNA viruses in samples using multiplex real-time PCR technique, in the course of which in changeable genetic surroundings taxonomically characteristic stable consensus and unique variable nucleotide sequences are detected simultaneously. For this purpose single-stranded oligonucleotide probes containing palindromic motifs are created. In these palindromic motifs semi-degenerate bases are placed suiting the variable regions of the virus to be currently detected, favourably suiting the variable sequences expressed in numbers in the international professional protocols, and with the said semi-degenerate bases multiple internal loops are formed to ensure the structural flexibility of the probes. The bridge abutment-like close proximity of the multiple internal loops are provided with nucleotide sequences, with which the probes are bound on stable nucleotide sequences of the template to be detected, accessible in international genetic databases, via thermodynamically strong hybridisation. With this strong hybridisation the multiple internal loops are opened up, by flexibly spanning the variable regions around the stable template sequences the probes are bound to the variable sequences, and on the template to be detected a sequence of a length of at least 25-30 nucleotides bound in a thermodynamically stable and selective way is detected. The specification also relates to a KIT for the practical realisation of the procedure.

Description

PROCEDURE FOR RAPID DETERMINATION OF VIRUSES USING NUCLEIC ACID-BASED MOLECULAR DIAGNOSTICS, AND A KIT

FOR THIS PURPOSE The invention relates to a procedure for rapid determination of viruses using nucleic acid-based molecular diagnostics, in the course of which qualitative and quantitative determination of DNA viruses or RNA viruses in samples is performed using multiplex realtime PCR technique in such a way that in changeable genetic surroundings taxonomically characteristic stable consensus and subtype characteristic unique variable nucleotide sequences are detected simultaneously with the help of flexible oligonucleotide probes sensitive for spatial conformation planned by us. Furthermore, the specification relates to a KIT for the practical realisation of the procedure.

The etiological screening and periodical testing of certain human diseases of viral origin is essential both in respect of public health and clinical medicine, as for example the presence of pathogenic viruses represents an increased risk not only to host organisms carrying the virus, but also to healthy persons potentially targeted by communal, therapeutic, environmental vectors transmitting viral pathogens. An example of being targeted by the latter type of vectors is the direct use of virus-infected (hepatitis, influenza, etc.) environmental waters or animal and plant-based nutrient sources, or the inadequacy of the hygienic conditions of certain medical interventions, or viral infections spread through insect bites (influenza, dengue fever, etc.). In established infection viruses spread onto further target organs and tissues in the host organism mostly through the blood stream and the nervous system, and they may also appear in different body excretions (e.g. nasal and throat excretions, saliva, sweat, urine), forming by this further sources of infection. For the above reasons it can be understood that the reliable detection of pathogens is essential for starting the appropriate therapeutic-hygienic actions, which calls for the use of sensitive, specific, rapid diagnostic methods or the combination of such methods in given cases.

In the present specification the abbreviation DNA is used for deoxyribonucleic acid, which can be double-stranded or single-stranded in structure. In the present specification the abbreviation RNA is used for ribonucleic acid, which can be partly double-stranded or single-stranded in structure. The term viral genome relates to the entire genetic material carried by viral DNA or viral RNA, all nucleotide sequences. The term viral genotype, genotype variant relates to all taxonomically characteristic stable consensus and subtype characteristic unique variable nucleotide sequences of the given viral genome variant.

In the present specification the term RT reverse transcription means the transcription of the initial RNA as template sequence into cDNA complementary DNA sequence using RNA- dependent DNA polymerase reverse transcriptase enzyme.

In the present specification the term PCR method is used to refer to the in vitro enzymatic amplification of single-stranded DNA chains, the widely known technique of polymerase chain reaction. In the case of an initial RNA template the RT reverse transcription and PCR are combined in the RT-PCR reverse transcription PCR technique, which, in the present procedure, is realised using the faster single-step, same-space RT-PCR reverse transcription PCR technique, which involves a lower risk of contamination.

The real-time PCR method according to the procedure is used to relate to a version of the above PCR and RT-PCR reverse transcription PCR nucleic acid amplification, in which nucleic acid amplification during the reaction is monitored with the help of PCR probe element (see below) fluorescent-labelled oligonucleotides, so-called internal probes, as a result of which at the end of the real-time PCR process the reaction kinetics can be summarised.

In the present specification the term PCR probe element(s) relates to short, single-stranded oligonucleotide DNA sequences delimiting, directing and determining the selective specific detectability of PCR amplification of single-stranded DNA chains, including: forward primer(s), reverse primer(s), primer pair(s) consisting of the two former ones together, and probe(s) labelled for the purpose of detection.

In the present specification the term hybridisation annealing is used to refer to the complex of complementary nucleotide sequences formed with secondary chemical bonds, hydrogen bonds, that is their hybridisation pairing.

In the present specification the physical-chemical term of thermodynamically strong hybridisation is used to refer to the hybridisation pairing of complementary natural nucleotides, nucleotide sequences.

In the present specification the physical-chemical term of thermodynamically weak hybridisation is used to refer to the hybridisation pairing of degenerate and semi-degenerate nucleotides, nucleotide sequences. In the present specification the basis for the interpretation of the term genetic code "wobble" is the biological phenomenon when in the genomic polynucleotide DNA or RNA chain consecutive trinucleotides, nucleotide base triplets built from A adenine, T thymine, G guanine, C cytosine or U uracil nucleotides provide the different genetic signals, genetic codes determining diverse amino acids. When reading the genetic code to protein synthesis, the messenger RNA nucleotide triplet codon complementary to the code is complementary to the transfer RNA anticodon nucleotide triplet sequence carrying the protein-building amino acid, that is it establishes hybridisation pairing with it. An amino acid can be determined by several different nucleotide triplet sequences, consequently an amino acid can have several different codons. What happens in nature is that in the first two reading positions of the codon base triplets there is full complementarity to the corresponding positions of the anticodon base triplet, there is strong hybridisation pairing, in the third position of the codon it changes because of degeneration (see below), which allows a thermodynamically weaker connection between the codon and the anticodon in this third reading position, that is the genetic code wobbles.

On the basis of the above, in the present specification the term degenerate sequence is used to refer to the nucleotide or oligonucleotide sequence, which, as the natural feature of the genetic code, is characterised by that the same amino acid can be coded by several different base triplets, that is nucleotide triplets. In accordance with this, in the present specification the term degenerate oligonucleotide is used to refer to an oligonucleotide, in the sequence of which any of the A adenine, T thymine, G guanine, C cytosine or U uracil nucleotide bases can occur in the individual positions. Consequently, the term degenerate oligonucleotide also relates to a synthetic nucleotide sequence, where any type of nucleotide can occur in any nucleotide position. According to the general degeneration rule of our specification, in the "n" positions of nucleotide sequences any of the A adenine, T thymine, G guanine, C cytosine nucleotides can occur.

In the present specification the term semi-degenerate oligonucleotide is used to refer to a synthetic nucleotide sequence, in which degenerations occur only in the given nucleotide positions and according to given rules. In the present specifications there are the following rules of semi-degeneration. Base position Possible nucleotides

b g + t + c

d g + a + t

h a + t + c

k g + t

m a + c

r g + a

s g + c

y c + t

V g + a + c

w a + t

In the present specification palindromic sequences are nucleotide sequences, which, when read from the two ends of a single-stranded polynucleotide, oligonucleotide chain, show the same nucleotide sequence.

A standard testing possibility in the indirect detection of the presence of viruses is immunoserology, when infection is indicated by the in vitro detection of immunoglobulins, antibodies specific for surface and internal antigens of the virus, produced by the infected organism. Sensitive and specific immunoserologic methods concentrate mainly on the detection of antibodies specific to virus envelope surface membrane antigen determinants, nucleocapsid core antigen determinants below them, and viral genome antigen determinants embedded in nucleoprotein within the nucleocapsid core, that is on the detection of serum IgM and IgG immunoglobulins specific to viral antigen determinants listed above. Such immunoserologic methods include for example HI hemagglutination inhibition, CF complement fixation, NA detection of the presence of neutralising antibodies, IF immunofluorescence detection, RIA radioimmunoassay or EIA enzyme-linked immunoassay [see: Wong's Virology: Diagnostic Methods in Virology http://virology- online.com/general/Tests.htm]. The above immunoserologic methods indirectly prove viral infection, such as for example in patent specification no. US 7595152, where the presence of the Influenza A virus and the Influenza B virus is proved by detecting antibodies specific to virus NS1 non-structural protein domain 1 epitopes. Another type of indirect detection is the immunodiagnostic antibody determination for the presence of the Hepatitis A virus described in the summarising work by Nainan O.V. et al. [Nainan O.V. et al (2006): Clin. Microbiol. Rev. 19: 63-79.]. A further example of the indirect immunoserologic demonstration of virus infection is the simultaneous detection of Hepatitis B and Hepatitis C virus specific antibodies with the help of a protein chip assay using nano-gold immuno- amplification [Duan L. et al.(2005): BMC Infectious Diseases 5:53, http://www.biomedcentral.eom/1471-2334/5/53]. Bichr S. et al. describe the detection of Hepatitis C virus neutralizing antibodies in their experimental model [Bichr S. et al. (2002): J.General Virology 83: 1673-1678.], in patent specification no. US 5759770 a new serotype determination of the Human immunodeficiency virus is summarised, while Gubler D.J. illustrates the seroepidemiology of the Dengue virus and the Dengue haemorrhagic fever for example using HI hemagglutination inhibition [Gubler D.J. (1998): Clinical Microbiology Reviews 1 1 : 480-496.]. In the case of numerous pathogenic viruses the detection of the presence of the viruses as above can be restricted by the window period in time, when following infection the clinical symptoms and the antibody response in the organism do not appear together, but significantly shifted in time (Murray P.R., Rosenthal K.S., Pfaller M.A.: Medical microbiology, 6th edition, Mosby, Elsevier, 2009; Robbins and Cotran Pathologic Basis of Disease: 8th edition, Saunders, Elsevier, 2010). Because of the above window period and the time consuming nature of the immunoserologic detections described above, the necessary public health and clinical procedures may suffer delay.

A classic standard testing possibility for the direct demonstration of the presence of viruses is the in vitro detection of the virus particles using qualitative (that is morphologic- immunologic typing of virus) and quantitative (that is virus number, virus titre determination) methods in blood serum, tissues and cells, and in the culture media and cells of the in vitro tissue culture experimentally infected with the virus [Roingeard P. (2008): Biol. Cell 100: 491-501.]. The direct detection of the presence of pathogenic viruses as above may be significantly influenced by the viral replication rate, the viral titre in blood serum, and the different cytopathogenic (that is leading to degenerative changes) nature of the virus in the infected target tissues.

To overcome the above factors of diagnostic uncertainty, beside the standard procedures, nucleic acid-based molecular diagnostic methods were introduced, such as nucleic acid amplification diagnostic tests using the widely known traditional PCR method [see patent specifications no. US 4683195, US 4683202, US 4889818, US 4965188]. The advantage of these diagnostic tests is that even in the case of a low virus numbers they are operating standard applications recognised all over the world, and in respect of their demand of chemicals, instruments and infrastructure they can be performed with the automated instrumental system of a clinical laboratory. The disadvantage of such diagnostic tests is their time-consuming nature, their demand for high-value instruments and their dependence on numerous external effects or user faults while preparing reactions. For example this is why endpoint assay is difficult in the case of the traditional nucleic acid amplification PCR or RT-PCR reverse transcription PCR method, or the optimisation of separation of products according to size by gel electrophoresis, as human factors have a significant influence on the result in final evaluation.

Traditional PCR and RT-PCR methods used in the in vitro diagnostic determination of the presence of viral nucleic acid have been described, and real-time RT-PCR studies using fluorescence or chemiluminescense oligonucleotide probes have also been published [for example for the detection of Influenza A virus patent specifications no. US 5187060, US 2006014142A1, US 7262292, US 20050037414A1, for the detection of Hepatitis B virus the studies by Ho S.K.N, et al. (2003):J.Med. Microbiol. 52: 397-402. or Erhardt A. et al. (1996): J.Clin.Microbiol. 34: 1885-1891., and another examples of detecting viral nucleic acid can be found in patent specifications no. US 7052878, US 20040126387A1, WO 0029613, US 5985544].

While the monoplex real-time PCR and the monoplex single-step real-time RT-PCR methods enable the examination of a template to be detected in a given sample, and through this the examination of a type of PCR product amplicon, multichannel real-time PCR instruments make it possible to obtain information about several templates to be detected in a testing system. With this multiplex method several detection reactions are performed and by using different fluorescent labels, the template sequences to be determined are detected according to the wavelengths of the different fluorescent channels. Consequently, in the same sample, in one step, in one reaction space several templates to be detected and several types of PCR product amplicons can be examined, as a result of which the reliability of the diagnostic result can be enhanced. These favourably repeatable multiplex multicolour realtime PCR methods are complex instrumental analytic solutions, which are equivalent to the standard immunoserologic and traditional nucleic acid amplification diagnostic tests in respect of different technical characteristics (e.g. specificity, the ability to evaluate the test results), but surpass them because of their shorter testing time. For this reason these favourably repeatable multiplex multicolour real-time PCR methods are very favourable applications in the rapid determination of pathogenic viruses of high genetic changeability using nucleic acid-based diagnostics [for example multiplex real-time PCR diagnostic virological optimisation and use in the work by Elnifro E.M. et al. (2000): Clin. Microbiol. Rev. 13: 559-570., or the simultaneous detection of HBV-HCV-HIV plasma samples using multiplex capture assay according to patent specification no. WO 0136442A1]. For the determination of the presence of viral nucleic acid in test samples with our procedure according to the invention, taxonomically characteristic stable consensus and subtype characteristic unique variable sequences are detected simultaneously using multiplex multicolour real-time PCR method having advantages described above.

To sum up the above it can be stated that during the rapid determination of the presence of viruses using in vitro nucleic acid-based molecular diagnostics, and when selecting the therapeutic possibilities of patients infected with a pathogenic virus it is essential

• to determine the virus titre indicating the progress of the disease,

• to determine the genotype of the virus of high genetic changeability. In the course of realising the above, the hit accuracy of the diagnostic methods, that is relative or clinical specificity %, sensitivity %, accuracy %, is significantly influenced by the relatively high genetic changeability of the viruses, which may be manifested for example in the different expression of a surface antigen determinant, or for example in the altered hybridisation annealing of the PCR probe elements - indicating hits in nucleic acid amplification diagnostic tests - to variable genetic target sequences. This is why we find it necessary to plan marker PCR probe elements, the hybridisation annealing of which, under the biochemical and physical circumstances of the diagnostic tests,

• is specific to the genotype variant of the virus to be detected,

• is not altered by genetic code "wobble",

· proves the presence and titre of the virus with a taxonomically reliable diagnosis.

When detecting the presence of viruses in test samples using real-time PCR method, fluorescent marking basically depends on the complex formed by the fluorescent-labelled PCR probe element and the complementary template sequences, that is on the thermodynamic stability of hybridisation annealing. This thermodynamic stability depends on the mutations in template sequence to be detected characteristic of the genotype of given viral strain, which makes it necessary to design sensitive PCR probe elements for specific diagnostic determination. An analysis of the genetic data lines of international bioinformatics databases [www.ncbi.nlm.nih.gov, www.embl.org, www.jdb.com] proves that when using standard or individually prepared nucleic acid amplification diagnostic tests the PCR probe elements cannot anneal in each case to viral template sequences to be detected, and by this they have a significant impact on the operation of PCR reactions. Generally the cause of this phenomenon is the high genetic changeability of viruses, as the errors of high rate nucleic acid replication supporting rapid multiplication, the distortion or lack of repairing-correcting enzymatic processes, and the characteristically small size of the viral genome all provide a favourable background for the fairly rapid accumulation of different types of mutations [Alberts B., Johnson A., Lewis J., Raff M., Roberts K., Walter P.: Molecular Biology of the Cell, 5^th edition, Garland Science, 2008; Murray P.R., Rosenthal K.S., Pfaller M.A.: Medical microbiology, 6^th edition, Mosby, Elsevier, 2009]. A diagnostically significant consequence of the high genetic changeability of viruses is that in the case of the same infectious viral strain, among the mature virions released from the infected host cell often there are genetic variants, in the detection of which PCR probe elements do not anneal to the right template positions, and this changed selectivity leads to a false positive result. At the same time a false negative result is obtained, if in the test sample, despite the presence of the viral nucleic acid to be detected, the specific hybridisation annealing of the PCR probe elements does not take place.

In our tests we also experienced that in the case of a classic fluorescent-labelled single-stranded linear oligonucleotide probe, such as the TaqMan probe [for a summarising publication see Kubista M. et al. (2006): Molecular Aspects of Medicine 27: 95-125.], the condition of its specific hybridisation annealing and marking is that there is no mutation in the complementary template sequence. However, in the rapidly changing world of viruses, up until now in in vitro diagnostics a mutation is determined as a marker, in the same positions of which always the same nucleotides can be detected. The surroundings of these small-sized sequence motifs of 6-8 nucleotides, as stable consensus positions, is very changeable, and these mutation hotspots basically do not enable the hybridisation annealing for example of a classic linear probe of the length of 25-30 nucleotides (e.g. TaqMan probe), or they enable it only with a very weak stability. Consequently, the proportion of false negative reactions increases significantly, which is not acceptable in in vitro diagnostics. The significance of this also appears in the in vitro diagnostics of infections caused by viruses causing human diseases, such as influenza viruses of considerably high genetic changeability. So due to the mutation hotspots mentioned above, within the same taxonomic unit or species several genetic variations can occur, which variations influence the accuracy of the diagnostic solutions or even the necessary therapy in some cases. For this reason, in the interest of selective and specific diagnostics the changeability of the viral genome makes it necessary to redesign classic PCR probe elements continuously. However, this demand for continuous redesigning significantly restricts the applicability of the classic single-stranded linear oligonucleotide probe.

In order to overcome the above limits "hairpin" type such as spatial stem-loop structured single-stranded oligonucleotide probes (e.g. Molecular Beacon, Scorpion) were created. The closed condition of these spatial stem-loop structured single-stranded oligonucleotide probes is ensured by the formation of the stem, that is by the template- independent semi-degenerate sequences of the two ends of the nucleotide chain hybridising to each other. The single internal loop held together by the stem contains the nucleotide sequence complementary to the template sequence to be detected. In the process of PCR amplification due to the strong hybridisation annealing of the single internal loop and the template sequence to be detected the less stable stem opens out, as a result of which at the two ends of the oligonucleotide chain the fluorescent donor and acceptor-quenching labels move away from each other in space, the emission and absorption wavelength spectra stop overlapping each other, and so the reporting fluorescent signal can be detected [see US Patent no. 6174670 on donor and acceptor oligonucleotide probes, and US Patent no. 5538848 on quenching fluorescence probes]. In this solution of fluorescence detection, the occasionally missing regeneration of the above spatial oligonucleotide probes at the beginning of the PCR cycles may represent a difficulty, which increases background fluorescence and deteriorates the reliability of the reaction. Another type of detection more favourable than the one above is accomplished with using complementary strand displacement hydrolysis probe [for the detailed description see study no. WO 9202638, and see studies no. US 5210015 and US 5487972 on fluorescence detection with the hep of the exonuclease activity of DNA polymerase], where the reporter fluorescent signal is generated by cleaving the fluorescent-labelled oligonucleotide probe at the 5 '-end due to the additional exonuclease enzymatic activity of thermostable DNA polymerase catalysing nucleotide chain extension (figure 1).

On the basis of the above it can be seen that the advantage of "hairpin" or single- loop spatial single-stranded oligonucleotide probes is that they surpass classic single- stranded linear oligonucleotide probes in that even in variable surroundings they are able to bind to template sequences through stable hybridisation annealing [see Bonnet G. et al. (1999): PNAS 96: 6171-6176., McChlery S.M., Clarke S.C. (2003): Molecular Biotechnology 25; 267-273., Strohsahl CM. et al. (2007): Biosensors and Bioelectronics 23: 233-240., Vet J.A.M. et al. (1999): PNAS 96: 6394-6399., and patent specifications no. US 6548254, WO 2004092408A2, WO 2005007815 A2]. For realising this binding, "hairpin" or single-loop spatial single-stranded oligonucleotide probes bind to sequence- sensitive regions, while they do not bind to template variable regions but bridge them over. On summarising the above it can be stated that by using single-loop spatial probes in diagnostics, the possibility of false negative results is reduced in the course of virus detection and genotyping. However, the use of such probes is restricted by the synthetic costs of the special structure and by the instability of detection caused by the additional exonuclease enzymatic activity of the thermostable DNA polymerase catalysing chain extension in the PCR reaction [Broude N.E.(2005): Encyclopedia of Diagnostic Genomics and Proteomics DOI: 10.1081 E-EDGP 120020717].

On the basis of the above it can be concluded that hybridisation annealing to template sequences to be detected is significantly influenced by the structure of the PCR probe elements. From this we came to the conclusion that in the rapid determination of highly changeable viruses using nucleic acid-based molecular diagnostics, the template to be detected itself also has a significant influence on the selective specificity of PCR reactions.

In the sequence of the template to be detected smaller modifications such as base substitutions or inversions change the interpretation of the code, the content of the information, but do not significantly affect the linearity of the nucleotide sequence. For example, with the help of genetic data lines obtained from international bioinformatics databases [www.ncbi.nlm.nih.gov, www.embl.org, www.jdb.com] we have a multitude of information about the individual genes and the sequence motifs in their vicinity, and the complete DNA sequence or cDNA sequence of numerous viruses can be found too.

As opposed to the above, in many cases we observed that according to the bioinformatics database the presence of hybridisation annealing positions for PCR probe elements does not clearly mean that there are homologous sequences in the template to be detected, as in the surroundings of the hybridisation annealing positions the base sequences may show significant differences. This latter phenomenon is illustrated in figure 2 with an example of the Hepatitis C virus, showing variable nucleotide cassettes surrounding stable sequences in the viral genome. The PCR reagents presently available on the market and the results published in this subject [Schroter M. es mtsai (2002): J. Clin. Microbiol. 40: 2046 - 2050., Stockton J. et al. (1998): J. Clin. Microbiol. 36: 2990-2995., Conway B. et al. (1995): Clin. Diagn. Virol. 3: 95-104., Khawsak P. et al. (2003): Southeast Asian J. Trop. Med. Public Health 34: 781-785., patent specification no. US 5527669, patent studies no. WO2007121247, WO2006013144, WO2004022784, WO1999029909, WO1996003528, WO1994029483, WO1993022440] typically contain solutions, in which the PCR probe elements delimiting and detecting the region to be amplified anneal and emit specific signals within a given sequence surroundings, the so-called inclusivity surroundings in the viral nucleic acid. In addition to our bioinformatics studies, on the basis of the above we performed comparative measurements with several PCR methods and modelled the changeable surrounding sequences of the annealing positions. However, in our measurements, despite taking into consideration inclusivity mentioned above, we observed a decrease in the specificity of the microbial reactions.

Considering the above we realised that for PCR-based diagnostic determination of the presence of viral nucleic acid, PCR probe elements containing degenerate - semi- degenerate sequences are needed, which indicate the hits of our PCR reactions reliably and selectively in changeable sequence surroundings. Suiting this diagnostic reliability we developed the oligonucleotide probe according to our procedure. A special feature of our oligonucleotide probe is that in the single-stranded chain planned by us palindromic motifs are created, and multiple internal loops are formed by thermodynamically weak hybridisation pairing of semi-degenerate bases placed in them (figure 3). With these internal loops the structural flexibility of the probe is ensured, as a result of which variable sequences can be bound with our probe. We recognised that the selective binding of these variable sequences can be founded by providing the close proximity of the multiple internal loops in our oligonucleotide probe with nucleotide sequences like bridge abutments, and with the latter nucleotide sequences thermodynamically strong hybridisation is realised on stable nucleotide sequences of the template to be detected. This strong hybridisation annealing opens up the semi-degenerate internal loops, as a result of which, by flexibly spanning the variable regions around the stable template sequences, our probe can be bound to variable sequences. A further characteristic feature of our oligonucleotide probe with multiple internal loops is that on the template to be detected the sequence of a length of at least 25-30 nucleotides suiting our expectations can be bound in a thermodynamically stable and selective way. Due to the multiple internal loops and the structural flexibility described above, we gave the oligonucleotide probe planned by us the attributives sensitive for spatial conformation and flexible, and we named it Camel probe (figure 3).

For the determination of the presence of viral nucleic acid using the multiplex multicolour real-time PCR method according to our procedure, in changeable genetic surroundings taxonomically characteristic stable consensus and subtype characteristic unique variable sequences are detected simultaneously with the help of flexible oligonucleotide probes sensitive for spatial conformation planned by us, the fluorescence detection of which is favourably solved with complementary strand displacement hydrolysis described above (figure 1, figure 3).

With the oligonucleotide probes planned by us and described above, in favourably set up buffer medium, such as the 5x MasterMix buffer medium according to table 1 for the determination of Influenza A virus according to our procedure, or for example the 5x MasterMix buffer medium according to table 4 for the determination of Hepatitis C virus according to our procedure, the target template sequences are detected reliably in changeable sequence surroundings. With our procedure, the genotype variations of given virus mutations are detected reliably because the changeable sequences surrounding template stable positions to be detected fit appropriately in our flexible probe sensitive for spatial conformation. Due to this, with our rapid nucleic acid-based molecular diagnostic procedure the specificity and sensitivity of detection can be reliably improved, false results caused by sequences carrying missing and deletion mutations can be avoided, and continuous genetic changes can be monitored too. The practical realisation of the rapid, sensitive and specific molecular diagnostic procedure according to the invention is supported by the KIT prepared for this purpose, a possible version of which is shown in figure 6.

The rapid determination of viruses using nucleic acid-based molecular diagnostics according to the invention is illustrated by examples of the following epidemiologically significant viruses, without restricting our scope of protection exclusively to the detection of the pathogenic viruses listed below. Influenza A virus (Orthomyxoviridae / Influenzaviridae) - this name is used to refer to the etiological factors of seasonal influenza. In the structure of the Influenza A virion we can distinguish the envelope, below it the M matrix region rich in protein, below it the internal helical nucleocapsid core assembly with the negative-strand RNA genome inside it embedded in NP nucleoprotein and arranged in eight segments. On the basis of the above, the M matrix protein and the NP nucleoprotein are immunological determinants, which antigens characteristically distinguish the entities of the Influenza A genus from the entities of the Influenza B and Influenza C genus. Besides, the immunoserological detection of the Influenza A viral strains is based on the characteristic unique combination of the HA haemagglutinin lectin and the NA neuraminidase enzyme glycoprotein antigens situated in the envelope of the virion, playing a role in the lifecycle and pathogenicity of the virus, specific to the given viral strain. In the present patent specification the term Influenza A positivity is used to refer to the presence of Ml matrix protein 1 and M2 matrix protein 2 coding sequences. In these Influenza A positive samples the variable regions coding the HA haemagglutinin lectin, the NA neuraminidase enzyme and the NP nucleoproteins are suitable for the genotype level classification of the Influenza A virus serotype combinations, such as: Orthomyxoviridae / Influenzaviridae / Influenza A virus / H1N1 variant / swine zoonosis origin, marked by us as AHlNlswl. In current standard virological practice mainly two-step endpoint measurement RT-PCR procedures (thermocycling + electrophoresis) are used for the nucleic acid-based determination of the presence and the titre of Influenza A virus, while the Sanger sequencing method is used for genotyping. Besides these golden standard techniques, as an additional test the alternative, sensitive and specific instrumental molecular diagnostic procedure according to our invention can be used for the fast preliminary screening of samples.

Deneue virus (Flaviviridae / Flavivirus) - this name is used to refer to the etiological factors of dengue fever, dengue haemorrhagic fever, dengue shock syndrome, the DV1, DV2, DV3, DV4 type viruses that are serologically related but have different immunodeterminants.The positive-stranded single-segment RNA genome of the Dengue virion codes several non-structural proteins and specific serologically dominant structural proteins of the viral envelope, the delimiting membrane below it and the internal nucleocapsid core. In the present specification the Dengue positivity relates to the presence of the CP capsid polyprotein coding sequences. In serological types 1 , 2, 3, 4 of the Dengue positive samples as above, with the help of four reverse oligonucleotides (reverse primers) sensitive to the variable sequences of CP coding genes, the genotype classification of four serotypes of the virus can be determined, such as: Dengue virus / DV1, DV2, DV3, DV4 genotypes. In current standard virological practice, for the nucleic acid-based determination of the presence and the titre of Dengue virus two-step endpoint measurement RT-PCR procedures (thermocycling + electrophoresis) are used, and nested PCR or the Sanger sequencing method is used for genotyping. Besides these golden standard techniques, as an additional test the alternative, sensitive and specific instrumental molecular diagnostic procedure according to our invention, can be used for the fast preliminary screening of samples raising the suspicion of Dengue infection, on the 1^st or 2^nd day following infection.

Hepatitis A virus (Picornaviridae / Hepatovirus) - this name is used to refer to viruses causing infectious hepatitis, jaundice, and showing the same serotype in biogeographic distribution. In Hepatitis A virus virion, which does not have an envelope, within the nucleocapsid core there is the single-segment positive- stranded RNA genome, which contains, for example, sequences coding the three main structural proteins of the nucleocapsid core (VP-1, VP-2, VP-3) and several non-structural proteins. In the present specification the Hepatitis A virus positivity relates to the presence of genetically stable region of the viral genome, gene region VP-1 coding the HsAg hepatitis A virus surface antigen determinant. In Hepatitis A virus positive samples as above, the genotype can be described with hsag-HA V variable sequences. In Hepatitis A virus positive samples indicating the same serotype there is no further genetic differentiation. In current standard virological practice, two-step endpoint measurement RT-PCR procedure (thermocycling + electrophoresis) or PCR colorimetry (Cobas Amplicor) is used for the nucleic acid- based determination of the presence and the titre of Hepatitis A virus, while the Sanger sequencing method is used for genotyping. Besides these golden standard techniques, as an additional test the alternative, sensitive and specific instrumental molecular diagnostic procedure according to our invention can be used for the fast preliminary screening of samples. Hepatitis B virus (Hepadnaviridae) - this name is used to refer to the etiological factors of viral hepatitis causing chronic liver disease progressing to liver cirrhosis or hepatocellular carcinoma, the hepatotropic viruses having different serotypes and genotypes in biogeographical distribution. The structurally entire viral particle, the Hepatitis B virus virion is covered by the viral envelope, below which the internal nucleocapsid core carries the single-stranded circular DNA genome. In the background of the serological variants of hepatitis B virus surface antigen expressed in the envelope there is the variability of the coding s gene region sequence. In the present specification the Hepatitis B virus positivity relates to the presence of the genetically stable c gene region coding the structural constituent of the virion's nucleocapsid core. In Hepatitis B virus positive samples as above, the variable sequences of the s gene region and the currently combined sequences of the c gene region, with the help of eight pairs (pair = forward primer + reverse primer) of oligonucleotides, are suitable for differentiating the eight main genotypes of the virus, genotypes A-H of Hepatitis B virus. In current standard virological practice, two-step endpoint measurement RT-PCR procedures (thermocycling + electrophoresis) or PCR colorimetry (Cobas Amplicor) is used for the nucleic acid-based determination of the presence and the titre of Hepatitis B virus, while the Sanger sequencing method is used for genotyping. Besides these golden standard techniques, as an additional test the alternative, sensitive and specific instrumental molecular diagnostic procedure according to our invention can be used for the fast preliminary screening of samples, for determining the virus titre and for genotyping the virus.

Hepatitis C virus (Flaviviridae) - this name is used to refer to the etiological factors of the so-called non-A non-B hepatitis, and cirrhosis or hepatocellular carcinoma developing from it. Having a varying genotype according to its biogeographic distribution, the entire virus particle is covered with the viral envelope, below which the internal nucleocapsid core contains the positive- stranded single-segment RNA genome. The envelope proteins, core proteins and NSP non-structural proteins supporting virus adsorption and penetration and specific for Hepatitis C virus are all proteolytic cleavage products of a common polyprotein. In the present specification the Hepatitis C virus positivity of the sample relates to the presence 5' UTR untranscribing non-coding stable genetic region of the viral genome. In Hepatitis C virus positive samples as above, the variable sequences of the region coding NSP non-structural proteins, with the help of six pairs (pair = forward primer + reverse primer) of oligonucleotides suiting the internationally recognised six genotypes are suitable for differentiating the genotypes that are the most significant from the aspect of the therapy, genotypes la/ lb, 2a, 3a, 4a, 5a, 6a of Hepatitis c virus. In current standard virological practice, two-step endpoint measurement RT-PCR procedures (thermocycling + electrophoresis) or PCR colorimetry (Cobas Amplicor) is used for the nucleic acid- based determination of the presence and the titre of Hepatitis C virus, while the Sanger sequencing method is used for genotyping. Besides these golden standard techniques, as an additional test the alternative, sensitive and specific instrumental molecular diagnostic procedure according to our invention can be used for the fast preliminary screening of samples, for determining the virus titre and for genotyping the virus.

Human immunodeficiency virus (Lentiviridae) - this name is used to refer mostly to retroviruses damaging CD4+ T lymphocytes, monocytes, macrophages, dendritic cells and neurocytes. Below the virion's envelope the nucleocapsid core containing p24 protein covers the positive-stranded two-segment RNA genome. The specific structural constituents of the envelope are virus adsorption and penetration supporting glycoproteins (e.g. gpl20, gp41) and the matrix between the envelope and the nucleocapsid core, rich in protein. In our specification the Human immunodeficiency virus positivity of the sample relates to the presence of nef envelope protein coding sequences. In two isotypes / genotypes of the Human immunodeficiency virus positive samples as above, the variable sequences inside the nucleocapsid coding gag (gag I, II) gene, on the basis of two reverse oligonucleotides, are suitable for the genotype level classification of serotype I and II of the virus, such as: Human immunodeficiency virus HIV-I, HIV-II serotype. In current standard virological practice, two-step endpoint measurement RT-PCR procedures (thermocycling + electrophoresis) or PCR colorimetry (Cobas Amplicor) is used for the nucleic acid-based determination of the presence and the titre of Human immunodeficiency virus, while the Sanger sequencing method is used for genotyping. Besides these golden standard techniques, as an additional test the alternative, sensitive and specific instrumental molecular diagnostic procedure according to our invention can be used for the fast preliminary screening of samples raising the suspicion of HIV infection in the first 30 days of infection, and then for examining the CD4+ lymphocyte buffy-coat layer isolated from peripheral blood drawn from patients.

Human papillomavirus (Papillomaviridae) - this name is used to refer to pathogenic viruses responsible for different malformations of epithelial origin. Presently more than 130 different types of Human papillomaviruses can be distinguished; their great diversity is due to the accumulation of frequent genetic mutations accompanying the increased propensity to multiply specific for viruses. In Human papillomavirus, not having a viral envelope, inside the nucleocapsid core there is the double-stranded DNA genome, which also codes early and late proteins ensuring settlement and multiplication in the host cell. Among Human papillomavirus infections so-called low-risk (HPV-L) and so-called high-risk (HPV-H) infections can be distinguished depending on whether the current infection is caused by the viral genotype inducing milder (for example warts) or more severe (for example tumours of epithelial origin) phenotype alterations. In the present specification the Human papillomavirus positivity of the sample relates to the presence of HPV-L genotype variant E6 early protein coding e6 sequences. In Human papillomavirus positive samples as above, the viral genotype can be differentiated with unique variable proto-oncogene nucleotide sequences coding proteins of Ll-L low-risk factors and proteins of Ll-H high-risk factors. In current standard virological practice, two-step endpoint measurement RT-PCR procedures (thermocycling + electrophoresis) or PCR colorimetry (Cobas Amplicor) is used for the nucleic acid-based determination of the presence and the titre of Human papillomavirus, while nested PCR or the Sanger sequencing method is used for genotyping. Besides these golden standard techniques, as an additional test the alternative, sensitive and specific instrumental molecular diagnostic procedure according to our invention can be used for the fast preliminary screening of samples, for determining the virus titre and for genotyping the virus.

The subject of the invention is a procedure for the rapid determination of the above viruses, and other DNA viruses or RNA viruses by extending our scope of protection, using nucleic acid-based molecular diagnostics, in the course of which qualitative and quantitative determination of DNA viruses or RNA viruses in samples is performed using multiplex realtime PCR technique in such a way that in changeable genetic surroundings taxonomically characteristic stable consensus and subtype characteristic unique variable nucleotide sequences are detected simultaneously with the help of flexible oligonucleotide probes sensitive for spatial conformation planned by us. For the simultaneous determination of several templates to be detected, favourably multiplex multicolour real-time PCR technique is used.

For the favourable realisation of the procedure according to our invention practically a buffer medium is set up, an example of which is the 5x MasterMix buffer medium for the determination of Influenza A virus shown in table 1 (also see implementation example 1), or the 5x MasterMix buffer medium for the determination of Hepatitis C virus shown in table 4 (also see implementation example 2).

According to a very favourable implementation of our procedure, for the determination of highly changeable viruses degenerate - semi-degenerate annealing positions are established in the PCR probe element oligonucleotides, as it can be observed for example in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 oligonucleotides designed for determining Influenza A virus and in SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50 oligonucleotides designed for determining Hepatitis C virus.

In our procedure we use multiplex multicolour real-time PCR technique, in which in changeable sequence surroundings taxonomically characteristic stable consensus and subtype characteristic unique variable nucleotide sequences are detected simultaneously with the help of oligonucleotide probes planned by us. In order to realise this flexible oligonucleotide probes sensitive for spatial conformation have been developed, the flexibility of which is ensured in such a way that palindromic motifs are created in the single-stranded oligonucleotide chain, and by the thermodynamically weak hybridisation pairing of the semi-degenerate nucleotide bases placed in them multiple internal loops are formed. Favourably, the nucleotide sequences in the bridge abutment-like close proximity of the multiple internal loops are planned in such a way that they are captured on the stable nucleotide sequences of the template to be detected, accessible in international genetic databases, via thermodynamically strong hybridisation. This strong hybridisation annealing opens up the internal loops, as a result of which, by flexibly spanning the variable regions around the stable template sequences to be detected, our probe can be bound to variable sequences. An example of our flexible oligonucleotide probe sensitive for spatial conformation as above is the SEQ ID NO 50 oligonucleotide probe, designed for determining Hepatitis C virus la/lb genotypes.

When planning our flexible probe sensitive for spatial conformation, the unique variable regions specific for the given viral genome subtype or genotype are favourably selected in accordance with the international professional protocols, by studying the international bioinformatics databases [for example www.ncbi.nlm.nih.gov, www.embl.org, www.jdb.com]. The probe designed by us contains multiple internal loops with semi- degenerate bases suiting the variable regions of the virus to be currently detected, favourably suiting the selected variable sequences expressed in numbers in the international professional protocols.

With the flexible probes sensitive for spatial conformation according to our invention we are able to bind sequences of a length of at least 25-30 nucleotides on the template to be detected in a thermodynamically stable and selective way. To sum up it is established that with our procedure, during the rapid determination of the presence of viruses using nucleic acid-based molecular diagnostics in different samples, despite the high genetic changeability specific for viruses in general, in changeable sequence surroundings the template sequences to be detected can be detected selectively, specifically and sensitively with the help of the flexible oligonucleotide probes sensitive for spatial conformation planned by us.

During the realisation of our procedure according to the invention, favourably the multiplex multicolour detection of the several types of PCR product amplicons is solved by attaching different fluorescent labels to the PCR probe element oligonucleotide probes planned by us, and by complementary strand displacement hydrolysis signal generation. During the above multicolour fluorescent detection we favourably work on the channels with different ranges of a multichannel device, which channels, depending on the fluorescent labels used, are favourably the following: 522-537 nm / 555-567 nm / 602-615 nm / 632-647 nm / 665-775 nm / 700-710 nm. We found it necessary to prepare a MasterMix reaction medium (see table 1, table 4), which enables the fluorescent signal generation of our fluorescent-labelled oligonucleotide probe on as many real-time PCR measuring instruments as possible. For this reason, in our procedure the 5x MasterMix buffer was favourably optimised with the efficient dilutions of the stock solution of DMSO dimethyl sulfoxide, FAME fatty acid methyl ester fractions C8-C10, ANS amino- naphthalenyl-sulfonic acid additives, of a concentration of 10-15 μ§/ηι1 (see tables 1 and 4), as a result of which the fluorescence spectra of the dyes applied was widened. This Fluorescence Shift extension towards the red spectrum is a safe solution from the aspect of quantitative evaluation, and by this the emission spectra of the individual real-time PCR dyes provide emission maximum in a wider range. In other words, the emitted light intensity peak, instead of the narrow wavelength range of ~10 nm, can be detected in a wider wavelength range between 20-35 nm. As a result of this our detection system can be repeated with appropriate precision on most real-time PCR platforms (Roche, ABI, BioRad, Corbett, Stratagene, etc.) available on the market, and it ensures a reaction with excellent fluorescence characteristics. On most devices the multichannel fluorescent detection according to our procedure can be performed without fluorescence background optimisation requiring the use of ROX carboxy-X-rhodamine reference dye.

An example of the realisation of our procedure is the triplex tricolour real-time PCR determination of the Influenza A virus genotype version A/2009HlNlswl of swine origin, which is described in detail in our first implementation example. For the triplex tricolour real-time PCR determination according to the invention, on the oligonucleotide probes designed for the templates to be detected favourably 5' end fluorophore reporter and 3' end acceptor-quencher (for example NFQ = non-fluorescence quencher or BHQ = black hole quencher) components are bound with covalent bonds. Favourable realisations involve the 5' end iso-fluorescein-amino-methyl and 3' end iso-tetramethyl-NFQ, the 5' end iso- carboxyl-dichloro-dimethoxyfluorescein and 3' end NFQ, and the 5' end iso-carboxyl- dichloro-rhodamin and 3' end NFQ labelling of the probes.

Another example of a further realisation of our procedure is the determination of genotype version la/lb of the Hepatitis C virus, which is described in detail in the second implementation example. For the duplex dual-colour real-time PCR determination according to the invention, on the oligonucleotide probes designed for the templates to be detected favourably 5' end fluorophore reporter and 3' end acceptor-quencher components are bound with covalent bonds. Favourable realisations involve the 5' end iso-fluorescein- amino-methyl and 3' end iso-tetramethyl-NFQ, and the 5' end iso-carboxyl-dichloro- dimethoxyfluorescein and 3' end NFQ labelling of the probes.

During the qualitative and quantitative detection of DNA viruses or RNA viruses according to our procedure, favourably the quantitative determination is based on subtype characteristic unique variable gene sequences occurring in one single copy in the viral genome examined. An example of this is presented in our second implementation example and in figure 5 describing the analysis and quantitative determination of genotype la/ lb of Hepatitis C virus using duplex dual-colour real-time RT-PCR method.

The invention relates to a procedure for the rapid determination of Influenza A virus using nucleic acid-based molecular diagnostics, in the course of which during the qualitative and quantitative detection of the viral R A in different samples taxonomically characteristic M2 matrix protein coding stable consensus m2 and 2009 swine zoonoses subtype characteristic HA/HI haemagglutinin lectin coding hl/sw, NP nucleoprotein coding np/sw unique variable nucleotide sequences are detected simultaneously using triplex tricolour real-time PCR technique. During the detection of the nucleotide sequences mentioned above, the PCR probe element oligonucleotide primer pairs (forward - reverse) and the fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation according to the invention favourably suit the SEQ ID NO 1 - SEQ ID NO 2, SEQ ID NO 4 - SEQ ID NO 5, SEQ ID NO 7 - SEQ ID NO 8 and the SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9 sequences planned by us (see sequence listing). The templates of the hybridisation annealing of the above PCR probe elements planned by us are contained by sequences SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12 (see sequence listing).

The invention also relates to a procedure for the rapid determination of Dengue virus using nucleic acid-based molecular diagnostics, in the course of which during the qualitative and quantitative detection of the viral RNA in different samples, during the joint examination of serotypes DV1, DV2, DV3, DV4 of the virus, CP capsid polyprotein coding taxonomically characteristic stable consensus and subtype characteristic unique variable nucleotide sequences are detected simultaneously using duplex dual-colour (four genotypes in 2 x dual-colour PCR reaction) real-time PCR technique. The detection of the above nucleotide sequences according to our procedure is performed with the help of PCR probe element oligonucleotide primer pairs (forward - reverse) planned by us and fluorescent- labelled flexible oligonucleotide probes sensitive for spatial conformation according to the invention. The templates of the hybridisation annealing of the above PCR probe elements planned by us are contained by sequences SEQ ID NO 13, SEQ ID NO 17, SEQ ID NO 21, SEQ ID NO 25, favourably they suit sequences SEQ ID NO 14 - SEQ ID NO 15, SEQ ID NO 18 - SEQ ID NO 19, SEQ ID NO 22 - SEQ ID NO 23, SEQ ID NO 26 - SEQ ID NO 27 and sequences SEQ ID NO 16, SEQ ID NO 20, SEQ ID NO 24, SEQ ID NO 28 (see sequence listing).

The invention also relates to a procedure for the rapid determination of Hepatitis A virus using nucleic acid-based molecular diagnostics, in the course of which during the qualitative and quantitative detection of the viral RNA in different samples, HsAg surface immunodeterminant coding taxonomically characteristic stable consensus VP-1 and subtype characteristic unique variable hsag-HA V nucleotide sequences are detected simultaneously using duplex dual-colour real-time PCR technique. The detection of the above nucleotide sequences according to our procedure is performed with the help of PCR probe element oligonucleotide primer pairs (forward - reverse) planned by us and fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation according to the invention. The templates of the hybridisation annealing of the above PCR probe elements planned by us are contained by sequences SEQ ID NO 29, SEQ ID NO 33, favourably they suit sequences SEQ ID NO 30 - SEQ ID NO 31, SEQ ID NO 34 - SEQ ID NO 35 and SEQ ID NO 32, SEQ ID NO 36 (see sequence listing).

The invention also relates to a procedure for the rapid determination of Hepatitis B virus using nucleic acid-based molecular diagnostics, in the course of which during the qualitative and quantitative detection of the viral DNA in different samples, for differentiating the eight most significant internationally recognised genotypes A-H of the virus, the current combination of the taxonomically characteristic core region and large S protein coding c stable consensus and subtype characteristic HB surface antigen coding s unique variable nucleotide sequences is detected simultaneously using duplex dual-colour real-time PCR technique. The detection of the above nucleotide sequences according to our procedure is performed with the help of PCR probe element oligonucleotide primer pairs (forward - reverse) planned by us and fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation according to the invention. The templates of the hybridisation annealing of the above PCR probe elements planned by us are contained by sequences SEQ ID NO 37, SEQ ID NO 41, favourably they suit sequences SEQ ID NO 38 - SEQ ID NO 39, SEQ ID NO 42 - SEQ ID NO 43 and SEQ ID NO 40, SEQ ID NO 44 (see sequence listing).

The invention also relates to a procedure for the rapid determination of Hepatitis C virus using nucleic acid-based molecular diagnostics, in the course of which during the qualitative and quantitative detection of the viral RNA in different samples, during the determination of the six different internationally recognised genotypes la/lb, 2a, 3a, 4a, 5a, 6a of the virus, taxonomically characteristic 5' end UTR untranscribing non-coding stable consensus sequences and subtype characteristic NSP non-structural protein coding sequences are detected simultaneously using duplex dual-colour real-time PCR technique. During the detection of the above nucleotide sequences the PCR probe element oligonucleotide primer pairs (forward - reverse) and the fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation according to the invention favourably suit sequences SEQ ID NO 45 - SEQ ID NO 46, SEQ ID NO 48 - SEQ ID NO 49 and sequences SEQ ID NO 47, SEQ ID NO 50 planned by us (see sequence listing). The templates of the hybridisation annealing of the above PCR probe elements planned by us are contained by sequences SEQ ID NO 51, SEQ ID NO 52 (see sequence listing).

The invention also relates to a procedure for the rapid determination of Human immunodeficiency virus using nucleic acid-based molecular diagnostics, in the course of which during the qualitative and quantitative detection of the viral RNA in different samples, for the differentiation of the two most significant internationally recognised genotypes HIV-I, HIV-II of the virus, taxonomically characteristic nef envelope protein coding stable consensus and subtype characteristic gag nucleocapsid coding unique variable nucleotide sequences are detected simultaneously using duplex dual-colour real-time PCR technique. The detection of the above nucleotide sequences according to our procedure is performed with the help of PCR probe element oligonucleotide primer pairs (forward - reverse) planned by us and fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation according to the invention. The templates of the hybridisation annealing of the above PCR probe elements planned by us are contained by sequences SEQ ID NO 53, SEQ ID NO 57, favourably they suit sequences SEQ ID NO 54 - SEQ ID NO 55, SEQ ID NO 58 - SEQ ID NO 59 and sequences SEQ ID NO 56, SEQ ID NO 60 (see sequence listing).

The invention also relates to a procedure for the rapid determination of Human papillomavirus using nucleic acid-based molecular diagnostics, in the course of which during the qualitative and quantitative detection of the viral DNA in different samples, taxonomically characteristic E6 early protein coding stable consensus e6 and subtype characteristic Ll-L low-risk factor protein coding or Ll-H high-risk factor protein coding unique variable proto-oncogene nucleotide sequences are detected simultaneously using duplex dual-colour real-time PCR technique. The detection of the above nucleotide W

24 sequences according to our procedure is performed with the help of PCR probe element oligonucleotide primer pairs (forward - reverse) planned by us and fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation according to the invention. The templates of the hybridisation annealing of the above PCR probe elements planned by us are contained by sequences SEQ ID NO 61, SEQ ID NO 65, SEQ ID NO 69, favourably they suit sequences SEQ ID NO 62 - SEQ ID NO 63, SEQ ID NO 66 - SEQ ID NO 67, SEQ ID NO 70 - SEQ ID NO 71 and sequences SEQ ID NO 64, SEQ ID NO 68, SEQ ID NO 72 (see sequence listing).

When using our multiplex real-time PCR method in a mono-colour system, the detection of the several different types of PCR products produced is solved with melting curve analysis following the well-known classic SybrGreen-I fluorescent-labelling.

Furthermore, the invention also relates to a KIT enabling the practical realisation of our ^' procedure aimed at the rapid determination of viruses using nucleic acid-based molecular diagnostics, a possible version of which is shown in figure 6.

Below a description is provided for the figures and the tables.

Figure 1 - Mechanism of fluorescent signal generation determined on the basis of the distance in space between PCR probe element fluorescent-labelled oligonucleotide probe 5' end R reporter and 3' end Q acceptor-quencher sections (complementary strand displacement hydrolysis). Due to the additional exonuclease activity of the thermostable DNA polymerase synthesising the new chain, the fluorescent-labelled oligonucleotide probe hybridised to the complementary template sequences is cleaved off the template hydrolytically, the 5' end R reporter and the 3' end Q acceptor-quencher labels move away from each other in space, and so the fluorescent signal is freed and can be detected by instruments.

Figure 2 - An example of the variable surroundings around stable sequences. In Hepatitis C viral genome (genotype HCV 2a) variable nucleotide cassettes can be observed around the stable sequences.

Figure 3 - The schematic drawing of the flexible oligonucleotide probe sensitive for spatial conformation according to the invention (see the description).

Figure 4 - Influenza A virus A/2009HlNlswl genotype determination using triplex tricolour single-step real-time RT-PCR technique (see the description and implementation example

1)· Figure 5 - Hepatitis C virus HCV la/lb genotype quantitative determination using duplex dual-colour single-step real-time RT-PCR technique (see the description and implementation example 2). In the figure 5' end UTR untranscribing non-coding stable consensus and subtype characteristic NSP non-structural protein coding sequences (NSP5 in this case) according to our procedure are detected simultaneously.

Top left insert: the process curve of the duplex dual-colour PCR reactions according to our procedure.

Bottom left insert: the quantitative determination according to our procedure is based on the subtype characteristic unique variable gene sequences occurring in one single copy in the viral genome examined (see the description). In accordance with this we use Hepatitis C virus RM (reference material=RM) reference nucleic acid dilutions of a known concentration (in our example 10⁵ IU/ml, 10³ IU/ml, where IU = international unit).

Top right insert: calibration curve produced with the help of instrument software for quantitative evaluation.

Bottom right insert: nucleic acid melting curves for checking the specificity of our reaction, la = Hepatitis C virus HCV la genotype presence,

lb = Hepatitis C virus HCV lb genotype presence.

Figure 6 - Example of a possible realisation of the KIT for the practical realisation of the procedure according to the invention.

Table 1 - Influenza A virus A/2009HlNlswl genotype determination, triplex tricolour single-step real-time RT-PCR reaction, 5x MasterMix components (see implementation example 1).

Table 2 - Influenza A virus A/2009HlNlswl genotype determination, triplex tricolour single-step real-time RT-PCR reaction components for a final volume of 20 μΐ (see implementation example 1).

Table 3 - Influenza A virus A/2009HlNlswl genotype determination, triplex tricolour single-step real-time RT-PCR reaction measuring parameters (see implementation example

1) ·

Table 4 - Hepatitis C virus HCV la/lb genotype determination, duplex dual-colour single- step real-time RT-PCR reaction, 5x MasterMix components (see implementation example

2) · W 201

26

Table 5 - Hepatitis C virus HCV 1 a/lb genotype determination, duplex dual-colour single- step real-time RT-PCR reaction components for a final volume of 20 μΐ (see implementation example 2).

Table 6 - Hepatitis C virus HCV la/ lb genotype determination, duplex dual-colour single- step real-time RT-PCR reaction measuring parameters (see implementation example 2). Table 7 - Measuring parameters of the procedure according to the invention.

LOD = limit of detection, analytical sensitivity, GU = genomic unit

IMPLEMENTATION EXAMPLES

The rapid determination of viruses using nucleic acid-based molecular diagnostics with multiplex real-time PCR technique, with PCR probe element oligonucleotide sequences planned by us. In the first example triplex tricoloui-, while in the second example duplex dual colour application is described.

The reactions described in the examples are reactions optimised for capillary real-time PCR instrument (Roche Light Cycler® 2.0), which can also take place on other platforms using the possibility of fluorescence shift (see the description).

Preliminary general steps / Sampling

Body excretions (e.g. nasal or throat excretion) or blood serum are the most suitable sample sources. Sampling takes place in compliance with the public health and epidemiological prescriptions.

Samples to be processed within 12 hours are stored in the dark, at a temperature between +2 °C and +8 °C. Samples to be processed later than this are to be stored at a temperature between -80 °C and -20 °C.

Preliminary general steps / Sample preparation

When sampling body excretions the sampler is rinsed with 0.5-1 ml traditional PBS phosphate buffered saline (pH=7.2). After intensive mixing (vortex) the liquid is put in a 1.5-2 ml centrifuge tube.

When sampling blood serum 1 ml of the sample is put in a 1.5-2 ml centrifuge tube.

The above samples are centrifuged at 10,000-12,000 rpm for 5 minutes. Remove the supernatant leaving a volume of 100 μΐ above the precipitate. Then the precipitate is mixed with vortex mixing, and the sample is ready for nucleic acid isolation. Preliminary general steps /Nucleic acid isolation

Nucleic acid isolation is performed using DNA or RNA isolating kits available in commercial distribution. In the implementation example the Influenza A virus and Hepatitis C virus nucleic acid extraction is realised with RNA isolating kits (for example Roche High Pure Viral RNA kit, QIAamp Viral RNA Mini Kit). The concentration and purity of the isolated RNA is examined before further applications (for example laboratory UV-visible spectrophotometry). The isolated RNA must be stored at a temperature between -80 °C and -20 °C.

For efficient isolation and the following amplification it is favourable to use the carrier nucleic acid, in a final concentration of 1 μg/μl.

The reliability of the real-time PCR reactions according to our procedure is tested with maize invertase DNA or maize invertase RNA detectability (internal control).

Example 1 / Influenza A virus A/2009HINlswl genotype detection using triplex tricolour single-step real-time RT-PCR technique

Ideal for screening the presence and genotype of the Influenza A virus (see figure 4).

a) From the planned, lyophilised SEQ ID NO 1 - SEQ ID NO 2, SEQ ID NO 4 - SEQ ID NO 5, SEQ ID NO 7 - SEQ ID NO 8 primer pairs and from the SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9 probes 100 pmol/μΐ stock solution is made by adding PCR grade water of a quantity determined on the accompanying synthesis report.

The probe is labelled with light-sensitive fluorescence dye. During the work processes, in order to make sure that the fluorescence shift takes place, special attention must be paid to that it is possibly exposed to light of a low intensity for a time as short as possible.

b) 10 μΐ of the 100 pmol/μΐ stock solution prepared as above is pipetted into a sterile Eppendorf tube, and it is diluted further by adding 90 μΐ of PCR grade water to obtain a working solution of a concentration of 10 pmol/μΐ. It is essential to prepare this latter working solution in order to avoid any contamination of the primer probe stock solution. The primer probe working solution prepared in this way is thoroughly homogenised with a pipette.

Prepare a 5x MasterMix buffer solution, of a composition determined in table 1, without enzymes, in such a way that the primer probe solution prepared in accordance with points a-b above is added to it subsequently, suiting the final concentrations stated in table 1.

The compulsory characteristics of steps a-c are the following:

o the stock solution is stored frozen;

o when preparing the stock solution the standard laboratory protocols relating to PCR procedures (EN-ISO 20838:2006, GLP) are strictly observed;

o work is strictly performed in a cleanroom environment (ISO 14644- 1:1999), at BSL3 Biosafety Level at least.

Table 2 shows how the triplex tricolour real-time PCR reaction of the Influenza A virus is set up, for a volume of 20 μΐ per reaction. Following the order determined in table 2 measure together PCR grade water, 5x MasterMix buffer solution prepared according to table 1 , without enzymes, a mixture of RT recombinant MMLV Moloney Murine Leukemia Virus reverse transcriptase and recombinant TAQ Thermus aquaticus thermostable DNA polymerase enzymes (enzyme blend), and finally template RNA isolated from the sample. Make sure that the final concentration of the template RNA does not exceed the critical 1-25 nM value.

Perform, run the reaction according to Influenza A virus taxon and genotype specific triplex tricolour single-step real-time RT-PCR determined in table 3.

The compulsory characteristics of steps d-e are the following:

o the reactions are measured together at a temperature between +2 and + 8 °C;

o while measuring together the reactions the standard laboratory protocols relating to PCR procedures (EN-ISO 20838:2006, GLP) are strictly observed;

Example 2 / Hepatitis C virus la/ lb genotype determination using duplex dual- single-step real-time RT-PCR technique

Ideal for screening the presence and genotype of the Hepatitis C virus (see figure 5). From the planned, lyophilised SEQ ID NO 45 - SEQ ID NO 46, SEQ ID NO 48 - SEQ ID NO 49 primer pairs and from the SEQ ID NO 47, SEQ ID NO 50 probes 100 pmol/μΐ stock solution is made by adding PCR grade water of a quantity determined on the accompanying synthesis report.

The probe is labelled with light-sensitive fluorescent dye. During the work processes, in order to make sure that the fluorescence shift takes place, special attention must be paid to that it is possibly exposed to light of a low intensity for a time as short as possible.

10 μΐ of the 100 pmol/μΐ stock solution prepared as above is pipetted into a sterile Eppendorf tube, and it is diluted further by adding 90 μΐ of PCR grade water to obtain a working solution of a concentration of 10 pmol/μΐ. It is essential to prepare this latter working solution in order to avoid any contamination of the primer probe stock solution. The primer probe working solution prepared in this way is thoroughly homogenised with a pipette. Prepare a 5x MasterMix buffer solution of a composition determined in table 4, without enzymes, in such a way that the primer probe solution prepared in accordance with points a-b above is added to it subsequently, suiting the final concentrations stated in table 4.

The compulsory characteristics of steps a-c are the following:

o the stock solution is stored frozen;

o work is strictly performed in a cleanroom environment (ISO 14644- 1 : 1999), at BSL3 Biosafety Level at least.

Table 5 shows how the duplex dual-colour real-time PCR reaction of Hepatitis C virus is set up, for a volume of 20 μΐ per reaction. Following the order determined in table 5 measure together PCR grade water, 5x MasterMix buffer solution prepared according to table 4, without enzymes, a mixture of RT recombinant MMLV Moloney Murine Leukemia Virus reverse transcriptase and recombinant TAQ Thermus aquaticus thermostable DNA polymerase enzymes (enzyme blend), and finally template RNA isolated from the sample. Make sure that the final concentration of the template RNA does not exceed the critical 1-25 nM value.

Perform, run the reaction according to Hepatitis C virus taxon and genotype specific duplex dual-colour single-step real-time RT-PCR determined in table 6.

The compulsory characteristics of steps d-e are the following:

o the reactions are measured together at a temperature between +2 and + 8 °C;

o work is strictly performed in a cleanroom environment (ISO 14644- 1:1999), at BSL3 Biosafety Level at least. With our invention we introduce a method for the rapid determination of human pathogenic and other viral strains using nucleic acid-based molecular diagnostics, which, as a measuring instrument application, supplements the standard methods, is more sensitive and quicker to realise as compared to them, it provides a specific result and has well-defined technical-measuring characteristics. These well-defined measuring parameters, included in the data of table 7, besides the commonly known rapidity of real-time PCR reactions, represent the technical and also economic advantages of our invention.

The KIT for the rapid determination of the above viral strains using molecular diagnostics is for the practical realisation of our procedure, and a possible construction of it is shown in example 6.

Practically the rapid nucleic acid-based molecular diagnostic method according to our procedure can be used on most real-time PCR platforms.

The areas of application of our procedure according to the invention: plant and animal hygiene and food industry quality control laboratories, food inspection agencies, general microbiology-virology laboratories, drinking water and wastewater inspection plants, waterworks control laboratories, workplace hygiene and labour and public health.

Claims

1. Procedure for rapid determination of viruses using nucleic acid-based molecular diagnostics, in the course of which qualitative and quantitative determination of DNA viruses or RNA viruses in samples is performed using multiplex real-time PCR technique, characterised by that in changeable genetic surroundings taxonomically characteristic stable consensus and subtype characteristic unique variable nucleotide sequences are detected simultaneously in such a way that oligonucleotide probes are made, in the single-stranded chain of the probes palindromic motifs are created, semi-degenerate bases are placed in them suiting the variable regions of the virus to be currently detected, favourably suiting the variable sequences expressed in numbers in the international professional protocols, with the said semi-degenerate bases multiple internal loops are formed to ensure the structural flexibility of the probes, the bridge abutment-like close proximity of these multiple internal loops are provided with nucleotide sequences, with which the probes are bound on stable nucleotide sequences of the template to be detected, accessible in international genetic databases, via thermodynamically strong hybridisation, with this strong hybridisation the multiple internal loops are opened up, by flexibly spanning the variable regions around the stable template sequences the probes are bound to the variable sequences, and on the template to be detected a sequence of a length of at least 25-30 nucleotides bound in a thermodynamically stable and selective way is detected.

2. Procedure as in claim 1, characterised by that the PCR probe element primer pairs, that is forward and reverse oligonucleotides, are made in such a way that in their sequence they favourably contain degenerate nucleotide annealing positions too.

3. Procedure as in claim 2, characterised by that the simultaneous detection of the stable consensus and subtype characteristic unique variable nucleotide sequences of the viral genotype according to our procedure is performed in one single step.

4. Procedure as in any of claims 1-3, characterised by that in the PCR analysis according to our procedure a multicolour system is used, in which the multiplex detection of differently sized products is solved with complementary strand displacement hydrolysis signal generation of 5' end and 3' end fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation.

5. Procedure as in claim 4, characterised by that in our multiplex multicolour PCR analysis, during the detection of differently sized products we work with several different colours, on several fluorescent channels, favourably within the ranges between 522-537 nm / 555-567 nm / 602-615 nm / 632-647 nm / 665-775 nm / 700-710 nm.

6. Procedure as in claim 5, characterised by that with our fluorescent-labelling system our procedure is used on optional real-time PCR devices.

7. Procedure as in any of claims 1-6, characterised by that favourably the quantitative determination of viruses is based on subtype characteristic unique variable sequences occurring in one single copy in the viral genome examined.

8. Procedure for the rapid determination of Influenza A virus using nucleic acid-based molecular diagnostics, favourably for the realisation of the procedure in any of claims 1-7, characterised by that during the determination of the Influenza A virus RNA, separated from Influenza B and Influenza C viruses, matrix protein coding m2 stable consensus and 2009 swine zoonoses subtype characteristic haemagglutinin lectin coding hl/sw, NP nucleoprotein coding np/sw unique variable nucleotide sequences determining phenotype HlNlswl of the year 2009 are detected simultaneously, in variable sequence surroundings.

9. Procedure for determining the presence of Influenza A virus as in claim 8, characterised by that our triplex tricolour real-time PCR technique, including the PCR probe element primer pairs, that is forward and reverse oligonucleotides, and the fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation, is favourably realised with sequences SEQ ID NO 1 - SEQ ID NO 2, SEQ ID NO 4 - SEQ ID NO 5, SEQ ID NO 7 - SEQ ID NO 8 and sequences SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9 planned by us.

10. Procedure for determining the presence of Influenza A virus as in claim 9, characterised by that in our triplex tricolour real-time PCR technique the templates of the hybridisation annealing of the PCR probe element primer pairs, that is forward and reverse oligonucleotides, and the fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation, planned by us are contained by sequences SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12.

11. Procedure for the rapid determination of Dengue virus using nucleic acid-based molecular diagnostics, favourably for the realisation of the procedure in any of claims 1-7, characterised by that during the joint examination of serotypes DV1 , DV2, DV3, DV4 of the Dengue virus, CP capsid polyprotein coding stable consensus and unique variable nucleotide sequences are detected simultaneously, in variable sequence surroundings.

12. Procedure for determining the presence of Dengue virus as in claim 11, characterised by that in our duplex dual-colour real-time PCR technique, 2 x dual- colour for four genotypes, the templates of the hybridisation annealing of the PCR probe element primer pairs, that is forward and reverse oligonucleotides, and the fluorescent- labelled flexible oligonucleotide probes sensitive for spatial conformation, planned by us are contained by sequences SEQ ID NO 13, SEQ ID NO 17, SEQ ID NO 21, SEQ ID NO 25, favourably suiting sequences SEQ ID NO 14 - SEQ ID NO 15, SEQ ID NO 18 - SEQ ID NO 19, SEQ ID NO 22 - SEQ ID NO 23, SEQ ID NO 26 - SEQ ID NO 27 and SEQ ID NO 16, SEQ ID NO 20, SEQ ID NO 24, SEQ ID NO 28.

13. Procedure for the rapid determination of Hepatitis A virus using nucleic acid-based molecular diagnostics, favourably for the realisation of the procedure in any of claims 1-7, characterised by that during the determination of the Hepatitis A virus RNA, viral surface immunodeterminant coding stable consensus VP-1 and unique variable hsag nucleotide sequences are detected simultaneously, in variable sequence surroundings.

14. Procedure for determining the presence of Hepatitis A virus as in claim 13, characterised by that in our duplex dual-colour real-time PCR technique the templates of the hybridisation annealing of the PCR probe element primer pairs, that is forward and reverse oligonucleotides, and the fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation, planned by us are contained by sequences SEQ ID NO 29, SEQ ID NO 33, favourably suiting sequences SEQ ID NO 30 - SEQ ID NO 31, SEQ ID NO 34 - SEQ ID NO 35 and SEQ ID NO 32, SEQ ID NO 36.

15. Procedure for the rapid determination of Hepatitis B virus using nucleic acid-based molecular diagnostics, favourably for the realisation of the procedure in any of claims 1-7, characterised by that during the determination of the Hepatitis B virus DNA, for differentiating the eight most significant internationally recognised genotypes A-H, the current combination of the viral core region and large S protein coding c stable consensus and HB surface antigen coding s unique variable nucleotide sequences are detected simultaneously, in variable sequence surroundings.

16. Procedure for determining the presence of Hepatitis B virus as in claim 15, characterised by that in our duplex dual-colour real-time PCR technique the templates of the hybridisation annealing of the PCR probe element primer pairs, that is forward and reverse oligonucleotides, and the fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation, planned by us are contained by sequences SEQ ID NO 37, SEQ ID NO 41, favourably suiting sequences SEQ ID NO 38 - SEQ ID NO 39, SEQ ID NO 42 - SEQ ID NO 43 and SEQ ID NO 40, SEQ ID NO 44.

17. Procedure for the rapid determination of Hepatitis C virus using nucleic acid-based molecular diagnostics, favourably for the realisation of the procedure in any of claims 1-7, characterised by that during the determination of the Hepatitis C virus RNA the sequences specific for the six different internationally recognised genotypes la/lb, 2a, 3a, 4a, 5a, 6a, 5' end UTR untranscribing non-coding stable consensus sequences and NSP non- structural protein coding unique variable nucleotide sequences are detected simultaneously, in variable sequence surroundings.

18. Procedure for determining the presence of Hepatitis C virus as in claim 17, characterised by that in our duplex dual-colour real-time PCR technique the PCR probe element primer pairs, that is forward and reverse oligonucleotides, and the fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation favourably suit sequences SEQ ID NO 45 - SEQ ID NO 46, SEQ ID NO 48 - SEQ ID NO 49 and SEQ ID NO 47, SEQ ID NO 50 planned by us.

19. Procedure for determining the presence of Hepatitis C virus as in claim 18, characterised by that in our duplex dual-colour real-time PCR technique the templates of the hybridisation annealing of the PCR probe element primer pairs, that is forward and reverse oligonucleotides, and the fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation, planned by us are contained by sequences SEQ ID NO 51, SEQ ID NO 52.

20. Procedure for the rapid determination of Human immunodeficiency virus using nucleic acid-based molecular diagnostics, favourably for the realisation of the procedure in any of claims 1-7, characterised by that during the determination of the Human immunodeficiency virus RNA, nucleotide sequences specific for two different internationally recognised genotypes HIV-I, HIV-II, nef envelope protein coding stable consensus and gag nucleocapsid coding unique variable nucleotide sequences are detected simultaneously, in variable sequence surroundings.

21. Procedure for determining the presence of Human immunodeficiency virus as in claim 20, characterised by that in our duplex dual-colour real-time PCR technique the templates of the hybridisation annealing of the PCR probe element primer pairs, that is forward and reverse oligonucleotides, and the fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation, planned by us are contained by sequences SEQ ID NO 53, SEQ ID NO 57, favourably suiting sequences SEQ ID NO 54 - SEQ ID NO 55, SEQ ID NO 58 - SEQ ID NO 59 and SEQ ID NO 56, SEQ ID NO 60.

22. Procedure for the rapid determination of Human papillomavirus using nucleic acid- based molecular diagnostics, favourably for the realisation of the procedure in any of claims 1-7, characterised by that during the determination of the Human papillomavirus DNA, E6 early protein coding stable consensus e6 and Ll-L low-risk factor protein coding or Ll-H high-risk factor protein coding unique variable proto-oncogene nucleotide sequences are detected simultaneously, in variable sequence surroundings.

23. Procedure for determining the presence of Human papillomavirus as in claim 22, characterised by that in our duplex dual-colour real-time PCR technique the templates of the hybridisation annealing of the PCR probe element primer pairs, that is forward and reverse oligonucleotides, and the fluorescent-labelled flexible oligonucleotide probes sensitive for spatial conformation, planned by us are contained by sequences SEQ ID NO 61, SEQ ID NO 65, SEQ ID NO 69, favourably suiting sequences SEQ ID NO 62 - SEQ ID NO 63, SEQ ID NO 66 - SEQ ID NO 67, SEQ ID NO 70 - SEQ ID NO 71 and SEQ ID NO 64, SEQ ID NO 68, SEQ ID NO 72.

24. Procedure as in any of claims 1-3, characterised by that during our multiplex PCR technique a mono-colour system is used, where differently sized products are detected using melting curve analysis following SybrGreen-I labelling.

25. KIT for the determination of viruses using nucleic acid-based molecular diagnostics, during this for the qualitative and quantitative determination of DNA viruses or RNA viruses in samples using multiplex real-time PCR technique, for the realisation of the procedure in any of claims 1-7, characterised by that by using a calibrator and a positive control nucleic acid of a known titre, besides detecting the viruses, the titre of the detected viruses is also determined.