WO1991011531A1

WO1991011531A1 - Hybridization probes for the detection of branhamella catarrhalis strains

Info

Publication number: WO1991011531A1
Application number: PCT/EP1991/000211
Authority: WO
Inventors: Rudi Rossau; Hugo Van Heuverswyn
Original assignee: N.V. Innogenetics S.A.
Priority date: 1990-02-02
Filing date: 1991-02-01
Publication date: 1991-08-08
Also published as: AU7188491A; CA2075186A1

Abstract

The invention relates to hybridization probes for detecting Branhamella catarrhalis strains. Representative probes of the invention are characterized by the following nucleotides sequences (I):

Description

HYBRIDIZATION PROBES FOR THE DETECTION OF BRANHAMELLA CATARRHALIS STRAINS

The invention relates to hybridization probes for detecting strains belonging to the species Branhamella catarrhalis. Hereafter, the word strains also encompasses isolates or organisms contained in a biological sample.

Branhamella catarrhalis is a fastidious Gram- negative bacterium which may be involved in serious infections in human beings. Although diagnostic test procedures for this organism exist, the speed and the specificity of the detection can be considerably improved by using deoxyribonucleic acid (DNA)-probe assays. These DNA probes can, for instance, be total geno ic DNA, plasmids, or synthetic oligonucleotides and these probes may target the genomic DNA or messenger or stable ribonucleic acid (RNA) species present in biological samples. Nevertheless, while not the only approach, the use of synthetic oligonucleotides is preferred. The limited length of the oligonucleotides provides extreme specificity; a few mismatches, induce a considerable decrease in stability of the probe-target duplex. Oligonucleotides can be rapidly synthesized in large amounts, have a long shelf-life, and are easily purified and labeled.

Species-specific probes have been described for a large number of organisms including Branhamella catarrhalis (Beaulieu and Roy, Abstract No. D-249, Annual Meeting of the American Society for Microbiology, 1989) .

However, species-specific probes derived from the ribosomal RNA (rRNA) genes of Branhamella catarrhalis have not been described. The aim of the invention is to provide probe sequences deduced from variable regions within the rRNA molecules.

Such probes have an increased sensitivity because ribosomal rRNA molecules are very abundant in a cell as compared with the genome.

However, because of the high conservation of the rRNA gene, it was not expected that specific rRNA derived probes could be obtained which would be specific so as to differentiate the organism concerned from its closest neighbors. That is why for each particular case, the specificity and sensitivity of the probes have to be verified experimentally. RNA-derived probes which could have been thought to be specific for Branhamella catarrhalis turned out to be nonspecific. For example, and unexpectedly, a species-specific probe for Branhamella catarrhalis could not be found in region III or V of the 16S rRNA molecule (as defined in Rossau et al., J. Gen. Microbiol. 135: 1735-1745, 1989) , whereas in this same region, specific probes for Neisseria qonorrhoeae could be derived. This is quite unexpected because in this respect it is known that there is no organism so similarly related to Branhamella catarrhalis as, for instance, Neisseria gonorrhoeae is to Neisseria meningitidis.

The sequence of the rRNA derived probes is preferably complementary to the rRNA sequence itself, but probes with a sequence identical to the rRNA sequence, and which subsequently target the DNA molecules, can be used as well for specific detection.

These rRNA-directed probes or their complements can also be used to detect fragments obtained by enzymatic amplification of the target sequence concerned. Thus, an object of the invention is to provide rRNA derived DNA probes for detecting most, if not all, Branhamella catarrhalis strains.

Another object of the invention is to provide DNA probes for detecting Branhamella catarrhalis strains with a simple hybridization test, such as a dot-spot, a strand-displacement, a sandwich hybridization test, or a competition hybridization, without resorting to any complementary analysis, such as the Southern blot analysis.

Still another object of the invention is to provide probes and a simple method for the in vitro diagnosis of Branhamella catarrhalis strains.

"rRNA-related" as used herein refers to the fact that the probes concerned hybridize with sequences normally present in ribosomal RNAs, no matter whether said probes are themselves formed of DNA or RNA fragments, or whether they consist of cloned fragments (in the case of DNA) or of synthetic oligonucleotides.

A hybridization probe of the invention for detecting Branhamella catarrhalis strains contains: either a sequence belonging to a nucleic acid selected from the following groups of nucleic acids and which includes itself with from 15 to the maximum number of nucleotides of the selected nucleic acid. Group BC1:

TATCAGAAGC AAGCTTCCTA ACTTCGTT BC1

AACGAAGTTA GGAAGCTTGC TTCTGATA BC1IC

AACGAAGUUA GGAAGCUUGC UUCUGAUA BC1ICR

UAUCAGAAGC AAGCUUCCUA ACUUCGUU BC1R

Group BC2:

TAGCTTGGGT TTCCCCAAGT T BC2

AACTTGGGGA AACCCAAGCT A BC2IC

AACUUGGGGA AACCCAAGCU A BC2ICR

UAGCUUGGGU UUCCCCAAGU U BC2R or a variant sequence which differs from any of the preceding sequences either by:

(i) addition to or removal from any of their respective extremities of one or several nucleotides, or by

(ii) changing of one or more nucleotides within any of said sequences, or by both (i and ii) , yet provided that in any of the above circumstances the said probe still hybridizes with the same RNA or DNA target as the corresponding unmodified sequence.

Probes targeting rRNA are advantageous over probes which target the genomic DNA such as the probe described by Beaulieu and Roy, since rRNA is single- stranded and consequently directly available for hybridization, and is present in multiple copies in bacterial cells.

In order to obtain the probe sequences of the invention, rRNA genes were enzymatically amplified using the polymerase chain reaction (PCR) . Conserved sequences in the 16S or 23S rRNA were used as primers. The amplified fragments were cloned in a plasmid vector and sequenced using the dideoxy chain termination method. This approach is considerably less tedious and time-consuming than the conventional cloning procedures using genomic banks or selected restriction endonuclease fragments. Although rRNA sequences are more rapidly obtained when the sequencing reactions are performed directly on purified rRNA or PCR fragments without cloning, the sequence information generated from cloned fragments is more accurate and complete. Since the strong secondary structure of rRNA molecules introduces many ambiguities within the nucleotide sequences obtained using the dideoxy chain termination method, it is advisable to sequence both strands, which is not possible with purified rRNA. In contrast to PCR fragments, cloned rRNA gene fragments can easily be purified in large amounts, which results in clearly -readable sequencing ladders. Since one mismatch in the probe sequence may result in useless probes, accuracy is highly preferred over speed when obtaining rRNA sequences.

In the sequences given in groups BC1 and BC2, the letters stand for the following nucleotides: A: Adenylic residue C: Cytidylic residue G: Guanidylic residue T: Thymidylic residue U: Uracylic residue

By the expression "target" is meant a sequence complementary to any of the seguences of groups BC1 and BC2, as heretofore defined. In the case where the probe of the invention would comprises nucleic acid elongations on either side or both of said above defined sequences — e.g. nucleic acid fragments of cloning vector or linker fragments resulting from the cleavage of said probe out of said cloning vector — it is understood that such elongations should be selected such as to avoid the possibility that they could themselves hybridize with any other corresponding complementary nucleic acid sequence outside of the above target in a DNA of any microorganism likely to be tested by the process of this invention as later defined. Such hybridization would be of a parasitical nature and reduce the specificity of the probe.

Preferred probes consist of nucleic acid fragments formed of any of the sequences of the groups defined above, with said fragments containing from 15 to the maximum number of nucleotides of the relevant nucleic acid sequence.

It is understood that in the above nucleotide sequences (and in the other ones referred to hereafter) , the left end of the formulae always corresponds to a 5' extremity and the right end to a 3• extremity of the sequence concerned.

When reference is further made therein to a "probe of group 'X"¹ — with 'X^« from BC1 or BC2 — it should be understood that such probe has a sequence included in one of the nucleic acids belonging to that group as defined above or defined hereinafter.

It is also understood that the word "nucleotide" as used herein refers indistinctly to ribonucleotides and deoxyribonucleotides and modified nucleotides such as inosine unless otherwise specified. The expression "nucleotides" also encompasses those which further comprise modification groups, e.g. chemical modification groups which do not affect their hybridization capabilities. Such modification groups aim, for instance, at facilitating their coupling, either directly or indirectly, with suitable markers or labels for the subsequent detection of the probes so marked or labeled particularly in their hybridization products with the relevant rRNA or DNA strand, e.g. that or those initially contained in a biological sample together with other DNA(s) and/or RNA(s) .

For instance, such modification groups are recognizable by antibodies which, in turn, can be recognized specifically by other antibodies, carrying a suitable enzymatic or fluorescent or chemiluminescent label. Possible labeling procedures will be exemplified later hereinafter.

The invention also relates to probes having any of the sequences defined above and in which some nucleotides are different, provided that the different nucleotide(s) do(es) not alter the specificity of the probes defined above. Some probes may consist of one of the nucleic acids belonging to any of the groups which are set forth above or of part thereof, with said probes however including nucleotidic elongation on either sides thereof to the extent that such elongations do no alter the specificity of said probes with the genetic material of Branhamella catarrhalis.

The invention thus provides for probes which are either replicas (those designated by numbers followed by "IC" or "ICR") in terms of nucleotide sequence of sequences contained in the RNAs or DNAs of most Branhamella catarrhalis strains with occasionally a few insignificant differences in nucleotide sequences or formed of sequences, those designated by bare numbers or by numbers followed by "R", complementary to sequences included in the natural RNAs of Branhamella catarrhalis. More particularly, it should be appreciated that the target sequences in the RNAs or DNAs concerned consist in any of the following successive sequences present in most, if not all, Branhamella catarrhalis strains, subject to possible insignificant natural differences from one strain to another, whereby such natural differences are not likely to affect the hybridization specificity of the probes of this invention which such targets: AACGAAGUUA GGAAGCUUGC UUCUGAUA AACUUGGGGA AACCCAAGCU A.

The probes of the invention can also be defined as being of at least 15 oligonucleotides for detecting one or more Branhamella catarrhalis strains of which the target comprises at least 15 contiguous nucleotides to the maximum number of oligonucleotides of one of the following nucleic acid sequences : TATCAGAAGC AAGCTTCCTA ACTTCGTT, or AACGAAGTTA GGAAGCTTGC TTCTGATA, or AACGAAGUUA GGAAGCUUGC UUCUGAUA, or UAUCAGAAGC AAGCUUCCUA ACUUCGUU, or TAGCTTGGGT TTCCCCAAGT T, or AACTTGGGGA AACCCAAGCT A, or AACUUGGGGA AACCCAAGCU A, or UAGCUUGGGU UUCCCCAAGU U.

The maximum length of the probes of the invention is such that there is no cross hybridization with other bacterial taxa and is preferably of about 20 to about 50 oligonucleotides, more preferably of about 30 to about 50 oligonucleotides.

The preferred rRNA derived probes are those which are complementary to the natural rRNAs concerned, for they hybridize both with said RNAs and the corresponding DNA. Yet, those which have sequences included in said rRNAs therefore which will only hybridize with the relevant natural DNAs and therefore are less sensitive than the preceding ones, are also part of the invention.

The probes according to the invention can be formed by cloning of recombinant plasmids containing inserts including the corresponding nucleotide sequences, if need be cleaving the latter out from the cloned plasmids upon using the adequate nucleases and recovering them, e.g. by fractionation according to molecular weights. The probes according to the invention can be also be synthesized chemically, for instance by the conventional phosphotriester method.

The variants defined here above included those hybridization probes for detecting Branhamella catarrhalis strains which target one of the sequences defined hereunder or their corresponding complementary sequence, when the hybridization medium, or the wash medium, or both as appropriate are the following: hybridization medium: containing about 3 x SSC, (SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) about 25 mM of phosphate buffer pH 7.1, 20% deionized formamide 0.02% ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, and about 0.1 mg/ml sheared, denatured salmon sperm DNA, wash medium: containing about 3 x SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide, wherein the target sequence and the corresponding relevant hybridization temperature (HT) and wash temperature (WT) respectively are as follows: AACGAAGUUA GGAAGCUUGC UUCUGAUA HT and/or WT: 50^βC AACUUGGGGA AACCCAAGCU A HT and/or WT: 35°C.

The invention also relates to a process for detecting Branhamella catarrhalis strains in a biological sample, wherein said process comprises contacting said biological sample in which the nucleic acids (DNAs and RNAs) have been made accessible to hybridization, if need be under suitable denaturation conditions, with a probe of the invention under conditions enabling hybridization between the probe and complementary nucleic acids of the strains, which may be present in the sample, and detecting the hybrids possibly formed.

The process of the invention enables the discrimination of Branhamella catarrhalis from any other organism such as yeast, fungi, protozoa, other bacterial strains, human cells which are liable to be present in the sample in which Branhamella catarrhalis is sought.

The process relates to the detection of Branhamella catarrhalis strains being directly in the sample or after the strain has been cultured. The detection of a hybrid can be interpreted as meaning that an infection due to Branhamella catarrhalis was present in the biological sample, when any of the probes of groups BC1 or BC2 is respectively being used.

According to an advantageous embodiment of the invention, in the process for detecting Branhamella catarrhalis strains, the probes used are the ones hybridizing both with DNA globally and RNA of the Branhamella catarrhalis strains, which may be present in the biological sample.

The hybridization conditions can be monitored by relying upon several parameters, e.g. hybridization temperature, the nature and concentration of the components of the media, and the temperature under which the hybrids formed are washed.

The hybridization and wash temperature is limited in upper value, according to the probe (its nucleic acid composition, kind and length) and the maximum hybridization or wash temperature of the probes described herein is about 35^βC to 50"C. At higher temperatures duplexing competes with the dissociation (or denaturation) of the hybrid formed between the probe and the target.

A preferred hybridization medium contains about 3 x SSC, (SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), about 25 mM of phosphate buffer pH 7.1, and 20% deionized formamide, 0.02% ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone and about 0.1 mg/ l sheared denatured salmon sperm DNA.

A preferred wash medium contains about 3 x SSC, 25 mM phosphate buffer pH 7.1 and 20% deionized formamide. Other hybridization or wash media can be used as well.

However, when modifications are introduced, be it either in the probes or in the media, the temperatures at which the probes can be used to obtain the required specificity should be changed according to known relationships, such as those described in the following reference: B.D. Hames and S.J. Higgins, (eds.). Nucleic Acid Hybridization: A Practical Approach, IRL Press, Oxford, U.K., 1985.

The general process for detecting Branhamella catarrhalis strains according to the invention can be carried out by suitably adjusting the hybridization temperature to a value at which hybridization is specific; in such a case, washing under more stringent conditions is not necessary.

According to another embodiment of the process of the invention, the hybridization temperature need not necessarily be adjusted to the value at which hybridization is specific and, in particular, can be lower than the temperature at which hybridization is specific, provided that washing is carried out at a temperature corresponding to the value at which hybridization is specific.

In a process embodiment for detecting Branhamella catarrhalis strains (and for distinguishing them from other bacterial taxa) with a probe of group BC1 the hybridization temperature is suitably adjusted to a range of about 50°C and/or the wash temperature to a range of about 50°C, the media being those defined above.

In another process embodiment for detecting Branhamella catarrhalis strains the probe used is any from group BC2 above defined, the hybridization temperature is suitably adjusted to a range of about 35°C and/or the wash temperature to a range of about 35°C.

The invention further relates to a kit for detecting specifically Branhamella catarrhalis strains containing: at least one probe selected from among any of those that are specific for Branhamella catarrhalis as defined above, i.e. a probe from groups BC1 or BC2; the buffer or components necessary for producing the buffer enabling an hybridization reaction between these probes and only the DNAs and/or RNAs of a strain of Branhamella catarrhalis to be carried out; and optionally containing means for detecting the hy¬ brids resulting from the preceding hybridization. In some particular applications, other probes as those defined above (group BCl and BC2) can be used for the detection of Branhamella catarrhalis strains by an hybridization procedure.

The following rRNA-derived oligonucleotide probes can be caused to hybridize to most, if not all,

Branhamella catarrhalis strains:

Group BC3:

TCTAATAGCG AGAGCTAAAA GCCCCC BC3 GGGGGCTTTT AGCTCTCGCT ATTAGA BC3IC GGGGGCUUUU AGCUCUCGCU AUUAGA BC3ICR UCUAAUAGCG AGAGCUAAAA GCCCCC BC3R

Group BC4:

GTCAGGGCTT ATGGGTATTA ACCATAAGCT T BC4 AAGCTTATGG TTAATACCCA TAAGCCCTGA C BC4IC AAGCUUAUGG UUAAUACCCA UAAGCCCUGA C BC4ICR GUCAGGGCUU AUGGGUAUUA ACCAUAAGCU U BC44R

Group BC5:

CGTCACTAAG TCTTTAAAAG ACCCAACGAC TG BC5 CAGTCGTTGG GTCTTTTAAA GACTTAGTGA CG BC5IC CAGUCGUUGG GUCUUUUAAA GACUUAGUGA CG BC5ICR CGUCACUAAG UCUUUAAAAG ACCCAACGAC UG BC5R

Group BC6:

CTTAATATGT CGCTTAAACA GTT BC6 AACTGTTTAA GCGACATATT AAG BC6IC AACUGUUUAA GCGACAUAUU AAG BC6ICR CUUAAUAUGU CGCUUAAACA GUU BC6R These probes are new and are also part of the invention.

The preferred hybridization and wash temperatures are 50°C, 50^βC, 50°C and 40^βC for BC3, BC4, BC5, and BC6, respectively, the media being those defined above.

Under the hybridization and wash conditions enabling hybridization with nucleic acids from Branhamella catarrhalis, the probes from group BC3 to BC6 also hybridize with nucleic acids from a limited number of non-Branha ella catarrhalis strains such as Moraxella nonliquefaciens, Moraxella lacunata, Neisseria ovis, and Neisseria caviae. The probes from group BC3 to BC6 can be used for the detection of Branhamella catarrhalis strains in specimens where the above mentioned organism are not likely to be found, or can be ruled out by using one or more additional test such as a Gram stain. Also in a sandwich-hybridization format as outlined below, these probes can be convenient for the specific detection of Branhamella catarrhalis.

The probes of the invention can be used in a sandwich hybridization system which enhances the specificity of a nucleic acid probe-based assay.

The principle and the use of sandwich hybridizations in a nucleic acid probe-based assay have been already described (e.g.: Dunn and Hassel, Cell, 12: 23-36; 1977; Ranki et al., Gene, 21: 77-85;. 1983). Although direct hybridization assays have favorable kinetics, sandwich hybridizations are advantageous with respect to a higher signal to noise ratio. Moreover sandwich hybridizations can enhance the specificity of a nucleic acid probe based assay. If properly designed, a sandwich hybridization assay indeed maximizes the specificity of a nucleic acid probe based test when using two probes recognizing two different nucleic acid stretches of one and the same organism. The only demands which must be met are that both probes (i) hybridize to the same nucleic acid molecule of the target organism and (ii) do not hybridize to the same non-target organisms.

For two given probes I and II, the sandwich hybridization system can be described as follows: Probe No. I hybridizes to nucleic acid from organisms A and B (not with C) ;

Probe No. II hybridizes to nucleic acid from organisms A and C (not with B) .

Since it is absolutely required that both probes hybridize to the target nucleic acid, a detectable signal will be generated only if nucleic acid from organism A is present in the sample. It is obvious that if one of the probes is specific for the organism to be detected, the other probe can be composed of any specific or non-specific sequence provided that it hybridizes to the same target molecule as the first probe.

The probes of the invention — groups BCl to BC6 — can be combined in a sandwich hybridization assay which is highly specific for Branhamella catarrhalis.

An advantageous combination of probes is constituted by probe BCl and probe BC2. other advantageous combinations of the probes are such that one of the probes is either BCl or BC2 and the second probe is chosen from among BC3, BC4, BC5, or BC6, and more preferably from among BC3, BC4, or BC5. In case BC6 is used as one of the probes, the target is genomic DNA.

Hereunder some advantageous combinations and their corresponding hybridization and wash temperatures (HT & WT) are given by way of example and not by way of limitation: Probe I

BCl BCl BCl BCl BC2 BC2 BC2

All these combinations have the 16S rRNA molecule as target.

In the sandwich hybridization process the probes can be added simultaneously or not to the biological sample in which the target DNA or RNA is sought.

The invention also relates to a kit for sandwich hybridization assay, for the detection in vitro of Branhamella catarrhalis strains in a biological sample, with said kit containing: at least one of the probes or one of the combinations of probes specific for the organisms of interest as above defined. the buffer or components necessary for producing the buffer enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Branhamella catarrhalis to be carried out, and optionally containing means for detecting the hybrids resulting from the preceding hybridization. The oligonucleotides of the invention can be used either as amplification primers in the polymerase chain reaction technique (PCR; Mullis and Faloona, Methods in Enzymology 155:335-350, 1987) to generate specific enzymatically amplified fragments and/or as probes to detect fragments amplified between bracketing oligonucleotide primers. The specificity of a PCR-assisted hybridization assay can be controlled at different levels.

The amplification process, or the detection process, or both can be specific. The latter case, giving the highest specificity, is preferred. Such a highly specific PCR-assisted test can be developed using the probes of the invention.

Using the probes of groups BCl and BC2 Branhamella catarrhalis strains can be specifically identified using a competition hybridization protocol. In such a test the target nucleic acid is allowed to hybridize either (i) with the detection probe in solution or with (ii) the capture probe bound to a solid support. By monitoring the amount of detection probe that can still be hybridized to the capture probe after the first hybridization has been carried out the presence of target molecules can be determined.

CONDITIONS OF THE USE OF PROBES:

The probes of the invention are advantageously labeled. Any conventional label can be used. The probes can be labeled by means of radioactive tracers such as ³²P, ³⁵S, ¹²⁵I, ³H, and ¹⁴C.

The radioactive labeling can be carried out according to any conventional method such as terminal labeling at the 3' or 5' position with the use of a radiolabeled nucleotide, a polynucleotide kinase (with or without dephosphorylation by a phosphatase) , a terminal transferase, or a ligase (according to the extremity to be labeled) . One of the probes of the invention can be the matrix for the synthesis of a chain consisting of several radioactive nucleotides or of several radioactive and nonradioactive nucleotides.

The probes of the invention can also be prepared by chemical synthesis using one or several radioactive nucleotides. Another method for radioactive labeling is a chemical iodination of the probes of the invention which leads to the binding of several ¹²⁵I atoms on the probes.

If one of the probes of the invention to be used for hybridization is made radioactive with a nonradioactive RNA or DNA, the method of detecting hybridization will depend on the radioactive tracer used.

Generally, autoradiography, liquid scintillation, gamma counting or any other conventional method enabling one to detect an ionizing ray issued by the radioactive tracer can be used.

Nonradioactive labeling can also be used by associating the probes of the invention with residues having: immunological properties (e.g. antigen or hapten) , a specific affinity for some reagents (e.g. ligand) , properties providing a detectable enzymatic reaction (e.g. enzyme, co-enzyme, enzyme substrate or substrate taking part in an enzymatic reaction) , or physical properties such as fluorescence or emission or absorption of light at any wavelength. Antibodies which specifically detect the hybrids formed by the probe and the target can also be used.

A nonradioactive label can be provided when chemically synthesizing a probe of the invention, the adenosine, guanosine, cytidine, thymidine and uracyl residues thereof being liable to be coupled to other chemical residues enabling the detection of the probe or the hybrids formed between the probe and a complementary DNA or RNA fragment.

However, the nucleotidic sequence of the probe when modified by coupling one or more nucleotides to other chemical residues, would be the same as the nucleotide sequence of one of the probes of the invention. The invention also relates to processes for detecting RNA and/or DNA with the probes of the invention by hybridization, which have been labeled and can be detected as described above. In this regard, conventional methods of hybridization can be used.

For detecting cells which originate from or are themselves constituting living organisms, the RNA and/or DNA of these cells are made accessible by partial or total lysis of the cells, if need be, using chemical or physical processes, and contacted with one or several probes of the invention which can be detected. This contact can be carried out on an appropriate support such as a nitrocellulose, cellulose, or nylon filter in a liquid medium or in solution. This contact can take place under suboptimal, optimal conditions or under restrictive conditions (i.e. conditions enabling hybrid formation only if the sequences are perfectly homologous at a specific stretch of the molecule) . Such conditions include temperature, concentration of reactants, the presence of substances lowering the optimal temperature of nucleic acid pairing (e.g. formamide, dimethylsulfoxide and urea) and the presence of substances apparently lowering the reaction volume and/or accelerating hybrid formation (e.g. dextran sulfate, polyethylene glycol or phenol) .

The elimination of probe of the invention which has not hybridized can be carried out by washing with a buffer solution of appropriate ionic strength and at an appropriate temperature, with or without treatment with SI nuclease or any other enzyme digesting single-strand DNA or RNA but not digesting DNA-RNA hybrids or double- strand DNA.

In a liquid medium, the hybrids of the probe of the invention paired to the cellular DNA or RNA fragments can be separated from the rest of the liquid medium in different ways, e.g. by chromatography over hydroxyapatite. Then the hybridized probes are detected by means of the label on the probe.

In order to target the chromosomal DNA fragments carrying the genes coding for the RNA fragments from which the labeled probes of the invention derive, after treating the DNA by one or several enzymes and denaturation of DNA fragments (i.e. separation of both chains) , one of the probes of the invention is contacted with the DNA fragments under the conditions enabling hybridization. After the time necessary to get complete the hybridization process, the non-hybridized fragments are separated from the hybridized fragments and the label is detected as has been described above for the detection of the cells.

Generally speaking, the different probes of the invention can also be contained in recombinant DNA, enabling their cloning, if the presence of a heterologous DNA is not a hindrance for the specificity of the probes in the encompassed uses.

Figures 1A and IB represent the partial nucleotide sequence of the cloned rRNA gene of Branhamella catarrhalis ITG 4197. More precisely, the linear conformation from 5• to 3' corresponds to the nucleotide sequence of the upper (sense) strand of the rRNA gene, with Figure 1A corresponding to a partial sequence of the 16S rRNA gene and Figure IB corresponding to a partial sequence of the 23S rRNA gene.

The strain of Branhamella catarrhalis used if Branhamella catarrhalis ITG 4197 which is available a the Institute of Tropical Medicine in Antwerp, Belgium. Probes of groups BCl to BC5 are derived from the 16S rRNA gene, whereas probes of group BC6 are derived from the 23S rRNA gene. EXAMPLES:

The examples hereafter relate to the preparation of the probes of the invention, the experimental results with respect to the specificity and sensitivity of the probes and their use for diagnostic purposes.

The methods used are essentially the same as described by Rossau et al., J. Gen. Microbiol.; 135: 1735-1745, 1989; or in the European patent application No. 8940/- 045.3 unless otherwise stated.

The enzymatic amplification of rRNA gene fragments of about 500 to 4500 basepairs was obtained by the polymerase chain reaction (PCR) technique performed according to the recommendations given in the "Gene Amp" kit of Perkin Elmer Cetus. Oligonucleotides corresponding to conserved or semi-conserved regions in the rRNA molecules were used as PCR primers.

Branhamella catarrhalis, also known as Moraxella catarrhalis or Neisseria catarrhalis, is a fastidious bacterium which is rather inert biochemically. Its important pathogenic potential has been recognized recently. Branhamella catarrhalis seems to be frequently involved in serious infections of the respiratory tract. The diagnosis of Branhamella catarrhalis requires culture of the organism, which may be hampered by overgrowth caused by less fastidious microorganisms, and a battery of phenotypical tests to distinguish this organisms from commensals such as Neisseria species present in the oral cavity.

In some instances the phenotypical test are inconclusive as to the identity of the presumptive Branhamella catarrhalis isolate since there are only a limited number of tests which differentiate Branhamella catarrhalis from phenotypically similar bacteria. The use of a DNA probe based assay may considerably simplify the laboratory diagnosis of Branhamella catarrhalis. Moreover, there is some heterogeneity within the species Branhamella catarrhalis which can be recognized using the probes of the invention.

1. Detection using dot-blot hybridization :

Part of the rRNA gene of Branhamella catarrhalis ITG 4197 was enzymatically amplified by the PCR technique and cloned in a plasmid vector. The fragment was subsequently sequenced by the dideoxy chain termination technique. The sequence is shown in Fig. 1. Oligonucleotide sequences of the non-conserved areas of the rRNA genes were selected. The following oligonucleotides were chemically synthesized: TATCAGAAGC AAGCTTCCTA ACTTCGTT BCl

TAGCTTGGGT TTCCCCAAGT T BC2

TCTAATAGCG AGAGCTAAAA GCCCCC BC3

The oligonucleotides were ³²P-labeled at their 5' ends or tailed at their 3' ends with digoxigenine labeled UTP using terminal transferase and used as hybridization probes.

As target, dot-spotted denatured genomical DNA or lysed cellular material (immobilized on nylon membranes) from a large number of Branhamella catarrhalis strains obtained from different locations as well as several strains of other bacterial taxa was used. The hybridization mixture used was either 3 X SSC, 25 mM potassium phosphate buffer, pH 7, deionized formamide (20%, v/v) , ficoll (0.02%, w/v) , bovine serum albumin (0.02%, w/v), polyvinylpyrrolidone (0.02%, w/v) and sheared, denatured salmon sperm DNA (0.1 mg ml^"1), or the solution given in the protocol sheet of the nonradioactive DNA labeling and detection kit (Boehringer Mannheim) except that 3 X SSC (1 X SSC is: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) instead of 5 X SSC was used and formamide was added up to 20% (v/v) . The wash solution contained 3 X SSC, 20% formamide, and 25 mM phosphate buffer pH 7.1.

The hybridization results with these probes are summarized below. The hybridization and wash temperatures were 50°C for BCl and BC3 and 35^βC for BC2 using the media defined above.

Taxon No. positive strains /No. strains tested

BCl BC2 BC3

Unequivocal B. catarrhalis strains 61/61 30/30 61/61

Aberrant B. catarrhalis strains:

strains 0/22 0/21 4/21

Unequivocal Branhamella catarrhalis strains are strains which exhibit phenotypical traits which do not deviate from those of Branhamella catarrhalis as described in Bergey's Manual of Systematic Bacteriology (Vol. I , Williams & Wilkins Co. Baltimore pp. 288-309) and which have high total DNA:DNA hybridization homologies with the Branhamella catarrhalis type strain (ATCC 25238) .

The aberrant Branhamella catarrhalis strains are those strains which were received as Branhamella catarrhalis strains but were found to be different from the unequivocal strains with respect to the genotype and phenotype. The exact taxonomic status of these strains remains to be determined. However, using some of the probes of the invention, the aberrant strains can be identified and distinguished from other organisms as indicated in the table below:

Groups Hybridization with

BCl BC2 BC3

1. Unequivocal

B. catarrhalis strains + + +

2. strain NCTC 4103 + +

3. strains N7, 018B and U33/U34 +

4. Non-B. catarrhalis strains - - -/+

The non-Branhamella catarrhalis strains tested are:

Moraxella lacunata ATCC 17967

Moraxella lacunata ATCC 17952

Moraxella bovis ITG 1601

Moraxella nonlicfuefaciens ATCC 19975

Neisseria cuniculi ITG 3388

Neisseria ovis NCTC 11227

Neisseria caviae ATCC 14659 Alysiella sp. ATCC 29468 Moraxella osloensis LMG 1043 Moraxella osloensis ATCC 17974 "Moraxella paraphenylpyruvica" LMG 5125 "Moraxella camembertii" LMG 7022 Psychrobacter immobilis LMG 6784 Acinetobacter calcoaceticus ATCC 23055 Escherichia coli B Escherichia coli MC 1061 Haemophilus influenza NCTC 8143 Eikenella corrodens NCTC 10596 Xanthomonas maltophilia LMG 958 Xanthomonas campestris LMG 568 Neisseria sp. D12 Neisseria sp. S17

2. Detection of rRNA using sandwich-hybridization rRNA extracted from Branhamella catarrhalis ITM 4197 was hybridized with an oligonucleotide probe with the following sequence: 5*-ACTGCTGCCTCCCGTAGGAGTCTGG-3' which was bound to the surface of a microtiter plate well. The hybridization was done for 1 1/2 h at 60"C in the hybridization mixture defined above (point l) from which formamide was omitted. The wells were washed 3 to 4 times with 300 μl phosphate buffered saline with 0.05% Tween-20 (PBS/Tween) . The 200 μl hybridization mix containing 2 pmol digoxigenine labeled probe BCl was added and hybridization was done at 60"C for 1 h. Washing was performed at the same stringency for 20 min. The detection protocol as described in the Boehringer labeling and detection kit was followed except that paranitrophenyl-phosphate (3.6 mg/ l) was used as substrate for alkaline phosphatase.

The results (OD reading at 405 nm) after 1 h of colordevelopment are given in the table below: f ol RNA

240

180

120

60

30

10

6

0 (blank)

Thirty fmol rRNA can be reliably detected using a nonradioactive sandwich hybridization format in microtiter plate wells.

3. Detection of Branhamella catarrhalis cells using a competition hybridization assay

Serial dilutions of cells of Branhamella catarrhalis ITM 4197 were made in Heart Infusion Broth (Difco) . The cells were lysed by addition of sodium dodecyl sulfate to a final concentration of 0.5%. Simultaneously aliquots of the same dilutions were plated on blood agar plates for cell count [expressed as CPU (Colony forming units) ] .

The composition of the solution was adjusted to 3 X SSC, 25 mM phosphate buffer pH 7.1 and 0.05 pmol of digoxigenine labeled probe BCl was added. The hybridization proceeded for 3 h at 60*C after which the mixture was transferred to a microtiter plate well in which a probe complementary in sequence to probe BCl was fixed to the surface. A further incubation was done at 60^βC for lh. After washing the wells, the detection was performed as described in point 2 here above. The results of the optical readings after 1 h of colordevelopment are shown in the table below:

Since a drop in signal of 10% or more is significant in this kind of test, 2 x 10⁵ CFU of Branhamella catarrhalis cells could be detected. No significant detection was obtained for 2 x 10⁴ CFU or less (ratios > 0.90).

4. Detection of Branhamella catarrhalis using PCR and competition hybridization.

One ng genomical DNA of Branhamella catarrhalis (ITM 4197) was enzymatically amplified with the following primers:

- 5'-TGGCTCAGATTGAACGCTGGCGGC-3^«

- 5'-TCTAATAGCGAGAGCTAAAAGCCCCC-3' in a total volume of 100 μl.

Thirty cycles consisting of 1 min denaturation at 95"C, 1 min annealing at 50"C and 1 min elongation at 72°C were performed.

The presence of specifically amplified material was determined using a competition hybridization protocol with digoxigenine labeled probe BCl as described in point 3. The results after 1/2 h of colordevelopment are shown in the table below:

μl of PCR mix used OP 405 ratio sample/control

0 5

10 15

From these figures it is obvious that a significant positive signal was obtained when 10 and 15 μl of the PCR mix was used.

Claims

1. Probe, for detecting one or more Branhamella catarrhalis strains, containing: either a sequence belonging to a nucleic acid selected from the following group of nucleic acids and which includes itself with from 15 to the maximum number of nucleotides of the selected nucleic acid:

Group BCl:

TATCAGAAGC AAGCTTCCTA ACTTCGTT BCl AACGAAGTTA GGAAGCTTGC TTCTGATA BC1IC AACGAAGUUA GGAAGCUUGC UUCUGAUA BC1ICR AUCAGAAGC AAGCUUCCUA ACUUCGUU BC1R

Group BC2:

TAGCTTGGGT TTCCCCAAGT T BC2 AACTTGGGGA AACCCAAGCT A BC2IC AACUUGGGGA AACCCAAGCU A BC2ICR UAGCUUGGGU UUCCCCAAGU U BC2R

or a variant sequence which differs of any of the preceding sequences either by:

* (i) addition to or removal from any of their respective extremities of one or several nucleotides; or by

* (ii) changing of one or more nucleotides within any of said sequences; or by

* both; (i and ii) yet provided that in any of the above circumstances the said probe still hybridizes with the same RNA or DNA target as the corresponding unmodified sequence.

2. Probe, according to Claim 1, for detecting one or more Branhamella catarrhalis strains containing a sequence belonging to a nucleic acid selected from the following group of nucleic acids, and which includes itself from 15 to the maximum number of nucleotides of the selected nucleic acids:

Group BCl:

TATCAGAAGC AAGCTTCCTA ACTTCGTT BCl AACGAAGTTA GGAAGCTTGC TTCTGATA BC1IC AACGAAGUUA GGAAGCUUGC UUCUGAUA BC1ICR UAUCAGAAGC AAGCUUCCUA ACUUCGUU BC1R

3. Probe, according to Claim 1, for detecting one or more Branhamella catarrhalis strains containing a sequence belonging to a nucleic acid selected from the following group of nucleic acids, and which includes itself from 15 to the maximum number of nucleotides of the selected nucleic acids:

Group BC2:

4. A nucleic acid probe of at least 15 nucleotides in length for detecting one or more Branhamella catarrhalis strains of which the target comprises at least 15 contiguous nucleotides to the maximum number of oligonucleotides of one of the following nucleic acid sequences:

TATCAGAAGC AAGCTTCCTA ACTTCGTT, or AACGAAGTTA GGAAGCTTGC TTCTGATA, or AACGAAGUUA GGAAGCUUGC UUCUGAUA, or UAUCAGAAGC AAGCUUCCUA ACUUCGUU. ^"

5. A nucleic acid probe of at least 15 nucleotides in length for detecting one or more Branhamella catarrhalis strains of which the target comprises at least 15 contiguous nucleotides to the maximum number of oligonucleotides of one of the following nucleic acid sequences:

TAGCTTGGGT TTCCCCAAGT T, or

AACTTGGGGA AACCCAAGCT A, or

AACUUGGGGA AACCCAAGCU A, or

UAGCUUGGGU UUCCCCAAGU U.

6. Process for detecting Branhamella catarrhalis strains in a biological sample from other organisms, wherein said process comprises contacting said biological sample in which the nucleic acids (DNAs and/or RNAs) of the strains have been made accessible to hybridization, if need be, under suitable denaturation conditions with a probe according to any one of Claims 1 to 5 under conditions enabling hybridization between the probe and complementary nucleic acids of the Branhamella catarrhalis strains, which may be present in the sample, and detecting the hybrids possibly formed.

7. Process for detecting Branhamella catarrhalis, according to Claim 6, wherein the preferred hybridization medium or the wash medium or both are the following ones:

* hybridization medium: containing about 3 x SSC, (SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) about 25 mM of phosphate buffer pH 7.1, 20% deionized formamide 0.02% ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, and about 0.1 mg/ l sheared, denatured salmon sperm DNA,

* wash medium: containing about 3 x SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide. The target sequence and the corresponding relevant hybridization temperature (HT) and wash temperature (WT) respectively are as follows:

AACGAAGUUA GGAAGCUUGC UUCUGAUA HT and/or WT: 50°C AACUUGGGGA AACCCAAGCU A HT and/or WT: 35°C.

8. Process for detecting Branhamella catarrhalis strains in a biological sample, according to anyone of Claims 6 or 7, wherein the probes used are the ones hybridizing both with DNA and RNA of Branhamella catarrhalis strains which may be present in the biological sample.

9. Process for detecting Branhamella catarrhalis in a biological sample, according to anyone of Claims 6 to 8, wherein:

* the hybridization medium contains about 3 x SSC, (SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) about 25 mM of phosphate buffer pH 7.1, 20% deionized formamide 0.02% ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, and about 0.1 mg/ml sheared, denatured salmon sperm DNA,

* the wash medium contains about 3 x SSC, 25 mM phosphate buffer pH 7.1, and 20% deionized formamide, and wherein the probe used is: anyone of the probes of Claims 2 or 4, the hybridization temperature being suitably adjusted to the range of about 50^βC and the wash temperature being suitably adjusted to the range of about 50"C, or anyone of the probes of Claims 3 or 5, the hybridization temperature being suitably adjusted to the range of about 35 " C and the wash temperature being suitably adjusted to the range of about 35*C.

10. Process for detecting Branhamella catarrhalis strains, wherein said process comprises contacting said biological sample, in which the nucleic acids (DNAs and/or RNAs) have been made accessible to hybridization, if need be, under suitable denaturation conditions with two probes, targeting the same nucleic acid molecule, and of which at least one is specific for Branhamella catarrhalis and which is selected from any one of the probes of Claims 1 to 5, or one of the probes defined hereunder:

Group BC3

Group BC4:

Group BC5:

Group BC6:

CTTAATATGT CGCTTAAACA GTT BC6 AACTGTTTAA GCGACATATT AAG BC6IC AACUGUUUAA GCGACAUAUU AAG BC6ICR CUUAAUAUGU CGCUUAAACA GUU BC6R

whenever required under hybridization and washing conditions, and adjusted so as to ensure specific hybridization with complementary nucleic acids of the Branhamella catarrhalis strains which may be present in the sample, yet not with complementary DNA or RNA of other organisms and detecting the hybrids possibly formed, one of the probes being preferably among the probes of group BCl or BC2 and the other probe being preferably chosen among the probes of group BC3 or BC4 or BC5, preferably in the following combination and under the following hybridization and wash temperatures (HT and WT) : probes of group BCl and probes of group BC2:

HT and WT: about 35 ° C probes of group BCl and probes of group BC3:

HT and WT: about 50 " C probes of group BCl and probes of group BC4:

HT and WT: about 50°C probes of group BCl and probes of group BC5:

HT and WT: about 50^βC probes of group BC2 and probes of group BC3:

HT and WT: about 35^βC probes of group BC2 and probes of group BC4:

HT and WT: about 35"C probes of group BC2 and probes of group BC5:

HT and WT: about 35°C

11. Process for detecting Branhamella catarrhalis strains using the polymerase chain reaction technique in which at least one of the probes of Claims 1 to 5 is used as amplification primer and/or detection probe of the amplified product.

12. Process for detecting Branhamella catarrhalis strains in a competition hybridization protocol using probes of groups BCl and BC2 of Claim 1, wherein :

- the target nucleic acid is allowed to hybridize either (i) with the detection probe in solution or with (ii) the capture probe bound to a solid support;

- monitoring the amount of detection probe that can still be hybridized to the capture probe after the first hybridization has been carried out which enables to determine the presence of target molecules.

13. Kit for the detection _in vitro of a large number, preferably all Branhamella. catarrhalis strains in a biological sample, with said kit containing: at least one probe selected among any of those according to Claims 1 to 5; the buffer or . components necessary for producing the buffer enabling a hybridization reaction between these probes and the DNAs and/or RNAs of a large number of, and preferably, all strains of Branhamella catarrhalis to be carried out;

- with appropriate means for detecting the hybrids resulting from the preceding hybridization.

14. Kit for sandwich hybridization assay, for the detection in vitro of Branhamella catarrhalis strains in a biological sample, with said kit containing: at least two probes targeting the same nucleic acid molecule, and of which at least one is specific for Branhamella catarrhalis and which is selected from any one of the probes of Claims 1 to 5, or one of the probes defined hereunder:

Group BC3:

TCTAATAGCGA GAGCTAAAAG CCCCC BC3 GGGGGCTTTTA GCTCTCGCTA TTAGA BC3IC GGGGGCUUUUA GCUCUCGCUA UUAGA BC3ICR UCUAAUAGCGA GAGCUAAAAG CCCCC BC3R

Group BC4:

Group BC5:

Group BC6:

CTTAATATGT CGCTTAAACA GTT BC6 AACTGTTTAA GCGACATATT AAG BC6IC AACUGUUUAA GCGACAUAUU AAG BC6ICR CUUAAUAUGU CGCUUAAACA GUU BC6R the buffer or components necessary for producing the buffer enabling hybridization reaction between these probes and the DNAs and/or RNAs of a strain of Branhamella catarrhalis to be carried out, and optionally containing means for detecting the hybrids resulting from the preceding hybridization.

15. Kit for detecting Branhamella catarrhalis strains using the polymerase chain reaction technique in which at least one of the probes of Claims 1 to 5 is used as amplification primer and/or detection probe of the amplified product.