WO2002004122A2 - Improved stability of hybridisation interactions in dipstick assays - Google Patents
Improved stability of hybridisation interactions in dipstick assays Download PDFInfo
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- WO2002004122A2 WO2002004122A2 PCT/GB2001/003047 GB0103047W WO0204122A2 WO 2002004122 A2 WO2002004122 A2 WO 2002004122A2 GB 0103047 W GB0103047 W GB 0103047W WO 0204122 A2 WO0204122 A2 WO 0204122A2
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- nucleic acid
- target nucleic
- probe
- capture
- detection
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6832—Enhancement of hybridisation reaction
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
Definitions
- the present invention relates to enhanced nucleic acid detection by dipsticks .
- Dipsticks of the invention are used to detect the presence of a target nucleic acid in a sample solution, for example to identify whether a patient is infected with a disease causing microorganism such as Chlamydia trachomatis .
- PCR polymerase chain reaction
- dipsticks detect unamplified target nucleic acid without the requirement for any specialist equipment and the results can be obtained much more rapidly than PCR- based methods . Patients can then be treated in the same visit. This is particularly advantageous where the patient is unlikely to, or cannot, return at a later date.
- a single stranded DNA capture probe is immobilised on a nitrocellulose filter at a capture zone remote from one end of the filter (the contact end) .
- Part of the sequence of the capture probe is complementary to the sequence of a first region of the target nucleic acid to be detected.
- a labelled single stranded DNA detection probe is immobilised on the nitrocellulose filter at a probe zone located between the capture zone and the contact end of the filter. The detection probe has sequence complementary to the sequence of a second region (distinct from the first region) of the target nucleic acid.
- the contact end of the nitrocellulose filter is contacted with the sample solution.
- the sample solution wicks up the filter by capillary action and passes the probe zone and the capture zone. As the sample solution passes the probe zone, it mobilises the detection probe and causes it to rise with the sample solution towards the capture zone. Mobilised detection probe can then hybridise to the second region of any target DNA present in the sample solution.
- the hybridised detection probe and target DNA arrive at the capture zone, the first region of the target DNA can hybridise to the immobilised capture probe .
- a ternary complex is thereby formed between the target nucleic acid, the capture probe and the labelled detection probe. Presence of label at the capture zone, therefore, indicates that target DNA is present in the sample solution.
- DNA detection probe is not immobilised on the nitrocellulose filter. Instead the detection probe is added to the sample solution under conditions allowing hybridisation of the detection probe to any target nucleic acid in the sample solution. The contact end of the nitrocellulose filter is then contacted with the sample solution and as the sample solution wicks up the dipstick, target nucleic acid which is hybridised to the detection probe is captured at the capture zone by the capture probe.
- the sensitivity of nucleic acid detection by conventional dipsticks can be low. If the target nucleic acid is double stranded, the sensitivity of dipstick detection can be particularly low. Consequently, the presence of target nucleic acid in a sample solution can sometimes be undetected. It is desired, therefore, to improve the sensitivity of target nucleic acid detection by dipsticks .
- a dipstick for testing for the presence of target nucleic acid in a sample solution which comprises : a chromatographic strip having a contact end for contacting the sample solution; and a capture probe immobilised at a capture zone remote from the contact end, the capture probe being capable of hybridising to the target nucleic acid or to a hook capture probe bound to the target nucleic acid, wherein one or both ends of the capture probe are coupled to one or more nucleotides, preferably at least three nucleotides, which do not hybridise to the target nucleic acid when the capture probe has hybridised to the target nucleic acid or which do not hybridise to the hook capture probe when the capture probe has hybridised to the hook capture probe.
- kits for testing for the presence of target nucleic acid in a sample solution which comprises a dipstick according to the invention and a detection probe capable of hybridising to the target nucleic acid thereby allowing detection of target nucleic utilising the detection probe.
- 'chromatographic strip' is used herein to mean any porous strip of material capable of transporting a solution by capillarity.
- the detection probe may instead be releasably immobilised at a probe zone located between the contact end and the capture zone of the dipstick.
- One or both ends of the detection probe may be coupled to one or more nucleotides, preferably at least three nucleotides, which do not hybridise to the target nucleic acid when the detection probe has hybridised to the target nucleic acid.
- a dipstick for testing for the presence of target nucleic acid in a sample solution which comprises : a chromatographic strip having a contact end for contacting the sample solution; a capture moiety, immobilised at a capture zone remote from the contact end, the capture moiety being capable of binding directly or indirectly to the target nucleic acid; and a detection probe, releasably immobilised at a probe zone located between the contact end and the capture zone, the detection probe being capable of hybridising to the target nucleic acid to allow detection of the target nucleic acid utilising the detection probe, wherein one or both ends of the detection probe are coupled to one or more nucleotides, preferably at least three nucleotides, which do not hybridise to the target nucleic acid when the detection probe has hybridised to the target nucleic acid.
- a kit for testing for the presence of target nucleic acid in a sample solution which comprises : a dipstick according to the second aspect of the invention in which the capture moiety is capable of binding to a capture ligand coupled to a capture probe bound to the target nucleic acid; and a capture probe capable of hybridising to the target nucleic acid or to a hook capture probe bound to the target nucleic acid wherein the capture probe is coupled to a capture ligand which can be bound by the capture moiety.
- One or both ends of the capture probe may be coupled to one or more nucleotides, preferably at least three nucleotides, which do not hybridise to the target nucleic acid when the capture probe has hybridised to the target nucleic acid or which do not hybridise to the hook capture probe when the capture probe has hybridised to the hook capture probe.
- kits for testing for the presence of target nucleic acid in a sample solution which comprises: i) a dipstick' comprising a chromatographic strip having a contact end for contacting the sample solution and a capture probe capable of hybridising to the target nucleic acid, or to a hook capture probe bound to the target nucleic acid, the capture probe being immobilised at a capture zone of the chromatographic strip remote from the contact end; and ii) a detection probe capable of hybridising to the target nucleic acid thereby allowing detection of the target nucleic acid utilising the detection probe, wherein one or both ends of the detection probe are coupled to one or more nucleotides, preferably at least three nucleotides, which do not hybridise to the target nucleic acid when the detection probe has hybridised to the target nucleic acid.
- a kit for testing for the presence of target nucleic acid in a sample solution which comprises : i) a dipstick comprising a chromatographic strip having a contact end for contacting the sample solution and a capture moiety immobilised at a capture zone of the chromatographic strip remote from the contact end, the capture moiety being capable of binding directly or indirectly to the target nucleic acid; ii) a capture probe capable of hybridising to the target nucleic acid, or to a hook capture probe bound to the target nucleic acid, wherein the capture probe is coupled to a capture ligand which can be bound by the capture moiety; and iii) a detection probe capable of hybridising to the target nucleic acid, wherein one or both ends of the detection probe are coupled to one or more nucleotides, preferably at least three nucleotides, which do not hybridise to the target nucleic acid when the detection probe has hybridised to the target nucleic acid.
- a kit for testing for the presence of target nucleic acid in a sample solution which comprises : i) a dipstick comprising a chromatographic strip having a contact end for contacting the sample solution and a capture moiety immobilised at a capture zone of the chromatographic strip remote from the contact end; ii) a capture probe capable of hybridising to the target nucleic acid, or to a hook capture probe bound to the target nucleic acid, wherein the capture probe is coupled to a capture ligand which can be bound by the capture moiety and one or both ends of the capture probe are coupled to one or more nucleotides, preferably at least three nucleotides, which do not hybridise to the target nucleic acid when the capture probe has hybridised to the target nucleic acid; and iii) a detection probe capable of hybridising to the target nucleic acid thereby allowing detection of the target nucleic acid utilising the detection probe.
- Dipsticks and kits of the invention may be used in methods for testing for the presence of target nucleic acid in a sample solution in which a detection probe capable of hybridising to the target nucleic acid is incubated with the sample solution under conditions for hybridisation of the detection probe to target nucleic acid.
- the contact end of the dipstick is contacted with the sample solution allowing sample solution to move up the dipstick by capillary action.
- Target nucleic acid hybridised to the detection probe in the sample solution can then be captured by the capture probe at the capture zone .
- the presence of target nucleic acid in the sample solution is then indicated by the presence of the detection probe at the capture zone.
- the contact end of the dipstick is contacted with the sample solution so that sample solution wicks up the dipstick by capillary action.
- the detection probe can hybridise to target nucleic acid in the sample solution and move with the target nucleic acid to the capture zone.
- Target nucleic acid hybridised to the detection probe is captured by the capture probe as the sample solution passes the capture zone. The presence of target nucleic acid in the sample solution is then indicated by the presence of the detection probe at the capture zone .
- the capture moiety may be capable of binding directly or indirectly to the target nucleic acid by base pairing or non base pairing interaction.
- the capture moiety may comprise a capture probe capable of hybridising directly to the target nucleic acid.
- the capture moiety may comprise a capture probe capable of hybridising- to a hook capture probe bound to the target nucleic acid.
- the capture moiety may be capable of binding to a capture ligand coupled to a capture probe bound to the target nucleic acid, thereby allowing indirect binding of the capture moiety to the target nucleic acid.
- the capture moiety may be an antibody or antibody fragment. If the capture probe is coupled to a capture ligand, the capture probe may be linked to a capture probe spacer which is linked to the capture ligand to space the capture ligand from the capture probe .
- the hook capture probe can be added to the sample solution so that it can bind to target nucleic acid in the sample solution and be captured by the capture probe as the sample solution wicks up the dipstick by capillary action.
- the capture probe, the hook capture probe and the detection probe may each comprise at least one nucleic acid or nucleic acid analogue. Where a probe comprises more than one nucleic acid or nucleic acid analogue, they are preferably hybridised together.
- the detection probe may be coupled to a label thereby allowing direct detection of target nucleic acid at the capture zone.
- the detection probe may be coupled to a detection ligand allowing indirect detection of target nucleic acid using a detection ligand binding moiety.”
- suitable labels include textile dyes, metal sol such as colloidal gold, and coloured particles such as coloured latex particles. Such labels can be coupled directly to the detection probe or, if the detection probe is coupled to a detection ligand, to the detection ligand binding moiety.
- Suitable capture or detection ligands include biotin (captured or detected for example by an anti-biotin antibody, avidin, streptavidin or a derivative thereof) , fluorescein (captured or detected for example by an anti- fluorescein antibody) and 2,4-dinitrophenol (DNP) (captured or detected for example by an anti-DNP antibody) .
- the detection probe may comprise a universal detection probe which is capable of hybridising to a hook detection probe bound to the target nucleic acid.
- the universal detection probe may be linked to a label or a detection ligand thereby allowing detection of the detection probe.
- kits of the invention may further comprise any reagent required for the kit to be used to detect target nucleic acid in a sample solution.
- kits of the invention which comprise a detection probe coupled to a detection ligand may further comprise a detection ligand binding moiety.
- the detection ligand binding moiety may comprise an antibody or antibody fragment, or a non antibody.
- the detection ligand comprises biotin
- the detection ligand binding moiety may comprise an anti-biotin antibody, streptavidin, avidin or a derivative thereof which retains biotin binding activity.
- the detection ligand binding moiety is labelled thereby allowing indirect detection of target nucleic acid utilising the detection probe and the detection ligand binding moiety.
- the invention relates to use of detection probes and/or capture probes linked to one or more non pairing' nucleotides and that the detection probe or capture probe may be immobilised to the dipstick or incubated with the sample solution depending on the method of detection of target nucleic acid.
- nucleotide or nucleobase is able to form stacking interactions with the base pairs formed between the capture probe or the detection probe and the target nucleic acid. Formation of the stacking interactions is thought to enhance the hybridisation of the capture probe or the detection probe to the target nucleic acid thereby improving the efficiency of capture or detection of the target nucleic acid at the capture zone of the dipstick.
- dipsticks and kits of the invention may be used in the following types of dipstick assay to test for the presence of a target nucleic acid in a sample solution:
- a dipstick which comprises a chromatographic strip having a contact end and a capture probe immobilised at a capture zone remote from the contact end, the capture probe being capable of hybridising to the target nucleic acid.
- a detection probe is contacted with the sample solution under conditions for hybridisation of the detection probe to the target nucleic acid.
- the sample solution is contacted with the contact end of the dipstick to cause sample solution to move by capillary action to the capture zone, thereby allowing target nucleic acid and the detection probe to move with the sample solution to the capture zone, and target nucleic acid to be captured at the capture zone.
- Detection probe can then be detected for at the capture zone. The presence of detection probe at the capture zone indicates that target nucleic acid was present in the sample solution.
- the detection probe may be releasably immobilised to the dipstick between the contact end and the capture zone instead of being separate from the dipstick.
- the detection probe is released into the sample solution so that released detection probe can hybridise to target nucleic acid in the sample solution as it moves to the capture zone.
- the detection probe may be separate from the sample solution and contacted with the capture zone of the dipstick. This will usually be done after the contact end of the dipstick has been contacted with the sample solution.
- the detection probe may be contacted directly with the capture zone, or the detection probe may be in a separate probe solution which is contacted with the contact end of the dipstick to cause the probe solution to move by capillary action to the capture zone .
- a dipstick which comprises a chromotographic strip having a contact end and a capture moiety immobilised at a capture zone remote from the contact end, the capture moiety being capable of binding a capture probe hybridised to the target nucleic acid.
- the capture probe is contacted with the sample solution under conditions for hybridisation of the capture probe to the target nucleic acid.
- the sample solution is contacted with the contact end of the dipstick to cause sample solution to move by capillary action to the capture zone, thereby allowing target nucleic acid and the capture probe to move with the sample solution to the capture zone, and target nucleic acid to be captured at the capture zone by binding of the capture moiety to the capture probe.
- Target nucleic acid can then be detected for at the capture zone.
- Target nucleic acid may be detected using a detection probe as described for assay (1) .
- the detection probe may be added to the sample solution with the capture probe or separately from the capture probe (in any order) .
- the detection probe may be releasably immobilised to the dipstick between the contact end and the capture zone, or may be contacted separately with the capture zone as described for assay (1) .
- the capture probe instead of being mixed with the sample solution, may be releasably immobilised to the dipstick between the contact end and the capture zone.
- the capture probe When the contact end of the dipstick is contacted with the sample solution causing the sample solution to move by capillary action to the capture zone, the capture probe is released into the sample solution so that released capture probe is released into the sample solution so that released capture probe can hybridise to target nucleic acid in the sample solution as it moves to the capture zone.
- Target nucleic acid may be detected for ' using a detection probe which may be contacted with the sample solution, releasably immobilised to the dipstick between the contact end and the capture zone, or contacted separately with the capture zone .
- the capture probe may be contacted with the capture zone before, (or exceptionally, at the same time as) the sample solution reaches the capture zone by capillary action. This will allow the capture probe to be bound by the capture moiety at the capture zone so that target nucleic acid may be captured.
- the capture probe may be in a separate capture probe solution which is contacted separately with the capture zone by directly applying it to the capture zone, or by contacting the capture probe solution with the contact end of the dipstick to cause the capture probe to move by capillary action to the capture zone. Subsequent contact of the contact end of the dipstick with the sample solution will allow target nucleic acid reaching the capture zone by capillary action to be captured there.
- target nucleic acid may be detected for using a detection probe which may be contacted with the sample solution, releasably immobilised to the dipstick between the contact end and the capture zone, or contacted separately with the capture zone.
- the target nucleic acid may be labelled directly in the sample solution, for example by covalent attachment of a label to the target nucleic acid. This may be achieved by contact of a precursor label with the sample solution and incubation of the sample solution and precursor label under conditions for covalent attachment of the label to target nucleic acid.
- the capture moiety of assay (2) may be a universal capture probe capable of hybridising to the capture probe, or the capture moiety may be capable of binding by non base pairing interaction to the capture probe.
- the capture probe comprises one or more capture ligands
- the capture moiety is a capture ligand binding moiety.
- dipstick assay uses more than one probe capable of hybridising to the target nucleic acid it is preferred that
- the sensitivity of detection of target nucleic acid using a one step hybridisation assay is about equal to the sensitivity of detection when hybridisation is carried out in multiple steps .
- Multiple step hybridisation may be carried out by sequential hybridisation of the different probes to the target nucleic acid in the sample solution, or by contacting the dipstick with different solutions each containing a
- the sample solution is of suitable composition to allow the hybridisation reactions to take place in a single hybridisation step and also to allow non base pairing interactions to take place (for example between a detection ligand and a detection ligand binding moiety and between a capture ligand and a capture ligand binding moiety) and transport a complex comprising target nucleic acid and one or more hybridised probes and (optionally) ligand binding moieties by capillary action up the dipstick.
- any ligand-ligand binding moiety interactions can take place, before the sample solution is contacted directly with the contact end of the dipstick (without the need to first dilute or alter the sample solution) .
- Ligand-ligand binding moiety interactions can additionally or alternatively take place on the dipstick if desired as the sample solution travels to the capture zone. Simple and rapid dipstick detection of target nucleic acid is thereby facilitated.
- sample solutions comprising a standard hybridisation buffer (such as SSPE buffer or Tris buffer) with salt, detergent and a blocking protein such as BSA or powdered milk.
- a standard hybridisation buffer such as SSPE buffer or Tris buffer
- salt such as SSPE buffer or Tris buffer
- detergent such as BSA or powdered milk.
- blocking protein such as BSA or powdered milk.
- a probe comprising sequence which is complementary to sequence of a target nucleic acid, thereby enabling the probe to hybridise to the target nucleic acid in a sample solution, and sequence ' which is non complementary to sequence of the target nucleic acid, wherein the non complementary sequence is at an end of the probe sequence and the non complementary sequence is not bound by components of the sample solution when the probe is hybridised to the target nucleic acid in the sample solution.
- the probe is a nucleic acid probe.
- the probe may be a nucleic acid analogue such as protein nucleic acid (PNA) .
- PNA protein nucleic acid
- the non complementary sequence is at least three nucleotides long, more preferably at least six nucleotides long.
- the complementary sequence is 10-100 nucleotides long, more preferably 20-35 nucleotides long.
- the non complementary sequence is at both ends of the probe sequence.
- a label or ligand is coupled to the probe.
- the label or ligand is covalently linked to the end of the non complementary sequence furthest from the comp1ementary sequence .
- a probe of the invention in a dipstick assay to test for the presence of a target nucleic acid in a sample solution.
- a dipstick or kit of the invention in a dipstick assay to test for the presence of a target nucleic acid in a sample solution.
- dipstick assay is used here to mean any assay using a dipstick in which sample solution is contacted with the dipstick to cause sample solution to move by capillary action to a capture zone of the dipstick thereby allowing target nucleic acid in the sample solution to be captured and detected at the capture zone, capture and/or detection of the target nucleic acid involving hybridisation of a capture/detection probe to the target nucleic acid.
- Figure 1 shows the chemical structure of non protein components suitable as components of the capture probe spacer or the detection probe spacer of the invention
- Figure 2 shows detection of Chlamydia trachomatis target nucleic acid using an embodiment of the invention
- Figure 3 shows the experimental setup for Example 1
- Figure 4 shows the experimental setup for Example 2
- Figure 5 shows the experimental setup for Example 3 ;
- Figure 6 shows the experimental setup for Example 4
- Figure 7 shows the experimental setup for Example 6
- Figure 8 shows the experimental setup for Example 7.
- FIG. 9 shows the structure of detection probes used in the examples .
- the examples relate to detection of a DNA fragment of Chlamydia trachomatis (CT) cryptic plasmid DNA.
- CT is one of the most common causes of sexually transmitted disease. CT infections can cause infertility and, during pregnancy, can result in spontaneous abortion, still birth or postpartum endometritis . In neonates, CT infection can cause blindness and chronic respiratory disease. Approximately 10% of infected men and upto 70% of infected women do not show symptoms of CT infection. Consequently, accurate diagnosis of CT infection is important so that early treatment of the disease can be initiated.
- a dipstick 10 is used to try to detect single or double stranded CT target nucleic acid 12 in a sample solution 14 (see Figure 2) .
- the dipstick 10 comprises a strip of nitrocellulose 16 having a contact end 18 for contacting the sample solution 14 and a capture probe 20 immobilised at a capture zone 22 of the nitrocellulose strip 16 remote from the contact end 18.
- An anti-biotin antibody- dye conjugate 24 (or an anti-fluorescein antibody-dye conjugate in example 5) is releasably immobilised at a conjugate zone 26 of the nitrocellulose strip located between the contact end 18 and the capture zone 22.
- the capture probe 20 is capable of hybridising to a first region of one strand (the first strand) of the target nucleic acid 12.
- the sample solution 14 is prepared by spiking 1ml urine with Chlamydia trachomatis bacteria then spinning the urine at 15K rpm for 30 minutes. The pellets are resuspended in lOO ⁇ l standard hybridisation buffer (including a blocking agent such as casein or BSA) .
- a detection probe 28 capable of hybridising to the target nucleic acid is then added (together with a helper probe capable of hybridising to the target nucleic acid adjacent the region recognised by the detection probe and/or the capture probe if used) .
- the detection probe 28 is coupled to biotin (or to fluorescein in example 5) (using methods well known to those of skill in the art) .
- the sample solution 14 is then heated to 100°C for 7 minutes and cooled.
- the contact end 18 of the dipstick 10 is then contacted with the sample solution 14.
- the sample solution 14 and any target nucleic acid 12 hybridised to the detection probe 28 moves up the dipstick 10 by capillary action.
- Complex formed between the anti-biotin antibody-dye conjugate 24, the detection probe 28 and the target nucleic acid 12 then moves up the dipstick 10 to the capture zone 22 where the target nucleic acid of the complex can hybridise to the immobilised capture probe 20.
- the capture probe 20 is immobilised at the capture zone 22 in such a way that it cannot be mobilised by the sample solution 14 as it moves past the capture zone 22. Consequently, the complex bound to the capture probe remains in the capture zone and can be detected by the presence of the dye of the anti-biotin antibody-dye conjugate at the capture zone.
- the detection probe 28 cannot be captured at the capture zone 22 and so no dye is visible at the capture zone. If there is CT target nucleic acid in the sample solution, but insufficient amounts of the target nucleic acid can be captured at the capture zone the presence of the target nucleic acid in the sample solution will not be detected.
- the sensitivity of detection of target nucleic acid can be reduced if the distance between the region of the target nucleic acid to which the capture probe hybridises and the region to which the detection probe hybridises is less than 26 nucleotides.
- the distance between these regions is at least 26 nucleotides and preferably at least 200 nucleotides .
- the capture of target nucleic acid described above is referred to as direct probe capture in the examples below.
- the strength of the detection of the target nucleic acid in the examples is recorded on a scale of 0 to 5, with 5 representing the strongest detection, and 0 no detection.
- sequences of the probes used in the following examples are:
- Detection probe (dp) Seq ID No 13 or Seq ID No 15 with biotin coupled directly to the 5 ' -end, or by a 3 nucleotide
- N 3 6 nucleotide (N s ) , S, or SS spacer, or SEQ ID No 13 with biotin coupled directly to the 3 '-end. 10 12 copies of each.
- Detection format anti-biotin antibody-dye conjugate
- Target DNA 73 or 76 nucleotide single stranded DNA fragments at ⁇ xlO 11 - 10 10 copies. Results dp Se ⁇ ID No 13
- N 3 , SS or S spacer was higher than the sensitivity using a detection probe with biotin coupled directly to the 5' -end.
- the N 3 and N s spacers were better than the S and SS spacers .
- the biotin (or other detection ligand) is coupled to one end of the detection probe.
- the biotin can be at the end of the detection probe proximal to the region of the target nucleic acid which hybridises to the capture probe (internally orientated) or, preferably, at the end of the detection probe distal to the region of the target nucleic acid which hybridises to the capture probe
- the detection probe does not have to be chosen so that the detection ligand is externally orientated in the complex captured at the capture zone.
- Capture format direct probe capture (cp) Seq ID No 14 immobilised on dipstick membrane; Detection probe: detection probe (dp) Seq ID No 13, Seq ID
- Detection format anti-biotin antibody-dye conjugate
- Target DNA 214 bp double stranded DNA fragments at 10 11 -
- the sensitivity of detection of double stranded target nucleic acid was improved more than five-fold using N 3 , N 6 , S or SS spacers .
- the sensitivity of detection was higher with the N s and
- the sensitivity of detection was higher with the N s spacer than the SS spacer, despite the fact that these spacers are of equivalent length, indicating that the physicochemical properties of the spacer are important for improved sensitivity of detection.
- the sensitivity of detection using only two detection probes each coupled at the 5 ' -end to biotin by an N 6 spacer (dpl3 non-lab + dpl5-N s -B + dpl6-N s -B) , in which the biotin is internally orientated in the complex captured by the capture probe, was greater than the sensitivity of detection using three detection probes each coupled at the 5 '-end directly to biotin (dpl3-B + dpl5-B + dpl6-B) in which the biotin of one detection probe (dpl3-B) is externally orientated in the complex captured by the capture probe .
- the sensitivity of target nucleic acid detection is increased by using a spacer to couple the detection ligand to the detection probe.
- spacers of equivalent length but with different physicochemical properties had different effects on the sensitivity of detection.
- spacers in which the non protein component consists only of nucleotides are better than spacers which include non nucleotide components . Possible explanations for this are:
- Nucleotide spacers may improve the sensitivity of detection by enhancing hybridisation of the detection probe to the target nucleic acid.
- the nucleotides of these spacers are not expected to base pair to nucleotides of the target nucleic acid when the detection probe hybridises to the target nucleic acid.
- the nucleobases of these nucleotides may form stacking interactions with the base pairs formed when the detection probe hybridises to the target nucleic acid. These stacking interactions may enhance the stability of the hybrid formed between the target nucleic acid and the detection probe, thereby enhancing the sensitivity of detection of target nucleic acid.
- Nucleotide spacers may be more rigid than the S and SS spacers .
- the ribose rings of the nucleotide spacers are expected to provide much more rigidity than the polyethylene glycol groups of the S and SS spacers . This greater rigidity might increase the availability of the detection ligand coupled to the nucleotide spacer for interaction with the detection ligand binding moiety.
- Polarity differences between the nucleotide and non nucleotide spacers may cause differences in the sensitivity of detection.
- Capture format direct probe capture (cp) Seq ID No 10 immobilised on the dipstick;
- Detection probe detection probe (dp) Seq ID No 13 coupled to biotin at the 5' -end either directly or by a N 6 , SS, (dS) 6 , (SC 3 ) S or SN 3 SN 3 S spacer. 10 12 copies of each.
- Detection format anti-biotin antibody-dye conjugate; Helper probes: SEQ ID No 5 and SEQ ID No 6 adjacent to SEQ ID No 10; SEQ ID No 1 and SEQ ID No 2 adjacent to SEQ ID No 13 at 10 12 copies;
- Target DNA 872 bp double stranded DNA fragment at 2X10 11 - 5xl0 10 copies.
- the SS, (dS) 6 , and (SC 3 ) S spacers are of equivalent length and had a similar effect on enhancing the sensitivity of detection of target nucleic acid despite their structural differences and properties .
- the N 6 spacer is of equivalent length to the SS, (dS) s , and (SC 3 ) S spacers.
- the N 6 spacer had the greatest effect on improving the sensitivity of detection of target nucleic acid.
- the sensitivity of target nucleic acid detection was greater using the N s spacer than the SN 3 SN 3 S spacer (the longest spacer tested) .
- a possible explanation for this could be that the S monomer reduces or eliminates stacking interaction between the nucleobases of the N 3 components of the spacer and the base pairs formed between the detection probe and the target nucleic acid. This data supports the conclusion that stacking interactions between unpaired nucleobases of the spacer and the duplex formed between the target nucleic acid and the detection probe are important in enhancing the sensitivity of detection of target nucleic acid.
- the membrane was then incubated with an anti-biotin antibody coupled to alkaline phosphatase (capable of converting a Nitro Blue Tetrazolium/5-Bromo-4- Chloro-3-Indolyl Phosphate (NBT/BCIP) chromogenic substrate) , washed, and incubated with the NBT/BCIP chromogenic substrate, and the membrane was observed to see if any colour formed at the capture zone.
- NBT/BCIP Nitro Blue Tetrazolium/5-Bromo-4- Chloro-3-Indolyl Phosphate
- Detection probe detection probe (dp) Seq ID No 13 coupled to biotin at the 5' -end either directly or by a nucleotide or non-nucleotide spacer. 10 12 copies of each.
- Detection format anti-biotin antibody-dye conjugate;
- Helper probes SEQ ID No 2 and SEQ ID No 3 adjacent to SEQ ID No 14 at 10 12 copies;
- Target DNA 416 bp double stranded DNA fragment at 5xl0 10 - 5xl0 9 copies.
- detection probe Seq ID No 13 coupled to biotin at the 5' -end, either directly or by a nucleotide or non-nucleotide spacer.
- Detection format anti-biotin antibody coupled to alkaline phosphatase, detection by NBT/BSIP chromogenic substrate.
- the sensitivity of detection with a six nucleotide spacer is marginally better than 3-5 nucleotide spacers.
- the sensitivity of detection with spacers consisting only of nucleotides was better than with spacers which include non-nucleotides .
- the N 3 spacer was better than the longest spacer SN 3 SN 3 S, equivalent to 15 nucleotides in length.
- the (dS) s spacer is slightly better than the SS spacer.
- the sensitivity of detection was highest with the longest spacers (SSSS and SN 3 SN 3 S) .
- the dS component has a similar structure to a nucleotide. Both have a ribose residue, expected to provide rigidity. However, the nucleotide has a nucleobase which is not present in the dS component .
- the different effect of the (dS) 6 spacer on the sensitivity of target nucleic acid detection compared to the N 6 spacer suggests that the greater effect on the improvement of sensitivity of detection using a nucleotide spacer is explained principally by the presence of the nucleobases which do not base pair with the target nucleic acid.
- the composition of the spacer appears to be more important than its length (compare the results for dp-N 3 -B s ' and dp-SN 3 SN 3 S-B 5 ' in the dipstick test of example 4) .
- the sensitivity of detection using a (dS) 6 spacer was greater than with an SS spacer.
- These spacers are of equivalent length. This suggests that the physicochemical properties of the spacer, such as rigidity or polarity, may also have an effect on the improvement of sensitivity of detection by the spacer.
- the dot blot analysis of example 4 shows that the length of the spacer is important for the availability of the biotin to the anti-biotin antibody.
- the sensitivity of biotin recognition by the anti-biotin antibody was similar whether the biotin was coupled to the immobilised probe by an N s , (dS) s or SS spacer.
- the effect of these spacers on the sensitivity of detection in the dipstick test of example 4 was different. This suggests that the composition of the spacer is more important in the hybridisation of the detection probe to the target nucleic acid than in the recognition of the biotin by the anti- biotin antibody.
- the length of the spacer appears to be more important than the composition of the spacer for recognition of the biotin (or other detection ligand) by the anti-biotin antibody (or other detection ligand binding moiety) .
- Detection probe detection probe (dp) Seq ID No 13 coupled to fluorescein. 10 12 copies.
- Detection format anti-fluorescein antibody-dye conjugate
- Target DNA 73 nt or 76 nt single stranded DNA fragments at 10 11 copies.
- Capture format direct probe capture (cp) Seq ID No 14 coupled at the 5' -end to BSA immobilised on the dipstick.
- the capture probe is coupled to the BSA by an N 6 or SN 3 SN 3 S spacer; Detection probes: detection probes (dp) Seq ID No 7, 8, 9,
- Detection format anti-biotin antibody-dye conjugate; Target : 872 bp double stranded DNA at 10 11 - 2.5xl0 9 copies.
- Capture format direct probe capture (cp) Seq ID No 15 coupled to BSA immobilised on the dipstick.
- the capture probe is coupled to the BSA by an N 6 spacer;
- the detection probe comprises a hook probe and a universal probe.
- the hook probe has sequence corresponding to Seq ID No 17 (capable of hybridising to the target nucleic acid) and sequence complementary to the sequence of a universal probe.
- the universal probe is coupled to a textile dye by an N 6 or SN 3 SN 6 spacer. There are 10 12 copies of the hook probe.
- Target 872 bp double stranded DNA at 10 11 and 10 10 copies.
- spacers comprising a protein immobilised to the dipstick and a non protein to couple the capture probe to the immobilised protein (examples 5 and 6) , or use of non protein spacers to couple the label to the detection probe (example 7) improve the sensitivity of detection of target nucleic acid.
- the sensitivity of nucleic acid detection was highest when the non protein component of the spacer consisted entirely of nucleotides .
- the sensitivity of detection of single and double stranded target nucleic acid by dipsticks has been found to be significantly improved by the use of non pairing nucleotides in accordance with the invention.
- the sensitivity of detection of double stranded circular target nucleic acid can also be increased by use of non pairing nucleotides in accordance with the invention.
- Single stranded target nucleic acid is known to form secondary structure by means of intramolecular base pairing interactions .
- Such secondary structure can inhibit binding of the capture probe and the detection probe to the target nucleic acid. Consequently, the region of the target nucleic acid to which the detection probe and capture probe bind is often chosen as a region predicted to be substantially free of secondary structure.
- the improved sensitivity of detection of double stranded target nucleic acid achieved by use of non pairing nucleotides in accordance with the invention means that the sensitivity of detection of single stranded target nucleic acid using a capture probe and/or a detection probe which recognises a region of the target nucleic acid involved in secondary structure will also be improved.
- An advantage of this is that the capture probe and/or detection probe may not have to be chosen based on predictions of the secondary structure formed by the target nucleic acid, thus simplifying the choice of capture and detection probe .
- Figure 9 represents nucleic acid of the detection probe
- B represents biotin coupled to a linker ' x represents the number of nucleotides y represents the number of hexaethyleneglycol phosphate monomers
Abstract
Description
Claims
Priority Applications (1)
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AU2001269292A AU2001269292A1 (en) | 2000-07-07 | 2001-07-06 | Improved stability of hybridisation interactions in dipstick assays |
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GB0016812.0 | 2000-07-07 | ||
GB0016836.9 | 2000-07-07 | ||
GBGB0016836.9A GB0016836D0 (en) | 2000-07-07 | 2000-07-07 | Improved dipstick assays (1) |
GB0016812A GB0016812D0 (en) | 2000-07-07 | 2000-07-07 | Enhancing nucleic acid hybridisation |
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WO2002004122A2 true WO2002004122A2 (en) | 2002-01-17 |
WO2002004122A3 WO2002004122A3 (en) | 2002-12-27 |
WO2002004122B1 WO2002004122B1 (en) | 2003-08-07 |
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PCT/GB2001/003047 WO2002004122A2 (en) | 2000-07-07 | 2001-07-06 | Improved stability of hybridisation interactions in dipstick assays |
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AU (1) | AU2001269292A1 (en) |
TW (1) | TWI286159B (en) |
WO (1) | WO2002004122A2 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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EP2313527A2 (en) * | 2008-07-15 | 2011-04-27 | Rapid Pathogen Screening Inc. | Lateral flow nucleic acid detector |
WO2011048227A1 (en) * | 2009-10-22 | 2011-04-28 | Biotools Biotechnological & Medical Laboratories, S.A. | Composition, method and kit for detecting bacteria by means of sequencing |
CN102154498A (en) * | 2011-03-21 | 2011-08-17 | 厦门大学 | Nucleic acid detecting method |
US8669052B2 (en) | 2008-06-10 | 2014-03-11 | Rapid Pathogen Screening, Inc. | Lateral flow nucleic acid detector |
US9121849B2 (en) | 2008-06-10 | 2015-09-01 | Rapid Pathogen Screening, Inc. | Lateral flow assays |
EP3103884A4 (en) * | 2014-02-05 | 2017-08-23 | Fuso Pharmaceutical Industries, Ltd. | Nucleic acid detection and assay method using mask oligonucleotide, and device for same |
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2001
- 2001-07-06 WO PCT/GB2001/003047 patent/WO2002004122A2/en active Application Filing
- 2001-07-06 TW TW90116567A patent/TWI286159B/en not_active IP Right Cessation
- 2001-07-06 AU AU2001269292A patent/AU2001269292A1/en not_active Abandoned
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EP0198662A1 (en) * | 1985-04-12 | 1986-10-22 | AMERSHAM INTERNATIONAL plc | Method for nucleic acid hybridisation |
US4868105A (en) * | 1985-12-11 | 1989-09-19 | Chiron Corporation | Solution phase nucleic acid sandwich assay |
US5310650A (en) * | 1986-09-29 | 1994-05-10 | Abbott Laboratoires | Method and device for improved reaction kinetics in nucleic acid hybridizations |
WO1990001564A1 (en) * | 1988-08-09 | 1990-02-22 | Microprobe Corporation | Methods for multiple target analyses through nucleic acid hybridization |
US5210015A (en) * | 1990-08-06 | 1993-05-11 | Hoffman-La Roche Inc. | Homogeneous assay system using the nuclease activity of a nucleic acid polymerase |
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Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
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US8669052B2 (en) | 2008-06-10 | 2014-03-11 | Rapid Pathogen Screening, Inc. | Lateral flow nucleic acid detector |
US8822151B2 (en) | 2008-06-10 | 2014-09-02 | Rapid Pathogen Screening, Inc. | Lateral flow nucleic acid detector |
US9121849B2 (en) | 2008-06-10 | 2015-09-01 | Rapid Pathogen Screening, Inc. | Lateral flow assays |
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JP2011528229A (en) * | 2008-07-15 | 2011-11-17 | ラピッド パトゲン スクリーニング,インク. | Lateral flow nucleic acid detector |
EP2313527A4 (en) * | 2008-07-15 | 2012-11-21 | Rapid Pathogen Screening Inc | Lateral flow nucleic acid detector |
WO2011048227A1 (en) * | 2009-10-22 | 2011-04-28 | Biotools Biotechnological & Medical Laboratories, S.A. | Composition, method and kit for detecting bacteria by means of sequencing |
CN102154498A (en) * | 2011-03-21 | 2011-08-17 | 厦门大学 | Nucleic acid detecting method |
EP3103884A4 (en) * | 2014-02-05 | 2017-08-23 | Fuso Pharmaceutical Industries, Ltd. | Nucleic acid detection and assay method using mask oligonucleotide, and device for same |
AU2015215750B2 (en) * | 2014-02-05 | 2021-02-25 | Fuso Pharmaceutical Industries, Ltd. | Nucleic acid detection or quantification method using mask oligonucleotide, and device for same |
US11193162B2 (en) | 2014-02-05 | 2021-12-07 | Fuso Pharmaceutical Industries, Ltd. | Nucleic acid detection or quantification method using mask oligonucleotide, and device for same |
Also Published As
Publication number | Publication date |
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TWI286159B (en) | 2007-09-01 |
WO2002004122A3 (en) | 2002-12-27 |
WO2002004122B1 (en) | 2003-08-07 |
AU2001269292A1 (en) | 2002-01-21 |
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