Modified primers for nucleic acid amplification and detection

Abstract

A method of nucleic acid amplification involving using a first modified primer which provides protection to the amplification product from exonuclease degradation and a second primer. The method provides a double stranded nucleic acid, one strand of which is degraded by a double strand nucleic acid specific exonuclease to form a single stranded nucleic acid, which is protected from exonuclease degradation.

Claims

1. A nucleic acid amplification method comprising steps of: obtaining a sample comprising a nucleic acid, the nucleic acid comprising a target nucleic acid; amplifying the target nucleic acid using a pair of primers complementary to the target nucleic acid, the pair comprising a first primer comprising the nucleotide sequence of SEQ ID NO: 1 and wherein nucleotides 1-4 are modified nucleotides that are not susceptible to hydrolysis by a 5′ to 3′ double stranded nucleic acid specific exonuclease and a second primer comprising SEQ ID NO: 2, wherein the amplification provides double stranded amplicons products comprising a first strand comprising the modified primer and a downstream amplified region and a second strand; incubating the double stranded amplicons with a 5′ to 3′ double stranded nucleic acid specific exonuclease which hydrolyses the second strand but does not hydrolyse the amplified region of the first strand, to provide single stranded nucleic acid products comprising the amplified region of the first strand; exposing the single stranded nucleic acid products to a plurality of electrochemically labelled probes designed to specifically hybridize to the single stranded nucleic acid products, thereby producing labelled hybridized nucleic acid products; exposing the labelled hybridized nucleic acid products to the exonuclease which hydrolyses the hybridized labelled probes causing a detectable electrochemical signal change from each of the hybridized labelled probes; and allowing a sufficient period of time for one after another one of the plurality of electrochemically labelled probes to hybridize with a same one of the single stranded nucleic acid products, thereby generating at least three detectable electrochemical signal changes from each of the single stranded nucleic acid products; and detecting each of the single stranded nucleic acid products multiple times.

2. The method of claim 1 further comprising steps of: incubating the sample comprising nucleic acid with uracil-N-glycosylase to hydrolyze nucleic acid containing uracil, thereby producing a sample free of contaminating nucleic acid containing uracil; and performing the nucleic acid amplification step with dUTP in the absence of dTTP.

3. The method of claim 1, wherein the nucleic acid amplification is achieved using PCR.

4. The method of claim 1, wherein the sample is a human sample, or a cellular sample.

5. The method of claim 1, wherein the at least one modified nucleotide comprises at least one modified sugar moiety, at least one modified intenucleoside linkage and/or at least one modified nucleobase.

6. The method of claim 5, wherein the at least one modified sugar moiety is a 2′-O-methyl sugar moiety; and/or the at least one modified intenucleoside linkage is a phosphorothioate linkage.

7. The method of claim 1, wherein the modified primer comprises at at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 modified nucleotides, wherein optionally the modified primer comprises 3 or 4 phosphorothioate linkages.

8. The method of claim 1, wherein the primers specifically hybridise to a Chlamydia trachomatis nucleic acid.

9. The method of claim 1, wherein the exonuclease is T7 exonuclease.

10. The method of claim 1, wherein the electrochemical label of the electrochemically labelled probes is a ferrocene label.

11. A method of detecting a nucleic acid of interest in a sample comprising nucleic acids, the method comprising: a) amplifying the nucleic acid of interest in the sample using a modified first primer comprising the nucleotide sequence of SEQ ID NO: 1 and wherein nucleotides 1-4 are modified nucleotides and a second primer comprising SEQ ID NO: 2, producing a double stranded nucleic acid having a first strand comprising the modified primer and the amplified nucleic acid of interest and a second strand; b) denaturing the double stranded nucleic acid with a 5′ to 3′ double stranded nucleic acid specific exonuclease, producing a single stranded nucleic acid product comprising the amplified nucleic acid of interest; c) hybridizing the single stranded nucleic acid product to more than one of a plurality of labelled probes complementary to the single stranded nucleic acid, producing a labelled nucleic acid product; d) exposing the labelled nucleic acid product to a 5′ to 3′ double stranded nucleic acid specific exonuclease to hydrolyze the labelled probe, thereby producing a signal and allowing a sufficient period of time for at least another two of the plurality of labelled probes to hybridize to the single stranded nucleic acid; and e) detecting multiple signals from the amplified nucleic acid of interest.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Overview of the amplification method of the invention.

(2) FIG. 2. Comparison of two C. trachomatis samples each amplified with standard and modified reverse primer (3 and 4 modified bases).

(3) FIG. 3. Amplification and detection of C. trachomatis target sequence using standard, 3-base modified and 4-base modified reverse primers using semi-rapid and rapid PCR protocols. Error bars are +/−1 standard deviation from the mean.

(4) FIG. 4. Exclusivity data for standard and 4-base modified reverse primer with 14 organisms using semi-rapid PCR of C. trachomatis target sequence. Error bars are +/−1 standard deviation from the mean.

(5) FIG. 5. Inclusivity data using standard and 4-base modified reverse primers with 14 serovars using semi-rapid PCR of C. trachomatis target sequence. Error bars are +/−1 standard deviation from the mean.

(6) FIG. 6. Comparison of standard and 4-base modified reverse primers using a range of C. trachomatis template concentrations and a consistent concentration of internal control nucleic acid. Error bars are +/−1 standard deviation from the mean.

(7) FIG. 7. Boxplot of C. trachomatis peak heights obtained with standard (ABR114) and 4-base modified (ABR115) reverse primers (n=10 for all detections) for outer chamber (CT1) and inner chamber (CT2) of a cartridge.

(8) FIG. 8. Amplification and detection of N. gonhorroeae target sequence using standard and 4-base modified forward and reverse primers using semi-rapid PCR.

(9) FIG. 9. Amplification and detection of internal control nucleic acid using standard and 4-base modified forward and reverse primers using semi-rapid PCR.

(10) FIG. 10. Amplification and detection of Mycoplasma genitalium target sequence using standard and 4-base modified forward and reverse primers using semi-rapid PCR.

MODES FOR CARRYING OUT THE INVENTION

(11) Comparison of Standard Primers and Modified Primers in C. trachomatis Assay

(12) Reverse (Rv) primers with 3 and 4 consecutive phosphorothioate modifications at the 5′ end were tested in several experiments together with an unmodified forward (Fw) primer and compared to peak heights obtained when using normal Rv and Fw primers together. Results are provided in FIG. 2. In the C. trachomatis assay, an electrochemically labelled probe hybridises to the strand extended from the reverse primer. Therefore, the reverse primer, rather than the forward primer is modified to prevent degradation, i.e. the first primer is the reverse primer and the second primer is the forward primer.

(13) A substantial increase in electrochemical signal was observed when the modified Rv primers were used compared with standard Rv primers. Four modifications provided a larger increase in peak height than three modifications. This increase was observed across a number of experiments using a range of template concentrations and amplification protocols. Experiments were also carried out to verify the system of primer protection against nuclease degradation including the use of a modified Fw primer.

(14) This is due to the fact that a single-stranded product is produced which is more amenable to probe hybridisation and therefore detection. Furthermore, the assay provides an increased signal by hydrolysing the probe after it hybridises to the first strand, but not allowing the first strand to be hydrolysed because it is protected by the modified nucleotides. In this way, multiple probe molecules are able to hybridise to a single copy of the first strand providing an increased signal.

(15) Compatibility of the Modified Rv Primer with Rapid Amplification

(16) Unmodified Rv primers and 3-base modified and 4-base modified Rv primers were used in amplification reactions with unmodified Fw primers. The PCR amplifications were performed using either a semi-rapid protocol (Baseline) or a rapid protocol (1-9 SLOW) in order to determine whether the modified primers could be used in the semi-rapid PCR protocol. The results of these experiments are provided in FIG. 3. All three primer sets had undetectable peak heights in the negative controls. Substantial increases in peak heights relative to the unmodified control were obtained for both amplification protocols (semi-rapid and rapid), with the Rv primer containing four modifications providing increased peak heights compared to three modifications. Therefore, modified primers were found to be compatible with rapid PCR.

(17) Effect of the Modified Rv Primer on the C. trachomatis Assay Inclusivity and Exclusivity.

(18) It is known in the art that it is possible for phosphorothioate modifications to affect a primer's annealing properties. Therefore an experiment was carried out to assess any potential effects of using a phosphorothioate-modified Rv primer on the C. trachomatis assay inclusivity and exclusivity. A number of organisms were selected to contain species that were clinically relevant, closely related to C. trachomatis and those that produced the highest signal outliers in previous exclusivity experiments (using standard primers). This panel was tested using both the standard unmodified Rv primer and the 4 base modified Rv primer using semi-rapid amplification. The results of these experiments are provided in FIG. 4. To establish inclusivity, 14 serovars of C. trachomatis were amplified using semi-rapid amplification in the presence of the standard C. trachomatis Rv primer or the 4-base modified Rv primer. The amplification products were detected electrochemically using electrochemically labelled probes. The results of these experiments are provided in FIG. 6. The data show that using Rv primers with 4 modified bases at the 5′ end does not affect the exclusivity or the inclusivity of the assay.

(19) Effect of the Modified Rv on Degradation of Carry-Over Amplicon by UNG

(20) UNG (Uracyl-N-Glycosylase) together with dUTPs may be used in the C. trachomatis assay to prevent false negative results from carry-over contamination by amplicon. It was tested whether use of modified primers would affect the mechanism or ability of UNG to degrade carry-over contamination.

(21) Amplification using standard and 4 base modified C. trachomatis Rv primer was carried out to generate test amplicon using dNTPs with dUTP instead of dTTP. Following this, a dilution of each amplicon was used in two subsequent PCRs (using standard Rv or a 4 base modified primer) in the presence or absence of UNG. Following amplification, amplification products were electrochemically detected.

(22) The results demonstrate that whilst using the modified Rv primer produces a greater electrochemical signal (as demonstrated above), amplification products containing the modified Rv primer are susceptible to UNG degradation in the same way that amplification products produced using the standard primer is. Therefore, using a C. trachomatis Rv primer with 4 phosphorothioate nucleosides at the 5′ end does not affect the ability of UNG to degrade carry-over contamination.

(23) Compatibility of the Modified Rv with the Internal Control

(24) The C. trachomatis assay uses an internal control which monitors the assay at each stage and verifies a negative result. Experiments were performed to test whether the use of a modified Rv primer adversely affected the amplification or detection of the internal control.

(25) An experiment was carried out that amplified a serial dilution of a C. trachomatis template with a consistent amount of internal control using standard unmodified reverse primers and 4-base modified reverse primers. The two amplified nucleic acids were detected electrochemically. Results of these experiments are provided in FIG. 7. FIG. 7 shows that the internal control is capable of being consistently amplified and detected using an assay involving standard unmodified Rv primer or modified Rv primer in the presence of a range of C. trachomatis templates. Therefore, using a C. trachomatis Rv primer with 4 phosphorothioate nucleosides at the 5′ end does not affect the amplification or detection of the internal control across a range of C. trachomatis template concentrations

(26) Compatibility of the Modified Rv with the Integrated Cartridge

(27) Cartridges were produced in which standard unmodified Rv primers (ABR114) or 4-base modified Rv primers (ABR115) were used to allow the performance of the different primers to be assessed on integrated cartridges. Ten replicates of each of 500 IFU and 0 IFU samples per cartridge type were amplified on the cartridge and detected in the cartridge using a cartridge reader. The results of these experiments are shown in FIG. 8 below. FIG. 8 shows that using the modified C. trachomatis Rv primer increased the mean peak height by ˜2.2 fold in the outer chamber of the cartridge and by ˜1.6 fold for the inner chamber of the cartridge for C. trachomatis positive samples compared to using standard unmodified primers. The 0 IFU samples were unaffected by the modified primer thereby increasing the signal-noise ratio.

(28) Comparison of Standard Primers and Modified Primers in N. gonhorroeae Assay

(29) In order to determine the different effects of using different combinations of modified and unmodified primers, experiments were performed in which 1000 copies of N. gonhorroeae were amplified and detected electrochemically. The results of these experiments are shown in FIG. 9.

(30) In the N. gonhorroeae assay, the probe hybridises to the strand extended from the forward primer. Therefore, the forward primer, rather than the reverse primer is modified to prevent degradation, i.e. the first primer is the forward primer and the second primer is the reverse primer. Where both forward and reverse primers are modified to include 4 phosphorothioate nucleosides, both strands are protected from degradation by the exonuclease. This prevents the probe from hybridising to the amplified target nucleic acid, resulting in a large signal decrease.

(31) Modifying the reverse primer rather than the forward primer means that the strand to which the probe binds is not protected from exonuclease degradation but the other strand is protected from degradation. Therefore, the majority of copies of the strand to which the probe binds will be degraded by exonuclease preventing hybridisation of the probe.

(32) Using unmodified forward and reverse primers provides a control level of amplification in the presence of the N. gonhorroeae target sequence.

(33) Modifying the forward primer, but not the reverse primer at the 5′ end provides protection from exonuclease degradation for the first strand to which the probe binds, meaning that only the other strand is degraded. Probe is able to bind to the first strand, and the probe is degraded to provide a signal. Degradation of the probe allows further probe molecules to bind to the first strand and provide an increased signal. This causes a substantial increase in the mean peak height compared to the peak height when using unmodified primers.

(34) Comparison of Standard Primers and Modified Primers in Assay for Internal Control

(35) In order to determine the different effects of using different combinations of modified and unmodified primers, experiments were performed in which 100 pg of internal control nucleic acid was amplified and detected electrochemically. The results of these experiments are shown in FIG. 10.

(36) In the internal control amplification reaction, the probe hybridises to the strand extended from the reverse primer. Therefore, the reverse primer, rather than the forward primer is modified to prevent degradation, i.e. the first primer is the reverse primer and the second primer is the forward primer. Where both forward and reverse primers are modified to include 4 phosphorothioate nucleosides, both strands are protected from degradation by the exonuclease. This prevents the probe from hybridising to the amplified target nucleic acid, resulting in a large signal decrease.

(37) Modifying the forward primer rather than the reverse primer means that the strand to which the probe binds is not protected from exonuclease degradation but the other strand is protected from exonuclease degradation but the other strand is protected from degradation. Therefore, the majority of copies of the strand to which the probe binds will be degraded by exonuclease preventing hybridisation of the probe.

(38) Using unmodified forward and reverse primers provides a control level of amplification in the presence of internal control nucleic acid.

(39) Modifying the reverse primer, but not the forward primer at the 5′ end provides protection from exonuclease degradation for the first strand to which the probe binds, meaning that only the other strand is degraded. Probe is able to bind to the first strand, and the probe is degraded to provide a signal. Degradation of the probe allows further probe molecules to bind to the first strand and provide an increased signal. This causes a substantial increase in the mean peak height compared to the peak height when using unmodified primers.

(40) Comparison of Standard Primers and Modified Primers in M. genitalium Assay

(41) In order to determine the different effects of using different combinations of modified and unmodified primers, experiments were performed in which varying copy numbers of M. genitalium were amplified and detected electrochemically. The results of these experiments are shown in FIG. 11.

(42) In the M. genitalium assay, the probe hybridises to the strand extended from the reverse primer. Therefore, the reverse primer, rather than the forward primer is modified to prevent degradation, i.e. the first primer is the reverse primer and the second primer is the forward primer. Where both forward and reverse primers are modified to include 4 phosphorothioate nucleosides, both strands are protected from degradation by the exonuclease. This prevents the probe from hybridising to the amplified target nucleic acid, resulting in a large signal decrease.

(43) Using unmodified forward and reverse primers provides a control level of amplification in the presence of M. genitalium target sequence.

(44) Modifying the reverse primer, but not the forward primer at the 5′ end provides protection from exonuclease degradation for the strand to which the probe binds, meaning that only the other strand is degraded. Probe is able to bind to the first strand, and the probe is degraded to provide a signal. Degradation of the probe allows further probe molecules to bind to the first strand and provide an increased signal. This causes a substantial increase in the mean peak height compared to the peak height when using unmodified primers.

REFERENCES

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