阅读说明：本技术 可切割合作引物和使用所述可切割合作引物扩增核酸序列的方法 (Cleavable partner primers and methods of amplifying nucleic acid sequences using the same ) 是由 J·B·马奥尼 S·崇 D·C·布里尔 于 2019-06-07 设计创作，主要内容包括：本发明涉及使用具有核糖碱基切割位点的可切割合作引物和温度稳定性聚合酶扩增核酸序列的改进方法。(The present invention relates to improved methods for amplifying nucleic acid sequences using cleavable partner primers having a nucleobase cleavage site and a temperature stable polymerase.)

1. A target-specific cooperative primer for amplifying a target polynucleotide region of a nucleic acid molecule, the primer comprising:

a 3 'to 5' buffer sequence fragment, and

a 5 ' to 3 ' inner primer sequence segment comprising a capture sequence at the 3 ' end of the inner primer sequence segment;

wherein the 5 'end of the buffer sequence fragment is linked to the 5' end of the inner primer sequence fragment.

2. The primer of claim 1, comprising a cleavage site located between the buffer sequence segment and the capture sequence segment.

3. The primer of claim 2, wherein the cleavage site comprises one or more ribonucleotides that can be cleaved by ribonuclease H.

4. The primer of claim 3, wherein said cleavage site comprises a single ribonucleotide.

5. The primer of any one of claims 1 to 4, wherein said capture sequence segment has a melting temperature (Tm) higher than that of said buffer sequence segment.

6. The primer of claim 5, wherein the Tm of the capture sequence segment is about 2 ℃ to 7 ℃ higher than the Tm of the buffer sequence segment.

7. The primer of claim 6, wherein the Tm of the capture sequence segment is about 5 ℃ to 7 ℃ higher than the Tm of the buffer sequence segment.

8. The primer of any one of claims 1 to 7, wherein said buffer sequence segment anneals to said target polynucleotide region upstream of the annealing of said capture sequence segment to said target polynucleotide region.

9. A kit for amplifying a target polynucleotide region of a nucleic acid molecule comprising in one or more containers at least two target-specific cooperative primers according to any one of claims 1 to 8; a thermostable polymerase; and a buffer.

10. The kit of claim 9, wherein the at least two target-specific cooperative primers comprise:

(a) a first primer that anneals to a first region of the target polynucleotide region; and

(b) a second primer that anneals to a region of the extension product of the first primer.

11. The kit of claim 10, wherein the nucleic acid molecule is double-stranded DNA, and wherein the second primer anneals to a second region of the target polynucleotide region on the strand complementary to the first region.

12. The kit of claim 9, wherein the nucleic acid molecule is double-stranded DNA, and wherein the at least two target-specific cooperative primers comprise:

(a) a first primer that anneals to a first region of the target polynucleotide region;

(b) a second primer that anneals to a second region of the target polynucleotide region on the complementary strand;

(c) a third primer that anneals to a third region of the target polynucleotide region; and

(d) a fourth primer that anneals to a fourth region of the target polynucleotide region on the complementary strand.

13. The kit of any one of claims 9 to 12, further comprising two loop primers.

14. The kit of any one of claims 9 to 13, wherein the buffer has a pH in the range of pH 6 to pH 9 and comprises a stabilizer selected from BSA, glycerol, a detergent, and mixtures thereof.

15. The kit of any one of claims 9 to 14, wherein the buffer comprises a monovalent salt at a concentration in the range of 0-500 mM.

16. The kit of any one of claims 9 to 14, wherein the buffer comprises a divalent metal cation at a concentration in the range of 0.5mM-10 mM.

17. The kit of any one of claims 9 to 13, wherein the buffer has a pH in the range of pH 6-pH 9 and comprises a monovalent salt at a concentration in the range of 0-500mM and a divalent metal cation at a concentration in the range of 0.5mM-10mM, and optionally, a stabilizer selected from BSA, glycerol, detergents, and mixtures thereof.

18. The kit according to any one of claims 9 to 17, wherein the thermophilic polymerase has strand displacement activity and is active at temperatures above about 50 ℃.

19. The kit of any one of claims 9 to 18, wherein the buffer further comprises single-stranded binding protein (SSB) in the range of 0.5ug to 2ug per reaction.

20. The kit of any one of claims 9 to 19, further comprising a ribonuclease (RNase).

21. The kit of claim 20, wherein the ribonuclease is ribonuclease H2.

22. The kit of any one of claims 9 to 21, further comprising a base repair enzyme.

23. The kit of any one of claims 9 to 22, further comprising deoxynucleotides (dntps).

24. A method of amplifying a target polynucleotide region of a nucleic acid molecule comprising:

contacting the nucleic acid molecule under conditions that promote strand displacement amplification with:

at least two target-specific cooperative primers according to any one of claims 1 to 8, and

a thermostable polymerase.

25. The method of claim 24, further comprising cleaving the cleavage site using ribonuclease H.

26. The method of claim 24 or 25, wherein the at least two target-specific cooperative primers comprise:

(a) a first primer that anneals to a first region of the target polynucleotide region; and

(b) a second primer that anneals to a region of the extension product of the first primer.

27. The method of claim 24 or 25, wherein the at least two target-specific cooperative primers comprise:

(a) a first primer that anneals to a first region of the target polynucleotide region;

(b) a second primer that anneals to a second region of the target polynucleotide region on the complementary strand;

(c) a third primer that anneals to a third region of the target polynucleotide region; and

(d) a fourth primer that anneals to a fourth region of the target polynucleotide region on the complementary strand.

28. The method of any one of claims 24-27, further comprising contacting the nucleic acid molecule with two loop primers.

29. The method of any one of claims 24 to 28, further comprising contacting the nucleic acid molecule with a single-stranded binding protein (SSB).

30. The method of claim 29, comprising:

(a) binding the single-stranded binding protein (SSB) to the thermostable polymerase, the at least two primers, and the nucleic acid molecule in a reaction buffer at a first temperature; and

(b) immediately or after a lag time at a temperature greater than 4 ℃ but less than 70 ℃, performing an isothermal strand displacement amplification reaction at a second temperature, wherein the increase is determined relative to the same mixture without the SBB.

31. The method of any one of claims 24 to 30, comprising performing PCR, qPCR, HDA, LAMP, RPA, TMA, NASBA, SPIA, SMART, Q-beta replicase, or RCA.

32. The method of any one of claims 24 to 31, further comprising isolating the amplified target polynucleotide region.

33. The method of any one of claims 24 to 32, further comprising detecting the amplified target polynucleotide region using a fluorescent probe, a DNA binding dye, a PNA or BNA probe, and a dye that recognizes a PNA/BNA-DNA complex, or a methylene blue dye for cyclic voltammetry.

34. The kit of claim 13, comprising:

(a) a first primer comprising SEQ ID No. 1;

(b) a second primer comprising SEQ ID No. 2;

(c) a first loop primer comprising SEQ ID No. 3; and

(d) a second loop primer comprising SEQ ID No. 4.

35. The kit of claim 13, comprising:

(a) a first primer comprising SEQ ID No. 5;

(b) a second primer comprising SEQ ID No. 6;

(c) a first loop primer comprising SEQ ID No. 7; and

(d) a second loop primer comprising SEQ ID No. 8.

36. The kit of claim 13, comprising:

(a) a first primer comprising SEQ ID No. 9;

(b) a second primer comprising SEQ ID No. 10;

(c) a first loop primer comprising SEQ ID No. 11; and

(d) a second loop primer comprising SEQ ID No. 12.

37. The kit of any one of claims 9 to 23, further comprising a ribonuclease inhibitor.

38. The kit of any one of claims 9-23 and 34-37, wherein the kit is a point-of-care diagnostic device.

Technical Field

The present invention relates to isothermal amplification and detection of DNA or RNA sequences, and in particular to isothermal amplification and detection using cooperative primers (co-operative primers).

Background

Nucleic Acid Amplification Tests (NAAT) have become the basis of microbiological laboratories, providing the day-of-the-day diagnosis of a wide range of infections. Although Polymerase Chain Reactions (PCR) have provided laboratories with good service since their inception, PCR tests have significant drawbacks because they are labor intensive and relatively slower compared to newer isothermal amplification methods. After the introduction of the first isothermal amplification method (strand displacement amplification and loop-mediated isothermal amplification), a number of other methods were introduced, and some of them can produce positive results in as little as 5-10 minutes. Point-of-care (POC) tests, which are designed to provide healthcare providers with quick and actionable results when and where patients first contact the healthcare system, require faster NAATs.

Traditional diagnostic tests for bacterial and viral infections involve virus isolation in cell culture, ELISA, serology, Direct Fluorescent Antigen (DFA) staining of specimens, and Shell Vial Culture (SVC) using a panel of monoclonal antibodies. The use of specific monoclonal antibodies against respiratory viruses during the early 1990 s allowed the detection of these viruses within 3 hours using DFA staining or slow growing viruses within 1-2 days using SVC. This is far superior to 8-10 days required for cell culture. The rapid EIA tests developed in the 1980 s and 1990 s for the immediate testing of bacteria and viruses lacked sensitivity; the clinical sensitivity of these tests ranges from 20% to 90%, varying widely with the patient population tested. Therefore, these rapid EIA tests are not recommended for use in an intensive care setting due to their low sensitivity.

Disclosure of Invention

Provided herein are improved methods and compositions for performing strand displacement amplification that employ cooperative primers comprising a ribonuclease (RNase) H cleavage site.

In one aspect, there is provided a target-specific cooperative primer for amplifying a target polynucleotide region of a nucleic acid molecule, the primer comprising:

3 'to 5' buffer (buffer) sequence fragments, and

a 5 ' to 3 ' inner primer sequence segment comprising a capture sequence at the 3 ' end of the inner primer sequence segment and a reverse complement sequence downstream of the capture sequence;

wherein the 5 'end of the buffer sequence fragment is linked to the 5' end of the inner primer sequence fragment.

In one embodiment, the primer comprises a cleavage site located between the buffer sequence segment and the capture sequence segment. In one embodiment, the cleavage site comprises one or more ribonucleotides that can be cleaved by ribonuclease H. In one embodiment, the cleavage site comprises a single ribonucleotide. In one embodiment, the capture sequence segment has a melting temperature (Tm) that is higher than the Tm of the buffer sequence segment. In one embodiment, the Tm of the capture sequence fragment is about 2 ℃ to 7 ℃, preferably about 5 ℃ to 7 ℃ higher than the Tm of the buffer sequence fragment. In one embodiment, the buffer sequence segment anneals to the target polynucleotide region upstream of where the capture sequence segment anneals to the target polynucleotide region.

In another aspect, a kit for amplifying a target polynucleotide region of a nucleic acid molecule is provided comprising in one or more containers at least two target-specific cooperative primers as described above, a thermostable polymerase, and a buffer.

In one embodiment, the at least two target-specific cooperative primers comprise: (a) a first primer that anneals to a first region of the target polynucleotide region; and (b) a second primer that anneals to a region of the extension product of the first primer.

In one embodiment, the nucleic acid molecule is double-stranded DNA, and wherein the second primer anneals to a second region of the target polynucleotide region on the strand complementary to the first region. In one embodiment, wherein the nucleic acid molecule is double-stranded DNA, the at least two target-specific cooperative primers comprise: (a) a first primer that anneals to a first region of the target polynucleotide region; (b) a second primer that anneals to a second region of the target polynucleotide region on the complementary strand; (c) a third primer that anneals to a third region of the target polynucleotide region; and (d) a fourth primer that anneals to a fourth region of the target polynucleotide region on the complementary strand.

In one embodiment, the kit further comprises two loop primers. In one embodiment, the buffer has a pH in the range of pH 6 to pH 9 and comprises a stabilizer selected from BSA, glycerol, detergents, and mixtures thereof. In one embodiment, the buffer comprises a monovalent salt at a concentration in the range of 0-500 mM. In one embodiment, the buffer comprises a divalent metal cation at a concentration in the range of 0.5mM to 10 mM. In one embodiment, the buffer has a pH in the range of pH 6 to pH 9 and comprises a monovalent salt at a concentration in the range of 0 to 500mM and a divalent metal cation at a concentration in the range of 0.5mM to 10mM, and optionally, a stabilizer selected from BSA, glycerol, detergents, and mixtures thereof. In one embodiment, the thermophilic polymerase has strand displacement activity and is active at temperatures greater than about 50 ℃. In one embodiment, the buffer further comprises a single stranded binding protein (SSB) in the range of 0.5ug to 2ug per reaction. In one embodiment, the kit further comprises a ribonuclease (RNase), preferably ribonuclease H2. In one embodiment, the kit further comprises deoxynucleotides (dntps).

In one embodiment, the kit comprises one or more of a fluorescent probe, a DNA binding dye, a PNA or BNA probe, and a dye that recognizes a PNA/BNA-DNA complex, or a methylene blue dye for cyclic voltammetry. In one embodiment, the kit comprises a ribonuclease inhibitor.

In one embodiment, the kit comprises: (a) a first primer comprising SEQ ID No. 1; (b) a second primer comprising SEQ ID No. 2; (c) a first loop primer comprising SEQ ID No. 3; and (d) a second loop primer comprising SEQ ID No: 4.

In one embodiment, the kit comprises: (a) a first primer comprising SEQ ID No. 5; (b) a second primer comprising SEQ ID No. 6; (c) a first loop primer comprising SEQ ID No. 7; and (d) a second loop primer comprising SEQ ID No: 8.

In one embodiment, the kit comprises: (a) a first primer comprising SEQ ID No. 9; (b) a second primer comprising SEQ ID No. 10; (c) a first loop primer comprising SEQ ID No. 11; and (d) a second loop primer comprising SEQ ID No: 12.

In another aspect, there is provided a method of amplifying a target polynucleotide region of a nucleic acid molecule comprising contacting the nucleic acid molecule under conditions promoting strand displacement amplification with: at least two target-specific cooperative primers as described above, and a thermostable polymerase.

In one embodiment, the method further comprises cleaving the cleavage site with ribonuclease H. In one embodiment, the method further comprises contacting the nucleic acid molecule with two loop primers. In one embodiment, the method further comprises contacting the nucleic acid molecule with a single-stranded binding protein (SSB). In one embodiment, the method comprises: (a) binding the single-stranded binding protein (SSB) to the thermostable polymerase, the at least two primers, and the nucleic acid molecule in a reaction buffer at a first temperature; and (b) immediately or after a lag time at a temperature greater than 4 ℃ but less than 70 ℃, performing an isothermal strand displacement amplification reaction at a second temperature, wherein the increase is determined relative to the same mixture without the SBB.

In one embodiment, the method comprises performing PCR, qPCR, HDA, LAMP, RPA, TMA, NASBA, SPIA, SMART, Q-beta replicase, or RCA. In one embodiment, the method further comprises isolating the amplified target polynucleotide region. In one embodiment, the method further comprises detecting the amplified target polynucleotide region using a fluorescent probe, a DNA binding dye, a PNA or BNA probe, and a dye that recognizes a PNA/BNA-DNA complex, or a methylene blue dye for cyclic voltammetry.

Drawings

These and other features of the preferred embodiments of the present invention will become more apparent in the following detailed description when taken in conjunction with the accompanying drawings, wherein:

figure 1 shows a schematic representation of a Cleavable Cooperative Primer (CCP) comprising two oligonucleotide sequence fragments with two different melting temperatures (Tm) and a single ribonucleotide positioned between the capture (F2) and buffer (F3) sequences. The CCP also has a region complementary to the target region of the nucleic acid molecule (F1C).

FIG. 2 shows a schematic diagram showing annealing of the F2 capture oligonucleotide sequence of the forward CCP (F-CCP) to the complementary sequence in its target (F2C). The arrow indicates the position to which F3 will anneal. The higher Tm of the F2 region of the partner primer binds first to its complement, anchoring the primer to the target. This facilitates the buffer primer with lower Tm (F3) to bind more easily to its complement even at reaction temperatures significantly higher than the Tm of the F3 sequence.

Figure 3 shows a schematic showing the annealing of the F3 buffer sequence to its complementary F3C sequence upstream of the F2 capture primer. The arrows indicate the direction of polymerization.

FIG. 4 shows a schematic diagram showing (A) extension of the F2 capture sequence in the 5 '-3' direction, and (B) displacement of the F2 capture sequence strand by F3 buffered primer extension (FIG. 4B). The arrows indicate the direction of polymerization.

FIG. 5 shows a schematic showing the displaced F2 strand, and the binding of capture and buffer sequences of a reverse cleavable cooperative primer (R-CCP) to the displaced F2 extended strand. The arrows indicate the direction of polymerization.

FIG. 6 shows a schematic showing the extension of the B2 capture sequence along the displaced F2 sequence strand. The arrows indicate the direction of polymerization.

Figure 7 shows a schematic showing that the B2 capture primer sequence strand extends continuously beyond the rnase H cleavage site between the F3 and F1C sequences. Next, B3 buffered primer extension and displaced the extended B2 capture primer sequence strand. The arrows indicate the direction of polymerization.

FIG. 8 shows a schematic diagram showing ribonucleotide cleavage sites (white arrows) formed by the extended B2 capture strand in FIG. 6 and the F2 capture strand in FIG. 4. After formation of the dsDNA, the dsDNA is cleaved by ribonuclease H2 on the strand containing the ribonucleotides. Black arrows indicate the direction of polymerization.

FIG. 9 shows a schematic showing displacement of the capture strand of B2 by the extension product of B3. White arrows indicate dsDNA cleavage sites.

FIG. 10 shows a schematic diagram showing the extension and release (indicated by arrows) of the F2 extended strand after RNase H cleavage at the forward cooperative primer cleavage site. This product can now extend around the reverse cooperative primer cleavage site, thereby forming a loop and further participating in amplification. The B2 extension product produces a loop product that activates the cleavage site within the R-CCP primer between B3 and B1C.

FIG. 11 shows a schematic diagram showing the release (bottom) of the F2 extension product after RNase H cleavage and F2C strand extension shown by the arrows in FIG. 10, which then forms a loop structure containing a cleavage site on the R-CCP strand.

FIG. 12 shows a schematic showing annealing of F1C and F1 sequences to form a loop structure extending in the 5 '-3' direction (top panel) and binding of the R-CCP primer to the released F2 extension product (bottom panel). The reverse partner primer binds to the F2 extension product to produce a double stranded product. The arrows indicate the direction of polymerization.

FIG. 13 shows a schematic showing the extension of the F1C/F1 loop around the R-CCP sequence on product 1 (top panel), thereby forming a cleavage site, and the extension of the B2 capture primer sequence on product 2 (bottom panel). White arrows indicate the formation of a dsDNA ribonuclease H2 cleavage site at the ribose site after extension of the F1 strand. The reverse cooperative primer bound to the F2 extension product produced a double stranded product. Black arrows indicate the direction of polymerization.

FIG. 14 shows a schematic showing that the reverse cooperative primer binds to the F2 extension product to produce a double stranded product and begin exponential amplification. The top panel of FIG. 13 shows the displacement (arrow) of the lower strand of the F2 extension product by extension of the B3 primer sequence. Product 2 is formed by ribonuclease H cleavage at the dsDNA site formed by the F-CCP and R-CCP primers. The B3 primer was extended and replaced the B2 strand (bottom) of product 2.

Figure 15 shows a schematic showing that F1C hybridizes with F1 of product 2 and forms a loop structure (first panel). The bottom F1 strand was then extended, forming a loop structure around the R-CCP primer cleavage site (second panel). Following cleavage at the R-CCP cleavage site, the F2C strand is extended and displaces the B1C strand (third panel). This substitution allows B1C to form a ring with B1, which is then replaced with B1C (fourth panel). White arrows indicate the nucleobases forming the ribonuclease H2 cleavage site on dsDNA. Black arrows indicate the direction of polymerization.

FIG. 16 shows a schematic showing that the F-CCP containing a cleavage site binds to F2C of the ring structure formed in FIG. 15 and extends in the 5 '-3' direction towards the B1C/B1 ring structure (upper panel). At the same time, the B1C sequence was extended and replaced with the B1C/B1 circular strand. The result is a long linear chain (lower chain in the lower panel) which is then cut (shown in fig. 17). The arrows indicate the direction of polymerization.

FIG. 17 shows a schematic diagram showing the F-CCP sequence extended, cleaved and replaced by the B2 extension product, resulting in product 3 (upper strand). Product 3 then enters exponential amplification by formation of the F1C/F1 and B1C/B1 loops and subsequent annealing and extension of F-CCP and R-CCP. When a double-stranded DNA is formed, the backbone is nicked (nicked) by ribonuclease H2 on the same strand of ribonucleotide.

FIG. 18A (influenza A10)⁴One copy) and 18B (beta actin 10)⁴One copy) shows 10⁴Single copy of influenza A/H1 and human beta-muscleTime for the kinetin to reach positive amplification of CCPCSDA.

FIG. 19 shows the time to reach amplification positivity for LAMP and CCPCA for 100 copies. CCPSDA amplification can detect 8/8 copies of beta-actin in duplicate experiments, whereas traditional LAMP only detects 1/8. Amplification was measured using a BioRad CXF96 instrument and an Eva green dye (Biotium, Inc.) detection of amplified DNA.

FIG. 20 shows the time to reach a positive amplification for traditional LAMP and CCPCA for 50 copies. CCPSDA amplification can detect 50 copies of beta-actin in 8/8 replicates (squares), whereas LAMP fails to detect 50 copies in 8 replicates (circles). Amplification was measured using a BioRad CXF96 instrument and an Eva green dye (Biotium, Inc.) detection of amplified DNA.

FIG. 21 shows the time to reach amplification positivity for LAMP and CCPCA for 25 copies. CCPSDA amplification can detect 5/8 replicates of 25 copies of β -actin (squares), whereas traditional LAMP fails to detect 25 copies. Improved heating LAMP detected 2/8 duplicate β -actin in the experiment (data not shown). Amplification was measured using a BioRad CXF96 instrument and an Eva green dye (Biotium, Inc.) detection of amplified DNA.

FIG. 22 shows the time to reach amplification positive for heating LAMP and CCPCSA for 10 copies. CCPSDA amplification can detect 10 copies of beta-actin in 3/8 replicates (squares), while modified heating LAMP detects 10 copies in 2/8 replicates (circles), and traditional LAMP fails to detect 10 copies. Amplification was measured using a BioRad CXF96 instrument and an Eva green dye (Biotium, Inc.) detection of amplified DNA.

FIG. 23 shows specific and non-specific amplification of the heating LAMP and CCPCSA amplifications. CCPSDA produces fewer non-specific products that appear late in the reaction than traditional LAMP, and these products appear only after 50 minutes of amplification. SP, a specific product; NSP, non-specific product; green squares, CCPSDA specific amplification products; red circle, LAMP specific product; blue circle, no template LAMP; orange squares, no template CCPSDA.

FIG. 24 shows the results for CCPCSA using two CCP primers and CCPCSA using four primers (two CCP primers and two loop primers). CCPSDA amplification using two CCP primers (right panel) and CCPSDA amplification using four primers (left panel).

Detailed Description

NAAT, especially real-time PCR, multiplex PCR and more recently isothermal amplification methods, have largely replaced conventional methods for detecting bacteria and viruses, since these molecular tests detect 30% to 50% more positives. The move to isothermal amplification tests allows for the development of POC diagnostic tests, which should improve the detection and diagnosis of infections in clinical settings (e.g., emergency rooms and outpatients) as well as non-clinical settings (e.g., home or outdoors).

Isothermal amplification

A variety of amplification techniques have been developed that require multiple steps and more than a single temperature. Transcription-mediated amplification (TMA) employs a reverse transcriptase having ribonuclease activity, an RNA polymerase, and a primer having a promoter sequence at the 5' end. Reverse transcriptase synthesizes cDNA from the primers, degrades the RNA target, and synthesizes a second strand after reverse primer binding. RNA polymerase then binds to the promoter region of the dsDNA and transcribes a new RNA transcript, which can serve as a template for further reverse transcription. The reaction is rapid and can produce 10E9 copies in 20-30 minutes. Such systems are not as powerful as other DNA amplification techniques. This Amplification technique is very similar to Self-Sustained Sequence Replication (3 SR) and Nucleic Acid Sequence Based Amplification (NASBA), but uses different enzymes. Single Primer Isothermal Amplification (SPIA) also involves multiple polymerases and ribonuclease H. First, reverse transcriptase extends the chimeric primer along the RNA target. Ribonuclease H degrades RNA targets and allows DNA polymerase to synthesize the second strand of cDNA. Ribonuclease H then degrades a portion of the chimeric primer to release a portion of the cDNA and open the binding site for the next chimeric primer to bind, and the amplification process proceeds through the cycle again. The linear amplification system can amplify very low levels of RNA target in about 3.5 hours. The Q-beta replicase method is a probe amplification method. The region of the probe complementary or substantially complementary to the selected target is inserted into MDV-1RNA (the naturally occurring template of Q-BETA replicase). Q-beta replicates the MDV-1 plasmid so that the synthetic product is itself a template for Q-beta replicase, resulting in exponential amplification, provided there is an excess of replicase for the template. Since the Q- β replication method is so sensitive and can amplify regardless of the presence or absence of the target, multiple washing steps are required to clear a sample of non-specifically bound replicated plasmids. This exponential amplification took approximately 30 minutes; however, the total time including all washing steps is about 4 hours.

A variety of isothermal amplification techniques have been developed to circumvent the need for temperature cycling. Strand Displacement Amplification (SDA) was developed by Walker et al in 1992. This amplification method uses two sets of primers, a strand displacing polymerase and a restriction endonuclease. The bumper primer is used to displace the initially extended primer to create a single strand for the next primer to bind. Restriction sites are present in the 5' region of the primer. Thiol-modified nucleotides are introduced into the synthesis product to inhibit cleavage of the synthesized strand. This modification forms a nicking site on the primer side of the strand, which the polymerase can extend. This method requires an initial heat denaturation step of the double stranded target. The reaction is then run at a temperature below the melting temperature of the double stranded target region. Products of 60 to 100 bases in length are typically amplified using this method in 30-45 minutes.

SDA is the first isothermal amplification method described, involving nicking an unmodified strand at a recognition site with a restriction endonuclease and then extending the nick at the 3' end with a strand displacing polymerase, which displaces the downstream strand. The displaced strand can then serve as a target for an antisense reaction, ultimately resulting in exponential amplification of DNA. Since its development, it has been improved using methods such as hyperbranched and the like, and applied to genome-wide analysis of genetic variation.

Rolling circle replication was first characterized as the mechanism by which the viral circular genome replicates. Subsequently, it has been used as an exponential DNA amplification tool (100-fold amplification of DNA) and rapid signal amplificationBoth tools (100-fold signal amplification). In this method, a small piece of circular DNA is primed by the target, after which the strand displacing polymerase proceeds around the circular DNA, displacing the complementary strand. Finally, as more DNA is produced, the synthesized DNA remains attached to the loop, producing 10 in 90 minutes⁹One or more copies of the loop. RCA has been applied to detect point mutations in human genomic DNA.

Recombinase Polymerase Amplification (RPA) is one of the more recent isothermal DNA amplification techniques, involving a mixture of three enzymes, recombinase, single-stranded DNA binding protein (SSB), and strand-displacing polymerase. The recombinase can scan the primers and target them to their complementary sequences in the double-stranded target DNA, at which point the SSB binds to the primer-target hybrid and stabilizes the hybrid, allowing the strand displacing polymerase to initiate DNA synthesis. Using this method, DNA amplification can be accomplished in 10 to 20 minutes, showing high sensitivity and specificity. RNA amplification is also possible as shown by reverse transcriptase RPA (RT-RPA) assay targeting coronaviruses. In a recent report, Wang et al showed that feline herpes Virus 1(FHV-1) was detected at a detection level of 100 copies within 20 minutes. These reports support that RPA is a powerful tool for rapid detection of DNA and RNA targets.

Helicase-dependent amplification (HDA) is a method of replicating DNA in vivo by combining a DNA polymerase with a number of helper proteins, including DNA helicases that unwind double-stranded DNA. In HDA, helicases are included in the amplification mixture so that thermal cycling is not required for amplification. The single-stranded DNA intermediate for primer binding is generated by helicase, as opposed to PCR, which requires a heat denaturation step. HDA has been applied to a variety of biosensors for detecting multiple pathogen detection and is expected to be used in disposable POC diagnostic devices, for example for detecting Clostridium difficile (Clostridium difficile).

LAMP is currently one of the most widely used and most powerful isothermal amplification techniques for amplifying DNA or RNA sequences, based on a combination of a strong strand displacement polymerase with four to six primers. These primers recognize multiple specific regions in the target DNA, while two of the primers form a loop structure to facilitate subsequent rounds of amplification. By passingIn this manner, efficient isothermal amplification is achieved. Because the LAMP reaction is very powerful, a very large amount of DNA is produced; pyrophosphate ions (a by-product of amplification) are thus produced, producing a cloudy precipitate (magnesium pyrophosphate) that can be used to determine whether amplification has occurred. Using this method, 1 to 10 copies of DNA can be amplified to 10 in 30 to 60 minutes⁹To 10¹⁰Single copy, showing excellent sensitivity and specificity. However LAMP suffers from poor specificity due to primer dimer formation and amplification of non-specific products. In addition, multiple LAMP assays (M-LAMP) can be established with rapid diagnosis and single genomic copy sensitivity as has been shown for influenza a/H1, influenza a/H3, and influenza B, as well as Respiratory Syncytial Virus (RSV) a and B.

In contrast to the DNA amplification methods discussed above, the simple SMART or RNA target amplification method is based on signal amplification after formation of a three-way junction (3WJ) structure; the actual DNA or RNA target is not amplified. Two oligonucleotide probes are included in the reaction, both of which have complementary sequences to the DNA or DNA target and a smaller region complementary to the other probe. The two probes approach when bound to their targets, at which point 3WJ is formed. After formation of the 3WJ, the polymerase can extend the target-specific oligonucleotide, thereby forming a double-stranded T7 promoter region; this results in the constant production of RNA in the presence of the target DNA, which can be detected in real time. SMART has been used clinically to detect DNA from marine cyanobacteria in marine and freshwater environments.

There are many factors that adversely affect the results of the amplification method, including inhibitors of polymerase activity and other components found in clinical samples, which reduce amplification efficiency due to the secondary structure of the primer or template, and template-independent amplification resulting from reduced amplification efficiency and specificity, leading to false positive primer dimer formation. This negative effect is amplified at room temperature after setting up the reaction mixture, before the reaction mixture is moved to the amplification temperature, thereby presenting a specific problem for batch processing of large numbers of samples in the laboratory. This can occur when a large number of reactions are prepared for a single run, resulting in the reaction pausing at room temperature. This is common in large laboratories that handle high sample volumes and require batch processing to obtain high throughput results. Thus, high throughput is often negatively impacted by setup at room temperature, and key requirements of molecular diagnostic tests (including consistency, repeatability, and accuracy) can be negatively impacted. A ribonuclease H2 primer has been used that contains a single ribonucleotide near the 3' terminus and contains a blocked phosphorothioate nucleotide.

To further speed up DNA detection assays, signal amplification methods are becoming increasingly common. This involves an early specific sequence detection step, followed by an exponential cascade of DNA production, which is no longer dependent on the presence of the original target. Examples of signal amplification include Nucleic Acid Sequence Based Amplification (NASBA), Transcription Mediated Amplification (TMA), and SMART.

These and other amplification methods are described, for example, in Van Ness. J, et al, PNAS 2003100 (8): 4504-; tan, E, et al, anal. chem.2005,77: 7984-; lizard, P. et al, Nature Biotech 1998,6: 1197-.

Primers comprising a single ribonucleotide that can be cleaved by ribonuclease H and a blocked 3' terminus have been used to reduce primer dimer formation and reduce non-specific amplification. Ribonuclease H binds to the RNA/DNA duplex and cleaves at the RNA base and blocking group from the end of the primer. The requirement for primers to hybridize to the target sequence prior to ribonuclease H cleavage and activation, thereby forming dsDNA, eliminates primer dimer formation and reduces non-specific amplification. Ribonuclease H-dependent PCR or rhPCR using these blocked cleavable primers has been used to detect Single Nucleotide Polymorphisms (SNPs).

Cooperative primers

Isothermal amplification of nucleic acid sequences requires specific binding index DNA amplification early in the amplification to achieve maximum sensitivity for DNA detection. However, although loop-mediated isothermal amplification (LAMP) has good sensitivity and specificity, it is adversely affected by primer-dimer formation, which reduces both sensitivity and specificity. Primer dimer formation can result in non-specific amplification products that lower the detection limit for both PCR and LAMP. A variety of hot starts have been used for PCR, cooperative primers have been used for PCR, ribonuclease H cleavable primers and SSB proteins have been used to reduce non-specific amplification products in both PCR and LAMP.

A cooperative primer comprising two nucleotide sequences linked by a polyethylene glycol linker and complementary to a target gene to be amplified can be used for PCR to prevent primer dimer formation and reduce the amount of non-specific amplification products. Cooperative primers containing probe sequences can also be used to generate higher fluorescent signals after amplification.

FIG. 1 is a schematic representation of exemplary target-specific cooperative primers (CCPs) for amplifying a target polynucleotide region of a nucleic acid molecule. In this example, a forward CCP is shown. The reverse CCP has similar structure and sequence regions as the forward CCP.

In some embodiments, the cooperative primer comprises a 3 'to 5' buffer sequence (F3, B3) linked to a 5 'to 3' inner primer sequence. The 5 'end of the buffer sequence is linked to the 5' end of the inner primer sequence such that the primers comprise 2 sequence segments in opposite directions to each other.

In some embodiments, the inner primer sequence has a target region that is complementary to a target sequence of a nucleic acid molecule. Examples of nucleic acid molecules to be amplified include single-and double-stranded DNA, and RNA. The 3' end of the inner primer sequence comprises a capture sequence (F2, B2). In some embodiments, the inner primer sequence comprises a reverse complement sequence (F1C, B1C) downstream of the capture sequence. The buffer sequence is in the 3 '-5' direction, as opposed to the capture sequence in the 5 '-3' direction. Thus, the cooperative primers have two 3' ends, one on the capture sequence (F2, B2) and one on the buffer sequence (F3, B3). Since the primer has two 3' ends, polymerization occurs from both ends of the primer.

The capture sequence has a melting temperature (Tm) higher than the buffer sequence. Since the capture sequence has a higher Tm, it will anneal to the target sequence of the nucleic acid molecule before the buffer sequence anneals to its complementary target sequence (see fig. 2). In some embodiments, the cooperative primers have a high Tm capture sequence and a low Tm buffer sequence. In one embodiment, the Tm of the capture sequence is 1 ℃ to 10 ℃, preferably 2 ℃ to 7 ℃, more preferably 5 ℃ to 7 ℃ higher than the Tm of the buffer sequence. The buffer sequence anneals to the target nucleic acid molecule upstream of the capture sequence. Since the primer contains 2 sequence segments in opposite directions to each other, the primer self loops back (loop back) to anneal both the buffer and capture sequences to the target nucleic acid molecule (see FIG. 3). As polymerization occurs from both ends, polymerization from the 3' end of the buffer displaces the capture sequence and its extension product (see fig. 4).

In some embodiments, the cooperative primer comprises a cleavage site comprising one or more ribonucleotides located between the buffer and capture sequences. The cleavage site can be cleaved by a ribonuclease (e.g., ribonuclease H). Examples of ribonucleases include ribonuclease H1 and ribonuclease H2. In one embodiment, the cooperative primer comprises one cleavage site consisting of a single ribonucleotide, while the remainder of the primer is a deoxynucleotide.

In some embodiments, a nucleic acid sequence is amplified by Isothermal Strand Displacement Amplification (iSDA) using a formulation comprising at least two cooperative primers (CCP), a thermostable strand displacement DNA polymerase, and a buffer. Since isothermal strand displacement amplification is mediated by the CCP primers, the amplification process is also referred to as CCPSDA. The amplified product is fed back into the iSDA to improve the lower limit of detection and shorten the time to positivity.

In one embodiment, the CCPDA uses one forward (F-CCP) and one reverse (R-CCP) cleavable partner primers. The F-CCP binds to a first target sequence of a target nucleic acid molecule (e.g., a strand of DNA). The R-CCP binds to a second target sequence on the extension product of the F-CCP. The R-CCP can also bind to a second target sequence on a complementary target nucleic acid molecule (e.g., the complementary strand of DNA).

In an alternative embodiment, four CCP primers (two F-CCPs and two R-CCPs) are used. Two forward primers bind to one strand and two reverse primers bind to the complementary strand, thereby generating additional product to enter the exponential amplification phase.

In some embodiments, CCP primers are used for target amplification along with two loop primers (LF and LB) and a thermostable strand displacement DNA polymerase. In some embodiments where two loop primers are used to accelerate the reaction, the loop primers increase the amount of target DNA that is exponentially amplified. Referring to fig. 3, in one embodiment, a first loop primer is complementary to the first replacement strand between the F2C and F1C regions and a second loop primer is complementary to the region between B2C and B1C. The use of two loop primers in addition to the CCP primer accelerates the reaction, unlike the CCP primer alone. Specific nucleic acid sequences of viral, bacterial, fungal pathogens or eukaryotic DNA (see table 1) can be amplified and specific products generated for detection using a wide variety of DNA binding dyes or DNA specific probes.

Table 1: examples of oligonucleotides for cooperative primers

F-CCP and R-CCP bind to a region of the target genomic DNA consisting of 45-75nt in length. Both CCP primers contained a 3' -capture oligonucleotide sequence (F2 or B2) and an upstream buffer oligonucleotide sequence (F3 or B3) separated by ribonucleotides (see fig. 1). The melting temperature (Tm) of the capture oligonucleotide sequence is 5-7 degrees higher than the Tm of the buffer oligonucleotide sequence. The capture sequences of the CCP primers (F2, B2) were bound before the buffer oligonucleotide sequences (F3, B3) were bound.

After the F2 capture sequence and the F3 buffer sequence anneal to the complementary sequences of the target genomic DNA, they are both extended in the 5 '-3' direction by a thermostable polymerase (fig. 3). The 3 ' end of F3 extended in the 5 ' -3 ' direction and displaced the extension product from the 3 ' end of F2, and the 3 ' end of F2 also extended in the 5 ' -3 ' direction (see arrows in fig. 3 and 4).

The R-CCP primer was then bound to the 3' end of the displaced strand in two stages (FIG. 5): 1) the B2 capture sequence bound first, then 2) the B3 buffer sequence with a lower Tm than B2 bound second.

The 3 ' end of B2 extended in the 5 ' -3 ' direction (fig. 6), and then the 3 ' end of B3 extended in the 5 ' -3 ' direction, thereby displacing the extension product from the 3 ' end of B2 (fig. 7).

The B2 extension product extended in the 5 '-3' direction along the length of the F2 extension product and beyond the nucleobases on the F-CCP primer sequence (FIG. 8). Polymerization was stopped when the B2 extension product reached 5 '-5' ligation on the F3 sequence. The full length of the B2 extension product was replaced by the B3 extension product, as it also extended until the 5 '-5' linkage of the F3 sequence (see fig. 9). As the B2 extension product extends beyond the nucleobase, the nucleobase serves as a rnase H cleavage site, while the dsDNA is cleaved by rnase H (see fig. 9), exposing a new 3 ' end for further extension in the 5 ' -3 ' direction, which displaces the F2 extension product (see fig. 10), releasing the F2 extension product (shown as the bottom strand in fig. 11).

Turning to fig. 12, B2 extension product forms a loop at the F2C sequence of B2 extension product by hybridization of the F1 sequence of B2 extension product with the F1C sequence of B2 extension product (product 1, top panel of fig. 12). This allows the 3 ' end of F1 to extend back in the 5 ' -3 ' direction along the length of the B2 extension product approximately more than the ribobases of the R-CCP (FIG. 13). Ribonuclease H then cleaves R-CCP of product 1, exposing the 3' end for extension, thereby displacing and releasing the looped B2 extension product (as shown in FIG. 13) for exponential amplification (not shown).

The second R-CCP binds to the released F2 extension product (product 2) and then extends in the 5 '-3' direction (lower panel of fig. 12), thereby forming a strand complementary to the released F2 extension product (lower panel of fig. 13).

As before, however, the B2 extension product (product 2) from the second R-CCP was displaced by extension of B3 in the 5 '-3' direction (see arrows in the lower panel of fig. 13), and this displaced B2 extension product (product 2, fig. 14) formed a loop at F2C by hybridization of the F1 sequence to the F1C sequence (upper panel of fig. 15). This loop extends beyond the ribonucleotide cleavage site of the second R-CCP in the 5 '-3' direction, resulting in cleavage by ribonuclease H (FIG. 15). The cleaved strand is then extended in the 5 '-3' direction from B3 (see arrow in the third panel of fig. 15), displacing the B2 extension product, which forms a loop at B2 by hybridization of the B1 sequence to the B1C sequence (lower panel of fig. 15).

The second F-CCP primer then binds to the F2C loop and extends into the B1C and B2 loops (FIG. 16). The extension product terminates at the 5' end of B1C and is displaced, forming long linear dsDNA (FIG. 17). The dsDNA is cleaved by ribonuclease H at the ribonucleotide cleavage site and is displaced by extension of the B1 terminal strand. This displaced strand then forms a loop at F2 by hybridizing F1C to F1, which serves as a template to initiate another round of amplification. The two displaced strands are then amplified with F-CCP and R-CCP primers and the cycle is repeated.

Kit and reagent

In some embodiments, a kit for amplifying a target polynucleotide region of a nucleic acid molecule includes at least two cleavable cooperative primers (at least one forward and one reverse cooperative primer), a thermostable polymerase, and a buffer in one or more containers. The thermostable polymerase has strand displacement activity and is active at a temperature range of 50-80 ℃. In one embodiment, the kit comprises two cleavable cooperative primers, while in other embodiments, the kit comprises two forward cooperative primers and two reverse cooperative primers. In some embodiments, the kit further comprises dntps, ribonuclease H, a loop primer, a single-stranded binding protein (SSB), or a combination thereof.

In one embodiment, a single-stranded binding protein (SSB) is added to reduce the background generated by primer dimer amplification. SSB may be provided in a buffer in the range of 0.5ug to 2ug per reaction.

For detection of amplified nucleic acid molecules, various DNA detection methods can be used. For example, the amplification product can be detected by fluorescent signal detection using a fluorescent probe. The amplification product can be visually detected using a DNA binding dye by specific visual detection of DNA using a PNA or BNA probe and a dye that recognizes the PNA/BNA-DNA complex. Other examples of detecting amplification products include the use of methylene blue dyes and cyclic voltammetry.

In some embodiments, the kit is used to amplify target DNA and/or RNA. In some embodiments, the kit has a ribonuclease inhibitor. In one embodiment, the ribonuclease inhibitor is from NEB^TM(RNase inhibitor, Murine cat # M0314L). In one embodiment, the ribonuclease inhibitor is from Promega^TM(RNase Native (cat # N2215) and RNase Recombinant (cat # N2515.) in the case of RNA amplification from different pathogens, it is prudent to include inhibitors of ribonucleases in the reaction mixture, ribonucleases are extremely common and found on contaminated surfaces and/or plastics used in production, or in samples that are roughly purified using ribonuclease inhibitors to prevent degradation of the RNA target (RNA genome or even RNA transcript) during amplification, if no ribonuclease inhibitor is present and the reaction is contaminated with ribonuclease, amplification of the RNA target is affected. The method includes a pretreatment with a ribonuclease inhibitor prior to introducing the primers described herein into a reaction mixture for amplification.

In one embodiment, the kit is a point-of-care diagnostic device. Examples of point-of-care diagnostic devices are found in WO2016/0004536(PCT/CA2015/050648) and WO2017/117666(PCT/CA2017/000001), the entire contents of which are incorporated herein by reference.

Examples

Example 1 detection of DNA Using CCPASDA

This example outlines a method to demonstrate the use of CCP primers in isothermal Strand Displacement (SDA) amplification reactions.

To assess the function of CCP primers in SDA, we amplified two different gene targets, including the β -actin gene from human genomic DNA and the influenza a/H1 gene. The primers (F-CCP, R-CCP, LF, LB) used for the assay to be used for this evaluation are detailed in Table 1. The CCPSDA reaction with each set of primers was maintained at 25 ℃ (room temperature) for 0 to 2 hours prior to testing to allow primer dimer formation. After this room temperature maintenance, all reactions were performed in BioRad CFX96 at 63 ℃ for 30 minutes. Signal amplification in all these reactions will be performed by adding 1 × Eva green to the reaction (see Biotechnology Letters, 12.2007, Vol.29, p.12, 1939-.

For CCPSDA amplification using CCP primers, the primer mixture and template are heated to 94 ℃ for 4 minutes, held at 66 ℃ for several minutes, and cooled to room temperature just prior to addition to the reaction mixture. The primer/template mixture was added to the reaction mixture containing dntps, Eva green, Bst 3.0, ribonuclease H2 and amplified for 30 minutes at 63 ℃ on BioRad CFX 96.

For the R-CCP and F-CCP primers, the titration included 0. mu.M, 0.2. mu.M, 0.4. mu.M, 0.8. mu.M and 1.2. mu.M/reaction. For the second set of primers LF and LB, the titration included 0. mu.M, 0.2. mu.M, 0.4. mu.M, 0.8. mu.M and 1.2. mu.M/reaction.

25 μ L of Eva green reaction mixture contained: 12.5 μ L of 2 × Master Mix (1 × 20mM Tris-HCl, 10mM (NH)₄)₂SO₄、150mM KCl、2mM MgSO₄0.1% Tween 20pH 8.8 for LAMP, and Isothermal Amplification Buffer II (NEB) for iSDA, 0.6mM dNTP, 0.8. mu. M F-CCP and R-CCP primers, 0.4. mu. LF and LB primers, 6U Bst 3.0 enzyme, 0.6mM ribonuclease H2(IDT) (for ribonuclease H2 control, Buffer D will be used), 2. mu.L sample (20ng/mL human gDNA or 2.5ng/mL human gDNA or influenza A RNA), and nuclease-free water to 25. mu.L.

Influenza A/H1 of 10⁴Individual target copies (FIG. 18A) and 10 of human beta-actin⁴The results for each target copy (FIG. 18B) are shown in FIG. 18.

Example 2 comparison of CCPDDA to CCPDDALAMP showed reduced time to reach threshold amplification levels

The following examples demonstrate the improved sensitivity of CCPSDA compared to traditional LAMP.

The following example demonstrates the reduction in time for CCPSDA amplification to reach a threshold amplification level compared to LAMP using six unmodified primers. The increase in amplification rate is measured by the time it takes for the threshold amplification to be reached.

The CCPSDA and LAMP reactions were performed at 63 ℃. Human β -actin CCP primers are listed in table 1 and LAMP primers are listed in table 2.

Table 2: human beta-actin LAMP primer

For CCPSDA amplification using CCP primers, the primer mixture and template are heated to 94 ℃ for 4 minutes, held at 66 ℃ for several minutes, and cooled to room temperature just prior to addition to the reaction mixture. The primer/template mixture was added to the reaction mixture containing dntps, Eva green, Bst 3.0, ribonuclease H2 and amplified at 63 ℃ for 30 min on BioRad CFX 96.

LAMP reactions were performed in 8 replicates at 63 ℃ using 1 × AMP Buffer II containing: 20mM Tris-HCl, 10mM (NH)₄)₂SO₄、150mM KCl、2mM MgSO₄、0.1％20, pH 8.8, at 25 ℃. The reaction was performed in a volume of 25 μ L and consisted of: 8U Bst 3.0 DNA polymerase (New England Biolabs, Ipswich, Mass.), 20 ng/5. mu.L human genomic DNA (Roche Cat. No. 11691112001), and 0.2. mu. M F3 and B3 primers, 0.4. mu.M LF and LB primers, 1.6. mu. M F1P and B1P primers, 1.4mM dNTP, and Eva Green dye as shown in Table 2.

Primers (Integrated DNA Technologies, Coralville, Iowa) were added to the amplification reaction (20 mM Tris pH 8.8, 10mM (NH) at 25 ℃₄)₂SO₄、2mM MgSO₄) And supplemented with additional 6mM MgSO₄0.01% Tween-20 and 1.4mM dNTP.

The results for 100 target copies are shown in fig. 19, for 50 target copies in fig. 20, for 25 target copies in fig. 21, and for 10 target copies in fig. 22.

The time to positivity and the number of positives/number of tests for various target copy numbers are summarized in tables 3 and 4. For 10 target copies, the time to positivity for CCPSDA was 12.4, 15.6, and 17 minutes compared to 16 and 23 minutes for heating LAMP. For all three replicates of 10 target copies, traditional LAMP without a heating step failed to show an amplification signal above the threshold level. For 50 target copies, the time to positivity for CCPSDA was 20 minutes (8/8 positive) compared to 17 minutes for heating LAMP (5/8 positive) and 0/8 for traditional LAMP (table 4). For 25 copies, 5/8 replicates were detected for CCPSDA, while 0/8 replicates were detected for both conventional and heat LAMP. For 10 copies, CCPSDA detected 3/8 replicates, whereas traditional LAMP detected 0/8 replicates and heating LAMP detected 2/8 replicates.

Table 3: comparison of amplification results of traditional LAMP, heating LAMP and CCPCSA

Time to positive is expressed in minutes past the detection threshold.^a1/8 repeat the experiment positive.^bAverage of 100 copies of 8 replicates.^c8/8 average of duplicate experiments.^dAverage of 5 replicates of 50 copies.^e8/8 average of duplicate experiments.^f5/8 average of duplicate experiments.^gAverage of two replicates.^h3/8 average of duplicate experiments. ND, not performed.

Table 4: amplification results of traditional LAMP, heating LAMP and CCPDA

Example 3 amplification of CCPCSDA increased amplification of non-specific products to threshold amplification levels compared to LAMP Time of

The following examples demonstrate the improved specificity of CCPSDA compared to LAMP.

Use 10⁴The CCPSDA and LAMP assays were performed on individual copies of the human β -actin gene target. The CCPSDA reaction was carried out at 63 ℃ in 25. mu.L and consisted of using 1 × AMP Buffer II.

1 × AMP Buffer II (containing 20mM Tris-HCl, 10mM (NH) was used₄)₂SO₄、150mM KCl、2mM MgSO₄、0.1％20, pH 8.8, at 25 ℃) the LAMP reaction was carried out for 1 hour at 63 ℃ either immediately or with the indicated fractions incubated at 25 ℃ for 2 hours. The reaction was performed in a volume of 25 μ L and consisted of: 8U Bst 3.0 DNA polymerase (New England Biolabs, Ipswich, Mass.), 20 ng/5. mu.L human genomic DNA, and 0.2. mu. M F3 and B3 primers, 0.4. mu.M LF and LB primers, 1.6. mu. M F1P and B1P primers, 1.4mM dNTP, Eva green dye.

As shown in fig. 23, the time for the non-specific amplification product of LAMP to reach the threshold amplification level was 34 minutes compared to 52 minutes for CCPSDA.

Example 4-CCPASDA working with only two cooperative primers

This example demonstrates that CCPSDA can work with only two CCP primers.

Use 10⁴CCPSDA assays were performed in triplicate for each copy of the β -actin gene target. 25 μ L of CCPSDA reaction was performed with two CCP primers alone or with two CCP primers and two loop primers at 63 ℃ and consisted of 1 × AMP Buffer II. The concentration of human β -actin CCP primer (Table 1) was 08 μ M F-CCP and R-CCP primers, and 0.4 μ M LF and LB.

For CCPSDA amplification using CCP primers, the primer mixture and template are heated to 94 ℃ for 4 minutes, held at 66 ℃ for several minutes, and cooled to room temperature just prior to addition to the reaction mixture. The primer/template mixture was added to the reaction mixture containing dntps, Eva green, Bst 3.0, ribonuclease H2 and amplified at 63 ℃ for 30 min on BioRad CFX 96.

The results are shown in FIG. 24. CCPCDA with four primers, two CCPs and two loop primers, exceeded the amplification threshold at 10.5 minutes, whereas a reaction with only two CCP primers was slower, but exceeded the threshold between 48 and 52 minutes.

Although preferred embodiments of the present invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein, including those in the following list of references, are incorporated by reference.

For example, the present invention contemplates that any feature shown in any embodiment described herein may be combined with any feature shown in any other embodiment described herein and still fall within the scope of the present invention.

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