CN111808929A - An improved homogeneous recombinase polymerase nucleic acid amplification detection method and kit - Google Patents

Abstract

An improved homogeneous recombinase polymerase nucleic acid amplification detection method and kit, using recombinase, single-stranded binding protein, strand displacement DNA polymerase, RNase H and the forward primers required by the recombinase polymerase reaction system, Reverse primers, fluorescent probes containing uracil-ribose oligonucleotides, buffer systems and substrates such as dNTPs, energy sources such as creatine kinase-phosphocreatine-ATP, and stabilizers or promoters. The system uses RNase H and RNA fluorescent probes as the specific hybridization reporter system, which has the characteristics of high sensitivity and good compatibility. It is not necessary to design 46-52 base probes like the TwistAmp exo or nfo system, which is difficult to select conserved regions. , prone to mismatch and missed detection. The present invention includes the following steps: synthesizing modified primers and r U fluorescent probes, both of which are 28-36 bp in length, adding RNase H to the system, reacting with nucleic acid templates at a constant temperature of 37-40 ° C for 5-20 minutes, multi-channel real-time fluorescence collection , read every 10-30 seconds to obtain qualitative or quantitative results of viral nucleic acid in the specimen.

Description

An improved homogeneous recombinase polymerase nucleic acid amplification detection method and kit

technical field

The invention relates to the detection of biological materials, in particular to a rapid detection method and kit for specific nucleic acids in biological samples based on isothermal amplification technology.

Background technique

PCR System、Enigma Diagnostics的

MiniLab、Cepheid 的

System、卡尤迪生物的Mini8Plus实时荧光定量PCR系统，但均存在上述缺陷。Nucleic acid amplification analysis techniques such as polymerase chain reaction (PCR), especially fluorescent PCR, have been used as one of the gold standard methods for diagnosing various diseases because of their ability to detect trace amounts of pathogens. Clinical inspection, veterinary medicine, food safety, biosafety, agriculture, scientific research and other fields. However, PCR requires a complex and expensive thermal cycler, high temperature and variable temperature processes are prone to aerosol generation, high site requirements, batch processing, high operation requirements, and relatively time-consuming inherent defects, which limit PCR in the field point-of-care detection (POCT, Point of Care Testing). At present, although there are molecular POCT products constructed by PCR technology at home and abroad, such as DxNA's GeneSTAT, IQUAUM's

PCR System, Enigma Diagnostics

MiniLab, Cepheid's

System and Cayudi Bio's Mini8Plus real-time fluorescence quantitative PCR system, but all have the above defects.

On the other hand, after years of development, isothermal nucleic acid amplification has gradually matured in technology. It is more suitable for real-time detection because it does not require complex thermal cyclers and batch processing concepts, and the lower constant temperature of amplification is not easy to cross-contaminate. Isothermal amplification mainly includes loop-mediated isothermal amplification method (LAMP), transcription-mediated nucleic acid amplification (TMA or NASBA), recombinase polymerase amplification (RPA) and other technologies. Japan's LAMP uses multiple pairs of primers. Without probes, it undergoes a constant temperature reaction with Bst DNA strand displacement polymerase at around 65°C, and the product is subjected to turbidimetric or nucleic acid dye analysis, which is not suitable for clinical molecular diagnosis. TMA or NASBA is isothermal amplification for RNA targets, while DNA requires isothermal amplification. Heat denaturation is performed first, and chemiluminescence or fluorescent molecular beacon or electrochemiluminescence detection is used. The advantage is that high-sensitivity qualitative detection can be performed, but the disadvantage is also obvious. Since transcription amplification involves multiple enzymes, the amplification multiple and efficiency per unit time There is no clear mathematical formula that can be derived. The reaction can be started at room temperature. It is difficult to synchronize different sample reaction tubes, and the quantification is not as scientific and reasonable as fluorescence quantitative PCR. Molecular diagnostic reagents based on this principle have been approved by the FDA by Gen-probe in the United States and Biomerieux in France. They are usually complex in design, low in versatility and time-consuming, and the system requires complex instrument support. RPA is the latest amplification technology and relies on three enzymes: recombinases that can bind single-stranded nucleic acids, single-stranded DNA-binding proteins, and strand-displacing DNA polymerases. Recombinase combines with primers to form protein-nucleic acid complexes, which can search for homologous sequences in double-stranded DNA templates. Once the primers locate the homologous sequences, a strand displacement reaction will occur to initiate DNA synthesis and exponentially amplify the target region. . The replaced DNA strand binds to the single-stranded DNA-binding protein, preventing further replacement. In this system, a synthesis event is initiated by two primers, and the entire process proceeds very quickly, typically obtaining a detectable level of amplification product within 10-20 minutes. Product detection methods include electrophoresis, direction-finding chromatographic test strips, and special fluorescent probe detection, and the latter two are the most commonly used. The FDA-approved ALERE ^TM i INFLUENZA A&B, RSA, STREP A series and even the CE-approved Q HIV products all use RPA technology (fluorescent probe detection). The current homogeneous RPA detection system is realized by adding exonuclease III (exo enzyme) or E. coli endonuclease IV (nfo enzyme) and specially labeled probes on the basis of the aforementioned three-enzyme cyclic amplification. Hybridization cleavage, the fluorophore and the quencher group are separated to emit light, the amount of product is proportional to the cleaved fluorescence, and the time to reach the initial detection limit of the fluorescence increment instrument has a certain relationship with the logarithm of the initial concentration of the sample. achieve quantitative detection. The TwistAmp exo and TwistAmp nfo systems are commercially available from Twist Dx Inc, Cambridge, UK, for fluorescence detection and lateral flow detection, respectively.

The current RPA still has some shortcomings, mainly reflected in the probe design. RPA generally requires forward and reverse primers of more than 30 bases, probes need 46-52 bases, and 30-32 at the 5' end. The 15-20 bases at the 3' end are separated by three special base positions, and the two ends are T, which respectively mark the fluorescent group and the quenching group, and the middle is any base and tetrahydrofuran (THF) in space. Position matching, so the middle can be (FAM-dt)-THF-(BHQ1-dt), (VIC-dt)-THF-(BHQ1-dt), (NED-dt)-THF-(BHQ2-dt), (ROX-dt)-THF-(BHQ2-dt), (CY5-dt)-THF-(BHQ3-dt), etc. The amplicon should be at least 106bp or more, generally in the range of 110-200bp, and the amplification sensitivity greater than 250bp is not good. However, in the highly variable viral genome, it is actually very difficult to find a constant probe sequence of 50 consecutive bases. According to practice, the real-time detection probe of RPA can tolerate about one base mismatch, and more A mismatch at one base can result in a complete lack of signal. Likewise, the requirement for overly long primer sequences is also a drawback. These defects are not a problem for the detection of constant nucleic acid sequence amplification, but a big problem for the detection of viral nucleic acid. The genetic variation of the virus is often random, and the longer the continuous sequence is conservative, the more difficult it is to ensure , the probe method RPA actually requires a continuous 110 bases to be basically constant, which is almost impossible for viruses with high variability. In addition, longer probes such as oligonucleotides larger than 36 bp are not easily stabilized, which is also a disadvantage for highly demanding molecular diagnostic reagents. In addition, THF raw materials are not cheap, which is even worse for fluorescent probes that are expensive in themselves, and most synthesis service providers cannot legally provide such services.

SUMMARY OF THE INVENTION

In order to solve the problems existing in the existing RPA probe detection technology, the present invention provides a homogeneous detection method with simple temperature control, sensitivity and specificity, and solves the problem of difficulty in primer probe design (requiring too long). Meanwhile, the present invention also provides a kit based on the method.

The technical scheme adopted in the present invention is as follows: a homogeneous amplification detection system composed of T4gp32 (SSB), Bsu strand displacement DNA polymerase, T4uvsX, T4uvsY recombinase, and RNase H is used, forward and reverse primers, and the probe are Uracil Taqman probe (U probe), RPA amplification product hybridizes with U-probe to form a hybrid of DNA and RNA, and the RNA part is cleaved by RNase H to generate fluorescence. In order to improve the fluorescence signal-to-noise ratio, a fluorophore and a quencher group can be labeled on both sides of several U.

The present invention also provides a kit component, which is composed of buffer, magnesium acetate, potassium acetate, dithiothreitol, etc., preferably the following components: A) 50mM Tris-HCl buffer (pH8.0) , 100 mM KOAc, 2 mM DTT, 14 mM Mg(OAc) 2, 3 mM ATP, 50 mM phosphocreatine, 5% PEG35000 (Carbowax 20M), 0.2 mM d NTPs, 0.3 μM forward primer, 0.3 μM reverse primer, 0.1 μM probe. In addition, the aforementioned enzyme mixture B) was added to each 50 μl reaction system: T4gp32 (SSB) 0.9 μg, Bsu strand displacement DNA polymerase 30 ng, T4uvsX/T4uvsY recombinase 130 ng, RNase H 0.5 U, 10 pg creatine kinase, appropriate amount of protective agent ( BSA and trehalose) and so on. Amplification starts immediately after the addition of templates for components A and B.

In order to achieve a similar effect similar to hot start, THF structure can usually be added to the end of the primer, and the 3' end can be blocked, such as phosphate or biotin blocking, etc., and E. coli endonuclease IV (nfo enzymes). The system can exercise a warm start function. After this reagent is mixed with the template, the amplification cannot start the reaction. After the template is combined with the blocking primer containing THF, E. coli endonuclease IV cuts the THF, and the blocking group falls off, and the strand displacement polymerization reaction occurs.

An improved homogeneous recombinase polymerase nucleic acid amplification detection method and kit of the present invention comprise the following steps:

1. Nucleic acid extraction and purification of the samples to be tested can be carried out by means of rapid one-step method, boiling method, column extraction, magnetic bead method and other nucleic acid extraction methods. Refer to the instructions for use of such methods.

2. Nucleic acid amplification and detection, design the forward and reverse primers and U probes for the recombinase polymerase reaction, both of which are 30-36 bp long, and the amplicons are 100-200 bp, and the probe is marked with an intermediate marker such as (FAM- dt)-U...U-(BHQ1-dt), between two U can be 0-6 bases. The final concentration of primers was 0.2-0.5 μM, and the final concentration of U probe was 0.05-0.2 μM. After the RPA buffer, enzyme mixture, and template are mixed, the homogeneous detection can be carried out at 37-40°C for 20 minutes.

3. Multiple reaction times can detect FAM, VIC, NED, ROX, CY5, etc. in multiple channels.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Now take the detection of influenza A (InfluA) and feline herpes virus (FHV) as examples to describe an improved homogeneous recombinase polymerase nucleic acid amplification detection method and kit, including the following steps:

1. Nasopharyngeal swabs or swab specimens from other parts, viral nucleic acid extraction and purification: The rapid magnetic bead method reagent is used to extract and purify the nucleic acid of the specimen.

2. Nucleic acid amplification: Take 25 μl of InfluA RPA reaction solution, 5 μl of enzyme mixture, add 20 μl of nucleic acid template, and put on the machine after brief centrifugation. The isothermal amplification program was 39°C for 20 minutes, the fluorescence was read every 20 seconds, and the FAM/VIC/ROX channel was analyzed.

The formula of InfluA RPA reaction solution RPA, enzyme mixture and primer probe sequences (including primer probes used for RPA-exo system control) are as follows:

Enzyme mix

Respectively with SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 combination and SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4 combination, SEQ ID NO.5, SEQ ID NO.6 , SEQ ID NO.7 combination and SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.8 combination.

The preferred embodiments of the present invention have been described above in detail. Those skilled in the art can make many modifications and changes according to the concept of the present invention without creative work. Therefore, any technical solutions that can be obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the prior art according to the concept of the present invention shall fall within the protection scope established by the claims.

Claims (5)

1. An improved homogeneous phase recombinase polymerase nucleic acid amplification detection method and a kit, which are characterized in that: the recombinase polymerase nucleic acid amplification comprises recombinase, single-strand binding protein, strand displacement DNA polymerase, RNase H, buffer solution, primer probe, substrate, energy source, stabilizer and the like required by the system.

2. The improved method of claim 1, wherein the nucleic acid amplification assay employs an RNA fluorescent probe or an oligonucleotide fluorescent probe partially containing RNA such as one or more rUs.

3. The improved method of claim 1, wherein the hot-start RPA is used by modifying the primer ends (instead of one base being tetrahydrofuran and blocking the end base) and adding an e.coli endonuclease iv to the mixed enzyme system, which cleaves the primer ends and initiates the strand displacement polymerase reaction once the modified primer binds to the template. Otherwise, the RPA reaction can not be started because the ends of the upstream primer and the downstream primer are closed, so that the generation of non-specific products such as primer dimer and the like is effectively reduced, and the detection sensitivity is improved.

4. The improved homogeneous recombinase polymerase nucleic acid amplification detection kit of claim 1 comprising the following components: 10-100mM Tris-HCI buffer (pH7.3-8.3), 20-200mM KOAc, 0.5-5mM DTT, 10-30mM Mg (OAc)2, 0.5-5mM ATP, 1-100mM creatine phosphate, 0-10% PEG35000,0.1-2mM d NTPs, 0.1-1. mu.M forward primer, reverse primer, 0.05-0.5. mu.M probe; the enzyme mixture contains: 0.1-10 mug of T4gp32(SSB), 1-100ng of Bsu strand displacement DNA polymerase, 50-500ng of T4uvsX/T4uvsY recombinase, 0.01-5U of RNase H, 1-100pg of creatine kinase, 0.01-5U of Escherichia coli endonuclease IV, a proper amount of protective agent and the like.

5. The improved method of claim 1, wherein the fluorescence detector is used in combination with a suitable qualitative or quantitative analysis software, such as a constant temperature real-time fluorescence instrument. In addition, the real-time fluorescent PCR instrument can be compatible for use.