SRDA isothermal nucleic acid amplification reagent kit and application thereof

Abstract

The invention provides an SRDA isothermal nucleic acid amplification reagent kit and an application thereof. The reagent kit comprises a protein composition, wherein the protein composition comprisesrecA recombinase, Ago protein, single-stranded DNA conjugated protein and strand displacement DNA polymerase. The reagent kit further comprises reverse transcriptase. The Ago protein is added, so thatthe isothermal amplification efficiency is significantly improved; and the reverse transcriptase is added, so that general-purpose detection of DNA and RNA is realized. The reagent kit disclosed by the invention is good in detection specificity and high in sensitivity, the sensitivity of amplifying the DNA reaches 50copies / [mu]L, the sensitivity of amplifying the RNA reaches 100copies / [mu]L, andthe reagent kit has extremely high theoretical value and clinical application value.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to an SRDA isothermal nucleic acid amplification kit and an application thereof. Background technique [0002] At present, commonly used nucleic acid detection methods mainly include classical PCR, real-time fluorescence quantitative PCR, gene chip and isothermal nucleic acid amplification technology, etc. Among them, as the gold standard of nucleic acid detection, classical PCR has the widest application range and the longest use time, and is the basis of various emerging nucleic acid detection methods, but there are problems such as insufficient specificity and easy contamination; The nucleic acid amplification method developed on the basis of PCR and capable of real-time monitoring of the PCR process has better specificity and sensitivity than classical PCR, and has become the most widely used nucleic acid quantitative technology in the nucleic acid diagnostic industry...

AI Technical Summary

Problems solved by technology

Among them, although LAMP and SAT no longer rely on expensive detection instruments, the operation is more complicated, and the amplification time still takes about 1 hour; RPA, as the recognized isothermal nucleic acid amplification technology with the shortest detection time and the most convenient operation, does not rely on Bulky and expensive fluorescent quantitative detection equipment, the reaction time is only 5-20 minutes, has become a hot research topic at home and abroad

[0004] RPA technology was first introduced by the British Twist DX company, and the products currently on the market are mainly the RPA isothermal nucleic acid detection kit of the British TwistDX company and the RAA isothermal nucleic acid amplification detection kit produced by China Hangzhou Zhongce Biotechnology Co., Ltd. However, their kits need to use two different systems for DNA and RNA detection, and at the same time, the sensitivity for RNA amplification is low, only about 1000copies / μL

[0005] CN 105525040 A discloses a real-time fluorescent RPA kit for rapid detection of porcine type 2 circovirus, a test strip RPA kit and uses thereof, the two kits respectively include those shown in SEQ ID NO.1 and SEQ ID NO.2 The primers and their respective probes, although aimed at the same target sequence, the modified bases and detection platforms of the primers and probes are different, and the experiments show that the sensitivity of the two kits of the present invention is 10 2 copy / reaction, and both can only specifically detect porcine type 2 circovirus, and the coincidence between the detection results and qPCR is 100%. The two kits can detect porcine type 2 circovirus quickly, efficiently and sensitively. Circovirus provides an effective technical means for the differential diagnosis of porcine type 2 circovirus, but the test kit adopts the method of test strips, and the result is judged by the brightness of the strip, so the judgment of the result is easily caused by the subjectivity of the experimenter. cause of misjudgment

[0006] CN 107828913 A discloses a RAA constant temperature fluorescence detection method and kit for white spot syndrome (WSSV) of prawns. The detection kit includes a forward primer SEQ ID NO.1, a reverse primer SEQ ID NO.2, and a specific fluorescent Probe SEQ ID NO.3, reaction solution, recombinant polymerase and reference substance; the kit has strong specificity, high detection sensitivity, which can reach 0.1fg / μL, high accuracy, reliability, simple and fast operation, and is suitable for on-site detection , has a wide range of application scenarios, but the kit also cannot be used for RNA detection

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