RT-RAP primer probe set and kit for detecting blood virus

Abstract

The application provides an RT-RAP primer probe set and kit for detecting blood viruses, and belongs to the technical field of molecular biology. The primer probe set comprises a primer probe set for detecting hepatitis B virus, and / or a primer probe set for detecting hepatitis C virus, and / or a primer probe set for detecting human immunodeficiency virus. The application adopts the detection means of RT-RAA and qPCR in sequence, and can simultaneously and rapidly detect hepatitis B virus, hepatitis C virus and human immunodeficiency virus in combination with the specific primer probe set, has higher sensitivity than ordinary qPCR, greatly shortens the reaction time, and has good specificity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to an RT-RAP (Reverse Transcription Recombinase Aided Real-time PCR) primer probe set and kit for detecting blood viruses. BACKGROUND

[0002] Hepatitis B is a life-threatening global public health problem caused by Hepatitis B Virus (HBV) infection. Acute hepatitis patients can develop acute liver failure, leading to death. A portion of chronic infected individuals develop into liver cirrhosis and liver cancer, and other late-stage liver diseases, with high morbidity and mortality. Hepatitis C is a liver inflammation caused by Hepatitis C Virus (HCV). It can cause acute or chronic hepatitis, with severity ranging from mild symptoms to lifelong serious illness. The acute phase of hepatitis C usually has no symptoms and is not life-threatening. However, about 70% of infected individuals develop chronic hepatitis C, and about 15%-30% of these patients develop cirrhosis. AIDS is a global class B infectious disease caused by Human Immunodeficiency Virus-1 (HIV) infection, which is extremely harmful. HIV-1 is the global main epidemic strain of AIDS, and the number of deaths due to AIDS accounts for 79.28% of the number of deaths due to infectious diseases in China. The above three viruses can be transmitted through blood and are highly prevalent worldwide, and are also the main pathogens of transfusion-transmitted diseases in China. Rapid and timely detection and diagnosis of these viruses can reduce the transmission of transfusion-transmitted diseases and ensure the safety of blood use by patients.

[0003] Currently, the detection of Hepatitis B Virus, Hepatitis C Virus and Human Immunodeficiency Virus is mainly carried out by enzyme-linked immunoassay for antigen screening detection, but the false positive rate is high and it is easy to miss detection, so it is not the best screening method. Nucleic acid detection has the advantages of high specificity and sensitivity, can shorten the detection time and the "window period" of blood screening, and detect the missed detection in the serological window period, which is the first molecular biology method for blood screening by blood stations at all levels, but there are many nucleic acid detection methods, such as ordinary PCR, fluorescent quantitative PCR, isothermal amplification PCR, multiplex PCR, nested PCR, etc., and the sensitivity and specificity of different nucleic acid detection methods differ greatly. However, there is no report on rapid and sensitive detection of Hepatitis B Virus, Hepatitis C Virus and Human Immunodeficiency Virus. SUMMARY

[0004] The application provides an RT-RAP primer probe set and a kit for detecting blood viruses.

[0005] To solve the above technical problems, the application provides the following technical solutions.

[0006] The application provides an RT-RAP primer probe set for detecting blood viruses, which comprises a primer probe set for detecting hepatitis B virus (HBV) and / or a primer probe set for detecting hepatitis C virus (HCV) and / or a primer probe set for detecting human immunodeficiency virus (HIV).

[0007] Preferably, the fluorescent group comprises FAM, HEX, ROX, TET, JOE, CY3, CY5, TAMRA or VIC.

[0008] The application provides a kit for detecting blood viruses for non-disease diagnosis purposes, comprising the primer probe set and reagents for RT-RAA and qPCR.

[0009] Preferably, the kit further comprises n-docosane.

[0010] Preferably, the reagents for RT-RAA comprise reaction buffer, magnesium acetate and DEPC water; and the reagents for qPCR comprise reaction buffer, DEPC water and hot-start Taq polymerase.

[0011] The application provides a RT-RAA method for detecting blood viruses, comprising the following steps: 1) extracting nucleic acid from a sample to be detected; 2) using the nucleic acid as a template, and using the primer probe set to sequentially perform RT-RAA amplification and qPCR amplification, and collecting fluorescence signals; the RT-RAA and qPCR amplification systems are separated by n-docosane; 3) if the fluorescence curve corresponding to hepatitis B virus, hepatitis C virus or human immunodeficiency virus peaks and shows S-shaped amplification, it is determined that the sample to be detected contains the corresponding hepatitis B virus, hepatitis C virus or human immunodeficiency virus.

[0012] Preferably, the reaction system for RT-RAA amplification comprises 280 mM magnesium acetate, a template and an RT-RAA reaction mixture; and the volume ratio of the magnesium acetate, the template and the RT-RAA reaction mixture is 0.2-0.8:0.5-1.5:6-12.

[0013] Preferably, the RT-RAA and qPCR amplification systems are separated by n-docosane, comprising the following steps: adding melted n-docosane to the qPCR amplification system and standing, the n-docosane floating on the qPCR amplification system, and after the n-docosane solidifies, adding the RT-RAA amplification system on the solidified n-docosane.

[0014] Preferably, the RT-RAA amplification and qPCR amplification are performed in a tube with a cover; and adding the RT-RAA amplification system on the solidified n-docosane comprises the following steps: adding the RT-RAA amplification system except for magnesium acetate on the solidified n-docosane, adding magnesium acetate into the cover, and using the magnesium acetate to start the RT-RAA reaction.

[0015] The application provides application of the primer probe set and the kit in detection of hepatitis B virus, hepatitis C virus and human immunodeficiency virus.

[0016] Compared with the prior art, the application has the following beneficial effects:

[0017] The application first uses a detection method of RT-RAA and qPCR combination (Reverse Transcription Recombinase Aided Real-time PCR, RT-RAP) to detect hepatitis B virus, hepatitis C virus and human immunodeficiency virus alone or simultaneously, and the detection sensitivity is obviously better than that of qPCR. The detection method is efficient and fast, and greatly shortens the reaction time.

[0018] The designed primer probe set of the application has high sensitivity and good specificity, and can realize the purpose of detecting hepatitis B virus, hepatitis C virus and human immunodeficiency virus alone or simultaneously. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a schematic diagram of RT-RAP detection principle and process.

[0020] Figure 2 It is a sensitivity diagram of HBV RT-RAP and ordinary qPCR.

[0021] Figure 3 It is a sensitivity diagram of HCV RT-RAP and ordinary qPCR.

[0022] Figure 4 It is a sensitivity diagram of HIV RT-RAP and ordinary qPCR.

[0023] Figure 5 It is a sensitivity diagram of HBV triple RT-RAP and ordinary qPCR.

[0024] Figure 6 It is a sensitivity diagram of HCV triple RT-RAP and ordinary qPCR.

[0025] Figure 7 It is a sensitivity diagram of HIV triple RT-RAP and ordinary qPCR. DETAILED DESCRIPTION

[0026] The application provides an RT-RAP primer probe set for detecting blood viruses, including a primer probe set for detecting hepatitis B virus and / or a primer probe set for detecting hepatitis C virus and / or a primer probe set for detecting human immunodeficiency virus. The primer probe set is shown in Table 1.

[0027] Table 1 primer and probe information

[0028]

[0029]

[0030] Note: 1 represents the probe modification: 5' VIC fluorescent group, 3' BHQ-1 quencher; 2 represents the probe modification: 5' CY5 fluorescent group, 3' BHQ-3 quencher; 3 represents the probe modification: 5' HEX fluorescent group, 3' BHQ-1 quencher; Degenerate base: R = A or G, Y = C or T.

[0031] In the present application, the primer for RAA amplification is prolonged to 30-35 bp on the basis of the existing qPCR primer according to the design principle of RT-RAA primer.

[0032] In the present application, the use concentration of each primer in the primer probe set is preferably 8-12 μmol / L, more preferably 10 μmol / L.

[0033] In the present application, the human immunodeficiency virus is preferably human immunodeficiency virus type 1 (HIV-1).

[0034] The present application provides a kit for detecting blood viruses, comprising the primer probe set, RT-RAA reaction reagents and qPCR reaction reagents. The RT-RAA reaction reagents of the present application comprise reaction buffer, magnesium acetate and DEPC water; the qPCR reaction reagents comprise reaction buffer, DEPC water and hot-start Taq polymerase.

[0035] In the present application, the kit further comprises n-dodecane. The n-dodecane of the present application is a white crystal with a melting point of 44℃, which is solid at room temperature, with a density of 0.7944 g / mL, and can be disassembled with temperature change, and does not affect the nucleic acid amplification reaction. In the present application, the RT-RAA and qPCR two reaction systems are separated by using n-dodecane due to the different temperatures required for the RT-RAA and qPCR reactions. The present application realizes rapid, accurate and high-sensitivity detection of hepatitis B virus, hepatitis C virus and human immunodeficiency virus by adding n-dodecane as a special material for separating the two systems to realize single-tube closed-tube two-stage one-step detection.

[0036] The application provides an RT-RAP method for detecting blood viruses, comprising the following steps: 1) extracting nucleic acid of a sample to be detected; 2) using the nucleic acid as a template, using the primer probe set, sequentially performing RT-RAA amplification and qPCR amplification, and collecting fluorescence signals; the RT-RAA and qPCR amplification systems are separated by using n-dodecane; 3) if the fluorescence curve of hepatitis B virus, hepatitis C virus or human immunodeficiency virus rises and shows S-shaped amplification, it is determined that the sample to be detected contains corresponding hepatitis B virus, hepatitis C virus or human immunodeficiency virus. The method for extracting nucleic acid of the sample to be detected is not particularly limited; the sample to be detected is preferably blood; and the nucleic acid of the sample to be detected is preferably extracted by using a viral nucleic acid extraction kit (Xi'an Tianlong Technology Co., Ltd.).

[0037] In the application, the reaction system of the RT-RAA amplification comprises magnesium acetate, a template and an RT-RAA reaction mixture; the volume ratio of the magnesium acetate, the template and the RT-RAA reaction mixture is 0.2-0.8:0.5-1.5:6-12, preferably 0.5:1:9; the RT-RAA reaction mixture, preferably, comprises the following components in 40 μl: reaction buffer 29.4 μl, DEPC water 6 μl, HBV-RAA-F 0.7 μl, HBV-RAA-R 0.7 μl, HCV-RAA-F 0.8 μl, HCV-RAA-R 0.8 μl, HIV-RAA-F 10.4 μl, HIV-RAA-R 10.4 μl, HIV-RAA-F 20.4 μl, HIV-RAA-R 20.4 μl, and 9 μl is used for RT-RAP; the reaction procedure of the RT-RAA amplification comprises 39-42 ℃, 15-20 min. The reaction buffer of the application uses Tris buffer as a solvent, and preferably comprises the following components at the following concentrations: Tris buffer 45 mM, potassium acetate 80 mM, dithiothreitol 4 mM, polyethylene glycol with a mass volume concentration of 7.28%, ATP 5 mM, dNTPs 0.24 mM, phosphocreatine 30 μg / U, single-strand binding protein 500 ng / μL, recombinase 400 ng / μL, UvsY protein 70 ng / μL, DNA polymerase 90 ng / μL and exonuclease 85 ng / μL.

[0038] In the present application, the reaction system of qPCR amplification is 40 μl, preferably including the following components: reaction buffer 8 μl, DEPC water 12.8 μl, hot start Taq polymerase 2 μl, HBV-PCR-F 1.4 μl, HBV-PCR-R 1.4 μl, HBV-PCR-P 0.7 μl, HCV-PCR-F 3.6 μl, HCV-PCR-R 3.6 μl, HCV-PCR-P 0.9 μl, HIV-PCR-F 11.2 μl, HIV-PCR-R 11.2 μl, HIV-PCR-P 10.4 μl, HIV-PCR-F 21.2 μl, HIV-PCR-R 21.2 μl, HIV-PCR-P 20.4 μl; the reaction procedure of qPCR amplification includes: 95℃, 3 min; 95℃, 2 s, 60℃, 13 s, 30 cycles.

[0039] In the present application, the RT-RAA and qPCR amplification system are separated by n-dodecane, preferably including: adding melted n-dodecane in the qPCR amplification system for standing, the n-dodecane floats on the qPCR amplification system, after the n-dodecane solidifies, adding the RT-RAA amplification system on the solidified n-dodecane. The standing procedure of the present application preferably includes: 60℃, 1 min, 4℃, 30 s.

[0040] In the present application, as an embodiment, the qPCR amplification system is divided into eight tubes, after adding liquid n-dodecane, it is rapidly cooled and solidified at room temperature, floating on the PCR system. After 1 min treatment at 60℃ and 30 s treatment at 4℃, the n-dodecane is re-melted and uniformly solidified on the PCR system. The RT-RAA amplification system is divided into the upper layer of the solidified n-dodecane, so that the RT-RAA reaction is realized in the same tube first, then in the second stage, heating to 95℃, the recombinase is inactivated and the n-dodecane is melted and floats on the uppermost layer, then the large amount of target product in the RT-RAA reaction in the first stage is mixed with the qPCR system, and the next PCR reaction and detection are carried out. In the first stage, the template can be enriched in a large amount within 20 min, and then the second stage qPCR is carried out. The whole amplification process can be completed within 50 min, realizing rapid amplification. The method has ultra-high sensitivity, up to single copy / reaction, which is superior to traditional qPCR. The primer probe set of the present application also has the advantage of high specificity.

[0041] The present application provides the application of the primer probe set and the kit in detecting hepatitis B virus, hepatitis C virus and human immunodeficiency virus.

[0042] In the present application, all raw material components are commercially available products well known to those skilled in the art unless otherwise specified.

[0043] The technical solutions in the present application will be described clearly and completely below in combination with the embodiments in the present application. All other embodiments obtained by those of ordinary skill in the art on the basis of the embodiments in the present application without any creative labor belong to the scope of protection of the present application.

[0044] Establishment of single RT-RAP for detecting hepatitis B virus, hepatitis C virus and human immunodeficiency virus

[0045] 1. Sample source and nucleic acid extraction

[0046] From February 2022 to September 2022, 40 hepatitis B virus positive sera, 40 hepatitis C virus positive sera and 40 human immunodeficiency virus positive nucleic acids stored in the laboratory of Hebei People's Hospital were collected.

[0047] The collected positive serum samples were extracted with a viral nucleic acid extraction kit (Xi'an Tianlong Technology Co., Ltd.) to extract nucleic acids, and stored at -80℃ until use.

[0048] A total of 120 nucleic acids were detected in parallel using the Guangzhou Daan Gene hepatitis B virus nucleic acid detection kit, hepatitis C virus nucleic acid detection kit and human immunodeficiency virus type 1 (HIV-1) nucleic acid detection kit.

[0049] 2. RT-RAP detection using the primer probe set for hepatitis B virus, hepatitis C virus and human immunodeficiency virus in Table 1.

[0050] The RT-RAA kit is a basic method RT-RAA amplification kit (Jiangsu Qitian Co., Ltd.), the qPCR kit is a FastOne-Step probe RT-qPCR Kit reagent kit (ABclonal Company), and the amplification instrument is a Kpeng ArchimedX6 fluorescent quantitative PCR instrument. The corresponding amplification system is configured according to Tables 2-3.

[0051] RT-RAP detection steps: The qPCR system was divided into eight tubes, and 25 μl of n-dodecane was added. The eight tubes were placed in a LongGene A300 (60°C for 1 min, 4°C for 30 s) to make the n-dodecane evenly float on the qPCR system. 40 μl of the RT-RAA reaction system was added to each RT-RAA reaction unit tube, and the dry powder was dissolved in the reaction system. The unit tube was mixed twice in a Qitian B6100 mixing instrument. 9 μl of the RT-RAA system was divided into the solidified n-dodecane, and 1 μl of the sample nucleic acid was added. 0.5 μl of magnesium acetate was added to the tube cap, and the tube cap was closed. The magnesium acetate was dropped into the RT-RAA system by using an Eastwin instant centrifuge, and the RT-RAA reaction was started. The eight tubes were placed in a PCR instrument, and the reaction program was carried out according to Table 4. The detection process is shown in Figure 1 .

[0052] Table 2 RT-RAA (basic method) kit HBV / HCV / HIV reaction system

[0053]

[0054] Table 3 qPCR kit HBV / HCV / HIV reaction system

[0055]

[0056] Table 4 Reaction program

[0057]

[0058] Example 2 Establishment of a triple RT-RAP for detecting hepatitis B virus, hepatitis C virus, and human immunodeficiency virus

[0059] The qPCR reaction system was configured according to Table 6, and the RT-RAA reaction system was configured according to Table 5.

[0060] The triple RT-RAP detection steps were the same as the RT-RAP detection steps described in step 2 of Example 1. The reaction program was carried out according to Table 7.

[0061] Table 5 RT-RAA (basic method) kit reaction system

[0062]

[0063] Table 6 qPCR kit reaction system

[0064]

[0065] Table 7 Reaction program

[0066]

[0067]

[0068] Example 3 Sensitivity evaluation of hepatitis B virus, hepatitis C virus and human immunodeficiency virus RT-RAP detection method

[0069] 1. Single RT-RAP sensitivity evaluation: The target gene sequence was synthesized into a hepatitis B virus, hepatitis C virus and human immunodeficiency virus positive plasmid DNA containing target gene using pUC57 as a carrier (synthesized by Beijing Qikeli Biological Co., Ltd.). The synthesized three virus positive plasmids were diluted by 10 times in a continuous concentration gradient, and the concentration range was 10 0 ~ 10 4 copies / μL. Different concentrations of positive plasmid DNA were used as templates for single RT-RAP detection (the detection method was the same as in Example 1, and when the amplification template was RNA, reverse transcription was required). The RT-RAA reaction system and the qPCR reaction system were configured according to Table 2 and Table 3, respectively, and the addition amount of positive plasmid DNA was 1 μL, and the final concentration of RT-RAA primer was 0.4 μmol / L. The detection results are shown in Figures 2-4 .

[0070] General qPCR detection sensitivity evaluation: The detection method, primers and probes and test steps were the same as in Example 1.

[0071] As shown in Figures 2-4 , Figure 2 A and Figure 2 B are respectively the RT-RAP detection sensitivity (LOD) graph and the general qPCR detection sensitivity graph for hepatitis B virus; as shown in Figure 2 A and Figure 2 B, the sensitivity of RT-RAP detection for hepatitis B virus is 10 0 copies / reaction, and the sensitivity of general qPCR method is 10 1 copies / reaction. Figure 3 A and Figure 3 B are respectively the RT-RAP sensitivity graph and the general qPCR detection sensitivity graph for hepatitis C virus; as shown in Figure 3 A and Figure 3 B, the sensitivity of RT-RAP detection for hepatitis C virus is 10 0 copies / reaction, and the sensitivity of general qPCR method is 10 2 copies / reaction. Figure 4 A and Figure 4 B are respectively the RT-RAP sensitivity graph and the general qPCR detection sensitivity graph for human immunodeficiency virus-1; as shown in Figure 4 A and Figure 4As shown in B, the sensitivity of RT-RAP for detecting human immunodeficiency virus is 10 0 copies / reaction, the sensitivity of the conventional qPCR method is 10 1 Copy / React.

[0072] 2. Triple RT-RAP sensitivity evaluation: The positive plasmids of the three viruses synthesized in step 1 of this example were diluted in a 10-fold continuous concentration gradient, with a concentration range of 10 0 ~10 4 Copies / μL, using different concentrations of positive plasmid DNA as template, triple RT-RAP experiment (detection method is the same as Example 2). RT-RAA reaction system and qPCR reaction system were configured according to Table 5 and Table 6, respectively, and the total amount of positive plasmid DNA added for the three viruses was 1 μL. The test results are shown in Figures 5-7 .

[0073] Evaluation of triple conventional qPCR sensitivity: The detection method was the same as the primers, probes and experimental steps of qPCR in Example 2.

[0074] like Figures 5-7 As shown, Figure 5 A, 6A and 7A are triple RT-RAP positive plasmid sensitivity amplification signal diagrams, Figure 5 B, 6B and 7B are triple common qPCR positive plasmid sensitivity amplification signal diagrams, in which the detection of hepatitis B virus uses VIC fluorescence signal, hepatitis C virus uses CY5 fluorescence signal, and human immunodeficiency virus uses FAM fluorescence signal. Figure 5 As shown in A, 6A and 7A, the sensitivity of triple RT-RAP for detecting hepatitis B virus is 10 0 Copies / reaction, HCV sensitivity is 10 1 copies / reaction, human immunodeficiency virus sensitivity is 10 0 Copy / React. Figure 5 Figure 5 As shown in B, 6B and 7B, the sensitivity of triplex conventional qPCR for detecting hepatitis B virus is 10 1 Copies / reaction, HCV sensitivity is 10 2 copies / reaction, human immunodeficiency virus sensitivity is 10 1 Copy / React.

[0075] Example 4 Specificity Evaluation of RT-RAP Detection Methods for Hepatitis B Virus, Hepatitis C Virus, and Human Immunodeficiency Virus

[0076] Using 8 blood related pathogen samples preserved in the laboratory, including EB virus, human cytomegalovirus, human herpes virus, herpes simplex virus type 1, herpes simplex virus type 2, Treponema pallidum, Staphylococcus aureus and Candida albicans. The RT-RAP method established in Examples 1 and 2 was used for parallel identification, and the results are shown in Table 8.

[0077] Table 8 Specificity evaluation results

[0078]

[0079] Note: N represents negative

[0080] As shown in Table 8: the single RT-RAP method for detecting hepatitis B virus, hepatitis C virus and human immunodeficiency virus established in Example 1 can specifically detect hepatitis B virus, hepatitis C virus and human immunodeficiency virus, and does not cross react with other pathogens.

[0081] The triple RT-RAP method for detecting hepatitis B virus, hepatitis C virus and human immunodeficiency virus established in Example 2 can specifically detect hepatitis B virus, hepatitis C virus and human immunodeficiency virus, and does not cross react with other pathogens.

[0082] The above is only the preferred embodiment of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. An RT-RAP method for detecting blood viruses for non-disease diagnosis purposes, characterized in that: The steps include: 1) Extracting nucleic acid from the sample to be tested; 2) using the nucleic acid as a template, sequentially performing RT-RAA amplification and qPCR amplification using an RT-RAP primer probe set, and collecting fluorescent signals; the RT-RAA amplification and qPCR amplification systems use n-docosane as a spacer; 3) If the amplification curve corresponding to the hepatitis B virus peaks and shows an S-shaped amplification, it is determined that the test sample contains the corresponding hepatitis B virus; if the amplification curve corresponding to the hepatitis C virus peaks and shows an S-shaped amplification, it is determined that the test sample contains the corresponding hepatitis C virus; if the amplification curve corresponding to the human immunodeficiency virus peaks and shows an S-shaped amplification, it is determined that the test sample contains the corresponding human immunodeficiency virus; The RT-RAP primer probe set includes a primer probe set for detecting hepatitis B virus, a primer probe set for detecting hepatitis C virus, and a primer probe set for detecting human immunodeficiency virus; The primer and probe set for hepatitis B virus detection includes: HBV-RAA-F, HBV-RAA-R, HBV-PCR-F, HBV-PCR-R and HBV-P; The nucleotide sequences of HBV-RAA-F, HBV-RAA-R, HBV-PCR-F, HBV-PCR-R and HBV-P are shown in SEQ ID NO. 1 to SEQ ID NO. 5, respectively; The primer and probe set for detecting hepatitis C virus includes: HCV-RAA-F, HCV-RAA-R, HCV-PCR-F, HCV-PCR-R and HCV-P; The nucleotide sequences of HCV-RAA-F, HCV-RAA-R, HCV-PCR-F, HCV-PCR-R and HCV-P are shown in SEQ ID NO.6 to SEQ ID NO.10, respectively; The primer probe set for human immunodeficiency virus detection includes: HIV-RAA-F1, HIV-RAA-R1, HIV-PCR-F1, HIV-PCR-R1, HIV-P1, HIV-RAA-F2, HIV-RAA-R2, HIV-PCR-F2, HIV-PCR-R2 and HIV-P2; The nucleotide sequences of HIV-RAA-F1, HIV-RAA-R1, HIV-PCR-F1, HIV-PCR-R1, HIV-P1, HIV-RAA-F2, HIV-RAA-R2, HIV-PCR-F2, HIV-PCR-R2 and HIV-P2 are shown in SEQ ID NO.11 to SEQ ID NO.20, respectively; The HBV-P, HCV-P, HIV-P1 and HIV-P2 are respectively labeled with different fluorescent groups; The RT-RAA amplification reaction system includes 280 mM magnesium acetate, template and RT-RAA reaction mixture; the volume ratio of magnesium acetate, template and RT-RAA reaction mixture is 0.2-0.8:0.5-1.5:6-12; The RT-RAA amplification and qPCR amplification systems are separated by n-docosane, comprising: adding molten n-docosane to the qPCR amplification system and allowing the n-docosane to stand, with the n-docosane floating on the qPCR amplification system; after the n-docosane solidifies, adding the RT-RAA amplification system on the solidified n-docosane; The RT-RAA amplification and qPCR amplification are performed in a tube with a cap; adding the RT-RAA amplification system to solidified n-dodecane includes the following steps: adding the RT-RAA amplification system except magnesium acetate to the solidified n-dodecane, adding magnesium acetate to the cap, and using magnesium acetate to start the RT-RAA reaction.

2. The RT-RAP method according to claim 1, wherein The fluorescent group includes FAM, HEX, ROX, TET, JOE, CY3, CY5, TAMRA or VIC.

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