A kind of rap gene detection kit, detection method and application and rap virus detection kit

A gene detection and kit technology, applied in the field of gene amplification, can solve the problems of difficulty in designing probes, long time-consuming, easy contamination of N-PCR, etc.

Active Publication Date: 2022-05-17
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, RAA technology requires very long probes (45-50bp in length), which makes it difficult to design probes
Polymerase chain reaction (PCR) is a nucleic acid molecular diagnostic technology widely used in diagnosis at present. Quantitative PCR (qPCR), a PCR-derived technology, is currently the gold standard for nucleic acid detection of many pathogens, but the sensitivity needs to be improved.
Nested PCR (N-PCR) can improve the sensitivity of PCR detection, but N-PCR has the disadvantages of easy contamination and long time consumption
A more efficient gene amplification method is still lacking

Method used

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  • A kind of rap gene detection kit, detection method and application and rap virus detection kit
  • A kind of rap gene detection kit, detection method and application and rap virus detection kit
  • A kind of rap gene detection kit, detection method and application and rap virus detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] (50) throat swab specimens from Hebei Provincial Children's Hospital, (403) throat swab specimens and (103) alveolar lavage fluid specimens from Hunan Province CDC.

[0048] The primers and probes of ADV3, ADV7 and RSV in Table 1 were used for detection. Configure the system according to Tables 2 and 3, the final concentration of the outer primer is 0.4 μmol / L, and the final concentration of the inner primer and probe is 0.2 μmol / L. The RAA kit is the basic method RAA amplification kit of Jiangsu Qitian Co., Ltd., and the PCR kit is the Entrans qPCR Probe SetV2 kit of ABI Company.

[0049] Table 1 Primer and probe information for detection of three viruses

[0050]

[0051] Table 2 and 3 are the amplification systems of RAP.

[0052] Table 2 Reaction system of RAA basic kit or RT-RAA basic kit

[0053]

[0054] Table 3 qPCR reaction system

[0055]

[0056]

[0057] Take 10 μl of the reaction system in Table 2 and add it to the cap of the PCR tube, and 4...

Embodiment 2

[0073] Using the kit provided by the present invention, the method of Example 1 was used to collect 19 virus samples for differential identification. The 19 samples were from the Institute of Viral Disease Prevention and Control, Chinese Center for Disease Control and Prevention. The identification results are shown in Table 7. It can be seen from Table 7 that the RAP method can distinguish and identify the listed samples, and no cross-reaction was observed. The test results show that the method of the present invention can only specifically detect ADV3, ADV7 or RSV, and does not cross-react with other viruses.

[0074] The specific detection analysis of kit described in table 7

[0075]

[0076]

Embodiment 3

[0078]According to the comparative analysis of Vector11.0 software, specific primers and probes were designed in the conserved regions of ADV3, ADV7 and RSV viruses, and corresponding recombinant plasmids (synthesized by Beijing Qingke Biological Co., Ltd.) were synthesized. 基于ADV3的保守序列Hexon基因,本发明首先提供一条用于检测ADV3的靶序列,其具有SEQ ID NO.16所示的序列(GATAGAGGTCCTAGCTTCAAGCCATATTCCGGCACAGCTTACAATTCACTCGCTCCTAAGGGCGCGCCCAATACATCTCAGTGGATAGTTACAACAAATGGGGACAATGCAGTAACTACCACCACAAACACATTTGGCATTGCTTCCATGAAGGGAGACAATATTACTAAAGAAGGTTTGCAAATTGGGAAAGACATTACCACTACTGAAGGAGAAGAAAAGCCCATTTATGCCG)或其特异性片段,该序列连接pUC57载体合成目的质粒; 基于ADV7的保守序列Hexon基因,本发明首先提供一条用于检测ADV7的靶序列,其具有SEQ ID NO.17所示的序列(AAGCCATATTCCGGCACAGCTTACAATTCACTGGCTCCTAAGGGCGCGCCTAACACATCTCAGTGGATAGTTACAACGGGAGAAGACAATGCCACCACATACACATTTGGCATTGCTTCCACGAAGGGAGACAATATTACTAAGGAAGGTTTAGAAATTGGGAAAGACATTACTGCAGACAACAAGCCCATTTATGCCGATAAAACATATCAGCCAGAGCCTCAAGTTGGAGAAGAATCATGGACTGATATTGATGGAACAAATGAAAAATTTGGAGGTAGAGCTCTTAAACCAGCTACTAAAATGAAGCCATGC)或其特异性片段,该序列连接pU...

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PUM

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Abstract

The invention relates to a RAP gene detection kit, a detection method and application thereof and a RAP virus detection kit, belonging to the technical field of gene amplification. The kit of the present invention includes a pair of outer primers, a pair of inner primers, a probe and a reaction solution. The pair of outer primers is designed based on a recombinase-mediated isothermal amplification method, and the pair of inner primers and probes are based on real-time fluorescence. The quantitative nucleic acid amplification method is designed; the reaction solution includes a recombinase-mediated isothermal amplification reaction solution and a real-time fluorescence quantitative nucleic acid amplification reaction solution. The kit of the invention has high detection specificity and ultrahigh sensitivity, and the detection is efficient and fast.

Description

technical field [0001] The invention relates to the technical field of gene amplification, in particular to a RAP gene detection kit, a detection method and application, and a RAP virus detection kit. Background technique [0002] Recombinase aided amplification (RAA) is a new type of molecular diagnostic technology that amplifies at a constant temperature of 37-39°C. RAA has high amplification efficiency, and a large amount of target fragments can be amplified in 30 minutes or less. However, the RAA technique requires very long probes (45-50 bp in length), which makes it difficult to design probes. Polymerase chain reaction (PCR) is a nucleic acid molecular diagnostic technique widely used in diagnosis. Quantitative PCR (qPCR), a PCR-derived technology, is currently the gold standard for nucleic acid detection of many pathogens, but the sensitivity needs to be improved. Nested PCR (N-PCR) can improve the sensitivity of PCR detection, but N-PCR has the disadvantages of eas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2521/507C12Q2563/107C12Q2537/1376C12Q2531/113C12Q2545/113Y02A50/30
Inventor 马学军申辛欣王佶张瑞卿何小周应清届凡国豪
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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