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RAP gene detection kit, detection method and application thereof, and RAP virus detection kit

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

Active Publication Date: 2021-05-07
中国疾病预防控制中心病毒病预防控制所 +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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  • RAP gene detection kit, detection method and application thereof, and RAP virus detection kit
  • RAP gene detection kit, detection method and application thereof, and RAP virus detection kit
  • RAP gene detection kit, detection method and application thereof, 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 and (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 outer primers is 0.4 μmol / L, and the final concentration of inner primers and probes 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 Set V2 kit of ABI Company.

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

[0050]

[0051]

[0052] Table 2 and 3 are the amplification systems of RAP.

[0053] Table 2 Reaction system of RAA basic kit or RT-RAA basic kit

[0054]

[0055] Table 3 qPCR reaction system

[0056]

[0057]

[0058] Take 10 μl of the reaction system in Table 2 and add it to the cap of the PCR ...

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] No. sample ADV3 ADV7 RSV 1 Influenza A Negative Negative Negative 2 Influenza B Negative Negative Negative 3 rhinovirus Negative Negative Negative 4 parainfluenza virus Negative Negative Negative 5 human bocavirus Negati...

Embodiment 3

[0077]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(GATAGAGGTCCTAGCTTCAAGCCATATTCCGGCACAGCTTACAATTC ACTCGCTCCTAAGGGCGCGCCCAATACATCTCAGTGGATAGTTACAACA AATGGGGACAATGCAGTAACTACCACCACAAACACATTTGGCATTGCT TCCATGAAGGGAGACAATATTACTAAAGAAGGTTTGCAAATTGGGAAA GACATTACCACTACTGAAGGAGAAGAAAAGCCCATTTATGCCG)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(AAGCCATATTCCGGCACAGCTTACAATTCACTGGCTCCTAAGGGCGC GCCTAACACATCTCAGTGGATAGTTACAACGGGAGAAGACAATGCCAC CACATACACATTTGGCATTGCTTCCACGAAGGGAGACAATATTACTAAGGAAGGTTTAGAAATTGGGAAAGACATTACTGCAGACAACAAGCCCATT TATGCCGATAAAACATATCAGCCAGAGCCTCAAGTTGGAGAAGAATCA TGGACTGATATTGATGGAACAAATGAAAAATTTGGAGGTAGAGCTCTTAAACCAGCTACTAAAATGAAGCCATGC)或其特异性片段...

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Abstract

The invention relates to an RAP gene detection kit, a detection method and application thereof, and an RAP virus detection kit, which belongs to the technical field of gene amplification. The kit comprises an outer primer pair, an inner primer pair, a probe and a reaction solution, the outer primer pair is designed based on a recombinase-mediated isothermal amplification method, and the inner primer pair and the probe are designed based on a real-time fluorescent quantitative nucleic acid amplification method; the reaction solution comprises a recombinase-mediated isothermal amplification reaction solution and a real-time fluorescent quantitative nucleic acid amplification reaction solution. The kit provided by the invention has high detection specificity and ultrahigh sensitivity, and is efficient and rapid in detection.

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 even 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 o...

Claims

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Application Information

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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 中国疾病预防控制中心病毒病预防控制所
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