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PCR target sequence, primers and probe for detecting infectious SARS-CoV-2 and application of PCR target sequence, primers and probe

A sars-cov-2, target sequence technology, applied in the field of SARS-CoV-2 nucleic acid detection, can solve the difference in PMA binding efficiency, reducing PCR amplification efficiency and sensitivity, GC content, base bias conformation characteristics, etc. problems, to achieve good application prospects, shorten the test cycle, reduce the requirements of scientific research conditions and equipment

Active Publication Date: 2021-07-23
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When PMA enters a virus with damaged capsid protein, it will bind to the viral nucleic acid at a certain stoichiometric ratio. In theory, the modification of the target gene by PMA can inhibit the PCR amplification of the target fragment, so the longer target gene amplification fragment It will increase the probability of binding to PMA, thereby improving the inhibitory effect of PMA, but longer amplification fragments may reduce the amplification efficiency and sensitivity of PCR reactions
In addition, different regions and positions in the viral genome have different binding efficiencies with PMA due to their different GC content, base preference, and conformational characteristics.

Method used

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  • PCR target sequence, primers and probe for detecting infectious SARS-CoV-2 and application of PCR target sequence, primers and probe
  • PCR target sequence, primers and probe for detecting infectious SARS-CoV-2 and application of PCR target sequence, primers and probe
  • PCR target sequence, primers and probe for detecting infectious SARS-CoV-2 and application of PCR target sequence, primers and probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Screening and optimization of primers and probes for detection of infectious SARS-CoV-2:

[0027] 1.1 Design of infectious SARS-CoV-2 primers and probes

[0028] According to the SARS-CoV-2 (ZY38-1) gene group, 1~15 (SEQ ID NO.1~SEQ ID NO.45) primer probe sets were synthesized, referring to and synthesizing the SARS-CoV-2 detection primer probe released by China CDC Group; China CDC-ORF1ab and China CDC-N (SEQ ID NO.46~SEQ ID NO.51), refer to and synthesize US CDC-N1~N3 (SEQ ID NO. ID NO.52~SEQ ID NO.60). The sequences of the primers and fluorescent probes are as follows (the above primers and probes were synthesized by Shanghai Sangon Biotechnology Co., Ltd.):

[0029]Table 1. SARS-CoV-2 primers and probe sequences

[0030]

[0031]

[0032]

[0033] 1.2qRT-PCR reaction system and reaction conditions:

[0034] Reaction system: Enzyme Mix 2μl, MP buffer 12μl, upstream and downstream primers at a final concentration of 400-1000pM each, probes at a final conc...

Embodiment 2

[0055] Optimization of PMA-Triton X-100-qRT-PCR method for detection of infectious SARS-CoV-2

[0056] 2.1 PMA usage concentration optimization

[0057] Pipette the test samples of inactivated samples in equal volumes, add different concentrations of PMA (final concentrations are 0, 5, 50, 100, 150, 200, 250 μM) and incubate at room temperature for 20 minutes in the dark by vortexing. After the incubation, use PMA- lite TM The LED photolysis instrument photolyzes the sample for 15 minutes. Nucleic acid was extracted with Tiangen automatic nucleic acid extractor and used as a template for qRT-PCR. Considering the use effect and use cost comprehensively, the lowest PMA concentration with large difference in ΔCt or Ct>40 of the sample to be sequenced and no typical amplification curve was selected as the optimal PMA working concentration. The results are shown in Table 5, and the optimal working concentration of PMA is 100 μM.

[0058] Table 5 Optimum PMA Concentration Explor...

Embodiment 3

[0071] Application of multiplex qRT-PCR kit for detection of SARS-CoV-2 infectivity

[0072] This example refers to the "Specific Site Disinfection Technical Plan" (National Health Office Disease Control Letter [2020] No. 156) and "National Food Safety Standard Food Cold Chain Logistics Hygienic Specifications" (GB31605-2020) to simulate high-temperature, alcohol-based disinfectants ( 75% ethanol), chlorine-containing disinfectant (84 disinfectant), and quaternary ammonium salt disinfectant (Germet) treated food packaging surface samples (negative by plaque test, as inactivation completed group), in addition SARS-CoV-2 plaque test-positive virus fluid was used as the non-inactivated group. The optimal detection of infectious SARS-CoV-2 primers and probes and processing methods obtained in Examples 1-2 were applied to the detection of infectious SARS-CoV-2 multiplex qRT-PCR kit.

[0073] Sample collection steps:

[0074] Refer to the sample collection section in "Sampling and...

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Abstract

The invention relates to the field of SARS-CoV-2 nucleic acid detection, in particular to a PCR (Polymerase Chain Reaction) target sequence, primers and a probe for detecting infectious SARS-CoV-2 and application of the PCR target sequence, the primers and the probe. The PCR target sequence, the primers and the probe for detecting the infectious SARS-CoV-2, which are provided by the invention, can be used for well distinguishing the infectivity of the SARS-CoV-2, and meanwhile, the PCR target sequence, the primers and the probe have the characteristics of high sensitivity and good repeatability. Compared with a conventional infectious SARS-CoV-2 detection test, the the PCR target sequence, the primers and the probe have the advantages that the test period of infectious SARS-CoV-2 detection is greatly shortened, the requirements on scientific research conditions and equipment are reduced, nucleic acid of virus inactivation caused by virus capsid protein damage and infectious virions are effectively distinguished, the detection accuracy is improved, and the false positive phenomenon occurring in actual detection is reduced.

Description

technical field [0001] The present invention relates to the field of SARS-CoV-2 nucleic acid detection, more specifically, to PCR target sequences, primers and probes and applications for detecting infectious SARS-CoV-2. Background technique [0002] Severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2, SAR S-CoV-2, referred to as new coronavirus) infected pneumonia (Corona VirusDisease 2019, referred to as COVID-19), referred to as new coronary pneumonia, is transmitted through the respiratory tract an infectious disease. [0003] At present, nucleic acid detection of the new coronavirus is the gold standard for diagnosing new coronary pneumonia, screening asymptomatic infections, investigating cold chain food and environmental pollution, and is a key point in epidemic prevention and control. At present, the nucleic acid detection method for the new coronavirus is mainly qRT-PCR method, which plays an important role in the detect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2561/113C12Q2563/107C12Q2545/114Y02A50/30
Inventor 金梅林高朔邹维华邹忠杨丽吕长杰回显峰孙小美
Owner HUAZHONG AGRI UNIV
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