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Double-stranded primer probe, kit and multiplex PCR method for detecting SARS-COV-2

A SARS-COV-2, double-stranded primer technology, applied in the field of PCR, can solve problems such as inability to be divided, multiple PCR primer design considerations, and increased computational space and time complexity, so as to reduce complexity and solve problems. Competitive inhibition and specificity enhancement

Pending Publication Date: 2020-10-02
THE AFFILIATED HOSPITAL OF TRADITIONAL CHINESE MEDICAL TO SOUTHWEST MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Primer design is the key to the success of multiplex PCR, but primer design is not a simple superposition of primer design for singleplex reactions: on the one hand, the mutual interference between primers and between primers and template sequences leads to increased considerations in multiplex PCR primer design , greatly increasing the computational space and time complexity; on the other hand, multiplex PCR primer design needs to be considered as a whole and cannot be divided into a simple combination of multiple PCR primer design problems

Method used

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  • Double-stranded primer probe, kit and multiplex PCR method for detecting SARS-COV-2
  • Double-stranded primer probe, kit and multiplex PCR method for detecting SARS-COV-2
  • Double-stranded primer probe, kit and multiplex PCR method for detecting SARS-COV-2

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Embodiment

[0051] 1. Experimental procedures and experimental conditions:

[0052] 1, the selection of positive control plasmid: comprise the plasmid of SARS-COV-2 virus ORF, E and N gene (obtained from Shanghai Bioengineering Co., Ltd.).

[0053] 2. Specific primer probes for SARS-COV-2 virus ORF1ab, N and E genes:

[0054] Table 1

[0055] name Sequence (5'-3') Numbering ORF1ab-F(FAM) 6-FAM-CCCTGTGGGTTTTACACTTAA SEQ ID NO.1 ORF1ab-F (BHQ1) TGTAAAACCCACAGGG-BHQ1 SEQ ID NO.2 ORF1ab-R ACGATTGTGCATCAGCTGA SEQ ID NO.3 N-F(HEX) HEX-GGGGAACTTCTCCTGCTAGAAT SEQ ID NO.4 N-F(BHQ1) AGCAGGAGAAGTTCCCC-BHQ1 SEQ ID NO.5 N-R CAGACATTTTGCTCTCCAAGCTG SEQ ID NO.6 E_Sarbeco_F(ROX) ROX-ACAGGTACGTTAATAGTTAATAGCGT SEQ ID NO.7 E_Sarbeco_F(Dabcyl) TTAACTATTAACGTACCTGT-Dabcyl SEQ ID NO.8 E_Sarbeco_R ATATTGCAGCAGTACGCACACA SEQ ID NO.9

[0056] 3. The PCR reaction system is as follows:

[0057] Table 2

[0058] ...

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Abstract

The invention belongs to the technical field of PCR, and specifically discloses a double-stranded primer probe, kit and multiplex PCR method for detecting SARS-COV-2. The double-stranded primer probeincludes the specific primer probe used for detecting ORFlab, N and E gene sequences of SARS-COV-2 viruses; and the kit includes the double-stranded primer probe. Through the adoption of the double-stranded primer probe, the probability of forming a stem-loop structure by the primer or forming dimer between the primers can be reduced, so that the consistency of PCR amplification efficiency can beachieved, therefore, the inhibition of competitiveness between multiplex PCR can be solved, and the simultaneous detection on single tubes of multiple target genes of ORFlab, N and E of SARS-COV-2 viruses can be realized. Through the double-stranded primer probe, the integrated design of the primers and probes can be realized, the complexity of a system can be reduced, and the specificity of primer and template binding can be enhanced; and the double-stranded primer probe is good in temperature compatibility and convenient for new users to operate.

Description

technical field [0001] The invention relates to the technical field of PCR, in particular to a double-strand primer probe, a kit and a multiplex PCR method for detecting SARS-COV-2. Background technique [0002] Laboratory testing methods for the novel coronavirus (SARS-COV-2) include virus isolation and culture, serological testing, and molecular biological testing. At present, the detection of new coronaviruses in the market is mainly based on molecular biology detection. Since the detection of fluorescent quantitative PCR method is simple and fast, high accuracy, and can be used for early diagnosis, it is the best choice for the new coronavirus detection kits that have been used so far. The main experimental principle. Fluorescent quantitative PCR is the same as ordinary PCR reaction, and both need to design amplification primers according to the target sequence, and then monitor the PCR process in real time through fluorescent signals during the amplification process; i...

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2563/107C12Q2545/113Y02A50/30
Inventor 姚娟张章
Owner THE AFFILIATED HOSPITAL OF TRADITIONAL CHINESE MEDICAL TO SOUTHWEST MEDICAL UNIV