Primer, probe and kit for detecting novel coronavirus

A technology of coronaviruses and kits, applied in biochemical equipment and methods, measurement/inspection of microorganisms, resistance to vector-borne diseases, etc., to achieve the effects of less templates, overcoming false negatives, and improving accuracy

Active Publication Date: 2020-05-26
GUANGZHOU LBP MEDICINE SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Some scholars believe that the current nucleic acid test h

Method used

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  • Primer, probe and kit for detecting novel coronavirus
  • Primer, probe and kit for detecting novel coronavirus
  • Primer, probe and kit for detecting novel coronavirus

Examples

Experimental program
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Effect test

Embodiment 1

[0059] The design of embodiment 1, specific primer and probe

[0060] In the design of primer probes, it is necessary to select regions that are conserved within the group and specific among groups for design, so as to avoid false negatives caused by missed detection, or false positives caused by poor specificity.

[0061] Through sequence comparison and literature search, the present invention finds that the structural genes S, M, ORF1ab, E and N of 2019-nCoV are highly conserved and can be used as target regions for the identification and detection of new coronaviruses.

[0062] In this example, the sequences of endemic human coronavirus (HKU1, OC43, 229E and NL63), SARS coronavirus, MERS coronavirus, bat coronavirus (MG772933_Bat_bat_SL_CoVZC45), SARS-like virus (MG772934_Bat_SARS_like_bat_SL_CoVZXC21) were selected for comparison, and the results are as follows figure 1 shown. Open reading frame 1ab (ORF1ab), nucleocapsid protein (N), envelope protein (E), surface glycopr...

Embodiment 2

[0113] Embodiment 2, comparative analysis of specific primers and probes of the present invention and CDC recommended sequences

[0114] In order to evaluate the preferred primers and probes of Example 1, the sequence recommended by CDC (Center for Disease Control and Prevention) was used as a comparison sequence for detection evaluation.

[0115] China CDC does not recommend primer and probe sequences for E gene, S gene and M gene, but only recommends primer and probe sequences for ORF1ab and N genes. The recommended primer and probe sequences for ORF1ab and N regions are as follows:

[0116] ORF1ab-F (SEQ ID NO: 16): 5'-CCCTGTGGGTTTTACACTTAA-3';

[0117] ORF1ab-R (SEQ ID NO: 17): 5'-ACGATTGTGCATCAGCTGA-3';

[0118] ORF1ab-P (SEQ ID NO: 18): 5'-CCGTCTGCGGTATGTGGAAAGGTTATGG-3';

[0119] N-F (SEQ ID NO:31): 5'-GGGGAACTTCTCCTGCTAGAAT-3';

[0120] N-R (SEQ ID NO: 32): 5'-CAGACATTTTGCTCTCAAGCTG-3';

[0121] N-P (SEQ ID NO: 33): 5'-TTGCTGCTGCTTGACAGATT-3'.

[0122] The US CDC ...

Embodiment 3

[0129] The optimization experiment of embodiment 3, RT-PCR method

[0130] There are two methods for detection of viral RNA based on fluorescent RT-PCR, that is, the two-step method of performing reverse transcription into cDNA first, and then performing PCR, and the one-step method system of simultaneously performing RT-PCR in the same system. Both have pros and cons. Using the two-step system, the cDNA obtained by reverse transcription is easy to preserve; while the one-step method is fast, simple, and reduces the chance of contamination. For multiplex PCR, interference between primers and probes needs to be considered.

[0131] The invention compares the detection effects of the two reaction systems, first evaluates the detection effect of a single pair of primers and probes, and then conducts a combination test of the synthesized sequences according to the reaction tubes.

[0132] In this example, Takara RNA PCR Kit (RR019A) and Takara Ex TAQ HS (RR390L) were used in the...

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Abstract

The invention provides a primer, a probe and a kit for detecting a novel coronavirus. The specific primer and the probe are designed according to an ORF1ab gene, an E gene, an N gene, an S gene and anM gene of the novel coronavirus 2019-nCoV and are used for identifying and detecting the novel coronavirus. The specific primer/probe and the kit for detecting the 2019-nCoV are high in sensitivity,precision and accuracy, good in specificity and stability, small in template amount required by detection and objective and accurate in detection result. The five-target design can well overcome falsenegative caused by variation in the RNA virus passage process, the positive detection rate can be effectively improved, and missing detection is avoided as much as possible. High clinical applicationvalues are realized.

Description

technical field [0001] The invention relates to the field of virus detection, and more specifically, to a primer, a probe and a kit for detecting a novel coronavirus. Background technique [0002] Nucleic acid test (RT-PCR) is the "gold standard" for the diagnosis of new coronary pneumonia. The State Drug Administration emergency approval of the first batch of 7 companies' new coronavirus 2019-nCoV nucleic acid detection kits 2020-2-11 Version 002], approved products are based on the open reading frame 1a / b (Openreading frame 1ab, ORF1ab), envelope protein (Envelope protein, E) and nucleocapsid protein (Nucleocapsid) in the new coronavirus genome protein, N) Three gene designs. [0003] However, the evolution of viruses is very rapid [Bartoszewicz J M, Seidel A, Renard B Y. Interpretable detection of novel human viruses from genome sequencing data [J]. BioRxiv, 2020.], through sequence comparison and literature search, it can be found that Related SNP mutations have been ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2521/107Y02A50/30
Inventor 陈绍宇何瑰李楚明刘慧李灵鸽黄颖
Owner GUANGZHOU LBP MEDICINE SCI & TECH
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