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Probe and primer composition for rapidly detecting seven coronaviruses and other respiratory tract pathogens

A primer composition, coronavirus technology, applied in recombinant DNA technology, biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as the inability to achieve simultaneous detection of multiple pathogens

Pending Publication Date: 2020-07-24
JIANGSU BIOPERFECTUS TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these published detection methods have high sensitivity, they are limited by the fluorescence channel and cannot realize the simultaneous detection of multiple pathogens in a single tube.

Method used

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  • Probe and primer composition for rapidly detecting seven coronaviruses and other respiratory tract pathogens
  • Probe and primer composition for rapidly detecting seven coronaviruses and other respiratory tract pathogens
  • Probe and primer composition for rapidly detecting seven coronaviruses and other respiratory tract pathogens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] The design of embodiment 1 specific primer and probe

[0157] Search and download N genes of OC43-CoV, NL63-CoV, 229E-CoV, HKU1-CoV, SARS-CoV, FluB, RSV, ORF1a, ORF1b, N genes of MERS-CoV, ORF1ab, N genes of 2019-nCoV through NCBI search , the M gene of FluA, the HEXON gene of HadV and the RNaseP gene, using the Primer 3 software, the specific amplification primers and Taqman or MGB probes of the above pathogens were designed respectively, and synthesized by Shanghai Shuoying Biotechnology Co., Ltd. The primers and probes used for detection are shown in Table 2:

[0158] Table 2 Primer and probe sequence information

[0159]

[0160]

Embodiment 2

[0161] Example 2 sample nucleic acid extraction

[0162] (1) Clinical sample collection, storage and transportation: suitable for throat swab and sputum sample collection. Throat swab samples were collected in accordance with the throat swab collection method in the "Clinical Nursing Practice Guidelines" (2011 Edition). Collect throat swab samples from patients within 3 days of onset, and use special sampling cotton swabs made of absorbent cotton and wooden sticks. Gently depress the tongue with a tongue depressor, and quickly wipe the palatine arches on both sides of the patient's mouth and the secretions of the pharynx and tonsils with a cotton swab to avoid touching the pharyngeal swab with other parts; tube, snap off the cotton swab stem near the tip, and screw on the tube cap to seal it from drying out. Sputum samples are collected in accordance with "Diagnostic Criteria for Pulmonary Tuberculosis" WS288-2008. It is advisable to take the first mouthful of sputum in the ...

Embodiment 3

[0164] Example 3 detects the extracted sample nucleic acid

[0165] The preparation of the amplification reaction system was carried out according to Table 3:

[0166] Table 3 Amplification reaction system preparation table

[0167]

[0168]

[0169] Carry out RT-PCR amplification according to the following procedure: 42-50°C for 10-30min; 93-95°C for 5-10min; 93-95°C for 10-15s, 55-60°C for 40-60s, for a total of 45 cycles;

[0170] Perform melting curve analysis according to the following procedures: 93-95°C for 10-15s; 40-45°C for 30-60s; start the melting curve analysis program from 40-85°C.

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Abstract

The invention discloses a probe and primer composition for rapidly detecting seven coronaviruses and other respiratory tract pathogens. A group of specific primers and the Taqman or MGB probe are designed mainly according to the specificity of N genes of OC43-CoV, NL63-CoV, 229E-CoV, HKU1-CoV, SARS-CoV, FluB and RSV, ORF1a, ORF1b and N genes of MERS-CoV, ORF1ab and N genes of 2019-nCoV, an M geneof FluA and an HEXON gene of HadV, and by utilizing a melting curve technology, CoV-OC43, HCoV-NL63, HCoV-229E, HCoV-HKU1, SARS-CoV, MERS-CoV, 2019-nCoV, FluA, FluB, HAdV and RSV are rapidly identified according to the change of the melting temperature. By means of the design, multiple pathogens can be detected in one fluorescent channel at the same time, and a relatively high sensitivity can be met.

Description

Technical field: [0001] The invention belongs to the technical field of nucleic acid detection, in particular to a method for detecting coronavirus OC43, coronavirus NL63, coronavirus 229E, coronavirus HKU1, SARS coronavirus, MERS coronavirus, novel coronavirus (2019-nCoV), influenza A virus , Influenza B virus, adenovirus, respiratory syncytial virus kits and detection methods. Background technique: [0002] Coronavirus (Coronavirus, CoV) belongs to Nidoviridae, Coronaviridae, is a class of enveloped positive-strand RNA viruses, with a total length of 26-32kb, and is divided into three genera: α, β, and γ. The α and β genera are only pathogenic to mammals, and the γ genus mainly causes infection in birds. CoV is mainly transmitted through direct contact with secretions or through aerosols and droplets, and there is also evidence that it can be transmitted through the fecal-oral route. The infection spectrum of coronaviruses in nature is very wide, and common mammals such a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/16C12Q2600/166C12Q2531/107C12Q2521/107C12Q2537/143C12Q2527/107C12Q2547/101C12Q2561/101C12Q2545/101Y02A50/30
Inventor 赵西浩张蓉李伟伟曹新文陈永红郭玉婉苏波张梅梅李秀林刘中华王国强
Owner JIANGSU BIOPERFECTUS TECH CO LTD
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