Novel coronavirus detection primer probe combination and kit and application thereof

A detection kit and coronavirus technology, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, and resistance to vector-borne diseases, etc., can solve the problems of false negatives, high cost, difficulty in popularization, etc., and achieve high detection sensitivity Effect

Pending Publication Date: 2020-09-11
广州达健生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the gene sequencing method is relatively time-consuming, expensive, and requires high equipment investment costs, making it difficult to popularize; the conventional real-time fluorescent RT-PCR detection method is time-consuming and relatively low in cost and cost, but the method requires high sample quality. Higher, and the sensitivity needs to be further improved, otherwise false negatives are prone to occur, which is not conducive to the early diagnosis and treatment of patients, and is not conducive to the prevention and control of new coronavirus pneumonia

Method used

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  • Novel coronavirus detection primer probe combination and kit and application thereof
  • Novel coronavirus detection primer probe combination and kit and application thereof
  • Novel coronavirus detection primer probe combination and kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Sample processing and RNA extraction

[0035] 1) Types of samples: upper respiratory tract specimens, such as throat swabs, nasal swabs, nasopharyngeal extracts; lower respiratory tract specimens, such as deep cough sputum, respiratory tract extracts, bronchial lavage fluid, alveolar lavage fluid, lung tissue biopsy specimen.

[0036] 2) RNA extraction: RNA was extracted by using the viral genome DNA / RNA extraction kit produced by Tiangen Biochemical Technology (Beijing) Co., Ltd.;

[0037] 2. Specific primers, probe screening and system optimization for the detection of novel coronavirus

[0038] 1) Screening of specific primers and probes: Design primers and probes according to the gene sequence of the novel coronavirus announced by the China National Center for Disease Control and Prevention, and send the designed primers and probes to Beijing Ruibo Xingke Biotechnology Co., Ltd. The company's synthesis, the specific sequence is shown in the table below:

[003...

Embodiment 2

[0056] 1. Performance verification of the kit

[0057] 1) Specificity test

[0058] The optimized digital PCR reaction system was used to test one case of throat swabs from patients with positive samples of new coronavirus and its interference cases (influenza A / B virus, mycoplasma pneumoniae, chlamydia pneumoniae, adenovirus, and respiratory syncytial virus). Test, and select throat swabs from normal people as a negative control to test the specificity of the established method.

[0059] serial number sample copy number 1 COVID-19 Positive Sample 5751 2 negative control - 3 Influenza A virus - 4 Influenza B virus - 5 Mycoplasma pneumoniae - 6 Chlamydia pneumoniae - 7 Adenovirus - 8 respiratory syncytial virus -

[0060] The results show that the novel coronavirus gene detection kit provided by the present invention can accurately detect the novel coronavirus samples containing the target gene, without ...

Embodiment 3

[0068] 1. Clinical sample testing:

[0069] Blind testing was carried out on 30 clinically collected samples, including 20 throat swab samples pathologically diagnosed as novel coronavirus and 10 normal control throat swab samples. RNA was extracted according to the experimental method, and the PCR amplification system and amplification conditions were established. Test and set up negative and positive controls. At the same time, the primer-probe combination qPCR detection method provided by the present invention was used as reference 1, and the primer-probe combination disclosed in the "Technical Guidelines for Laboratory Detection of Novel Coronavirus Infected Pneumonia" was used for qPCR detection as reference 2 to compare the detection effects of the kits.

[0070] The results are shown in the table below

[0071]

[0072] The results show that the detection kit for detection of novel coronavirus digital PCR provided by the present invention has a detection sensitivity...

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Abstract

The invention provides a novel coronavirus detection primer probe combination and kit and an application thereof. A specific primer is designed according to a conserved sequence in novel coronavirus genetic sequences; the novel coronavirus nucleic acid detection kit is developed by matching with a digital PCR technology and optimizing a series of tests; the kit can be absolutely quantified withoutdepending on a reference substance or a standard substance, has higher detection sensitivity, specificity and stability, can reach the detection sensitivity 1-2 orders of magnitude higher than that of fluorescent quantitative PCR, can discover infected persons in the early stage or asymptomatic stage of virus infection more timely, is beneficial to prevention and control of novel coronavirus pneumonia, and realizes early diagnosis and early treatment of patients suffering from the novel coronavirus pneumonia.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a novel coronavirus detection primer-probe combination, kit and application thereof. Background technique [0002] The novel coronavirus (SARS-CoV-2), a novel coronavirus not previously identified in humans, can cause acute infectious pneumonia. The initial symptoms of infected people are mostly fever, fatigue and dry cough, and then gradually develop dyspnea. Most patients have a good prognosis, and some severe cases may develop acute respiratory distress syndrome or septic shock, and even die. At present, there is no effective antiviral drug against the pathogen, and isolation treatment, symptomatic and supportive treatment are the main methods. [0003] The incubation period of novel coronavirus infection pneumonia is generally 1-14 days, mostly 3-7 days. Due to the long incubation period of the new coronavirus infection pneumonia, and the strong infectiv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/166C12Q2563/159C12Q2545/113C12Q2545/101C12Q2563/107Y02A50/30
Inventor 邵琦
Owner 广州达健生物科技有限公司
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