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Primer, probe, test kit and method for real-time fluorescent quantitative PCR detection of novel coronavirus 2019-nCoV

A 2019-ncov, real-time fluorescence quantitative technology, applied in biochemical equipment and methods, microorganism-based methods, and microbial determination/inspection, etc. The effect of saving diagnosis time

Active Publication Date: 2020-04-24
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Since the new coronavirus 2019-nCoV is a new type of coronavirus identified and discovered in 2020, there is currently no specific detection method, and it is relatively common for this virus to cause severe respiratory infections in humans, and it is contagious to a certain extent. Diagnosed patients need isolation and treatment, so it is urgent to develop a molecular method that can quickly and accurately identify this new coronavirus 2019-nCoV, so as to effectively control the spread of the epidemic and achieve the purpose of rapid diagnosis, isolation and treatment of patients

Method used

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  • Primer, probe, test kit and method for real-time fluorescent quantitative PCR detection of novel coronavirus 2019-nCoV
  • Primer, probe, test kit and method for real-time fluorescent quantitative PCR detection of novel coronavirus 2019-nCoV
  • Primer, probe, test kit and method for real-time fluorescent quantitative PCR detection of novel coronavirus 2019-nCoV

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Experimental program
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Effect test

Embodiment 1

[0033] The development of embodiment 1 real-time fluorescent quantitative PCR detection kit

[0034] 1. In this embodiment, the E gene sequence encoding the envelope protein specific to the new coronavirus 2019-nCoV is selected to design a pair of specific primers and a specific fluorescent probe, and a pair of internal standard gene Rnase P is selected to design Specific primers and a specific fluorescent probe are used to construct real-time fluorescent quantitative PCR technology to detect the new coronavirus 2019-nCoV. The E gene is an important structural gene of the coronavirus, and the primers and probes designed in the present invention are only 100% similar to the E gene of the new coronavirus 2019-nCoV, while other coronaviruses including SARS, MERS, 229E, OC43, NL63 and HKU1 No match, so it can specifically bind to the E gene of the new coronavirus 2019-nCoV and initiate amplification, but not amplify other types of coronavirus. The primer sequences and probe seque...

Embodiment 2

[0073] The performance measurement of the real-time fluorescent PCR kit of embodiment 2 new coronavirus 2019-nCoV

[0074] 1. Verification of accuracy

[0075] The gold standard for viral nucleic acid detection is genome sequencing. The test results of this kit are compared with viral genome sequencing to analyze its accuracy. In this example, 4 specimens determined to be novel coronavirus 2019-nCoV by genome sequencing were selected, and the results after detection with the kit provided by the present invention are shown in Table 4 below. It can be seen from the results that all 4 positive samples were detected, indicating that the accuracy of the real-time fluorescent PCR of the novel coronavirus 2019-nCoV provided by the present invention is 100%.

[0076] Table 4 Accuracy analysis of the present invention

[0077]

[0078] 2. Specificity verification

[0079] The specificity of the kit is evaluated by detecting other pathogens. In this example, 32 positive samples of...

Embodiment 3

[0094]Embodiment 3 clinical detection

[0095] Alveolar lavage fluid, nasal swabs, throat swabs, whole blood, serum, plasma, urine and stool from patients with positive confirmed cases of novel coronavirus 2019-nCoV were tested with the above method, and the results are as follows: Figure 6 As shown, the alveolar lavage fluid, nasal swabs, throat swabs, whole blood, serum, plasma, urine and stool from patients with a positive diagnosis of the new coronavirus 2019-nCoV all had amplification curves, and the alveolar lavage fluid, The virus content in nasal swabs, throat swabs and stool samples was high, and the virus positive control and virus negative control were normal. At the same time, the amplification curves of the internal reference gene Rnase P of these samples were normal, as shown in Figure 7 As shown, it shows that the extraction and amplification process of this experiment is normal, and the positive and negative results are accurate. This proves that this metho...

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Abstract

The invention belongs to the technical field of virus detection, and particularly relates to a primer, probe, test kit and method for real-time fluorescent quantitative PCR detection of novel coronavirus 2019-nCoV. A single-tube double-fluorescence channel is adopted to simultaneously detect the existence of the novel coronavirus 2019-nCoV and reference gene Rnase P, and the existence of the novelcoronavirus 2019-nCoV RNA in specimens such as alveolar lavage fluid, nasopharyngeal swab, whole blood, serum, feces and tissues can be detected. The method is short in detection time period, and issuitable for clinical and bedside rapid detection of diagnosis; the virus detection specificity is high, and the accuracy is high; virus qualitative analysis and quantitative analysis are carried out,and the quantitative linear range is good; the detection sensitivity is high; the experimental result is good in repeatability and high in precision; and the reference gene is added into a detectionsystem, so that the quality of the whole process of extracting and amplifying the sample can be monitored through the detection result of the reference gene.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a novel coronavirus 2019-nCoV real-time fluorescent quantitative PCR detection primer and probe, a kit and a method. Background technique [0002] The novel coronavirus 2019-nCoV belongs to the Coronaviridae family and the betacoronavirus genus, and is a new type of coronavirus. The new coronavirus 2019-nCoV can cause human disease and can be transmitted between humans and animals. The symptoms of the new coronavirus 2019-nCoV infection include fever, wheezing and pneumonia. [0003] Since the new coronavirus 2019-nCoV is a new type of coronavirus identified and discovered in 2020, there is currently no specific detection method, and it is relatively common for this virus to cause severe respiratory infections in humans, and it is contagious to a certain extent. Diagnosed patients need to be treated in isolation, so it is urgent to develop a molecular method ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2561/113C12Q2563/107C12Q2531/113
Inventor 刘为勇孙自镛张敏宋慧娟
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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