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Novel coronavirus 2019-nCoV fluorescent RPA detection primers, probe, kit and method

A 2019-ncov, detection kit technology, applied in the field of biotechnology and molecular diagnosis, can solve the problems of cumbersome experimental procedures of thermal cyclers, difficult to meet the detection needs, etc., to reduce experimental time, short detection time period, and high precision Effect

Pending Publication Date: 2020-08-07
湖南润美基因科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the main method of nucleic acid detection is the PCR method. Conventional PCR detection requires a special thermal cycler and cumbersome experimental procedures, which is difficult to meet the detection needs of the field or other conditions outside the laboratory.

Method used

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  • Novel coronavirus 2019-nCoV fluorescent RPA detection primers, probe, kit and method
  • Novel coronavirus 2019-nCoV fluorescent RPA detection primers, probe, kit and method
  • Novel coronavirus 2019-nCoV fluorescent RPA detection primers, probe, kit and method

Examples

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Effect test

Embodiment 1

[0029] The development of embodiment 1 fluorescent RPA detection kit

[0030] 1. In this example, select the specific open reading frame ORF1ab gene sequence of the new coronavirus 2019-nCoV to design a pair of specific primers and a specific fluorescent probe, and select the internal standard gene RNase P to design a pair of specific primers And a specific fluorescent probe, the construction of fluorescent RPA technology can detect the new coronavirus 2019-nCoV. The ORF1ab gene is an important structural gene of coronavirus, and the primers and probes designed in the present invention are only 100% similar to the ORF1ab 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 and amplify the ORF1ab gene of the new coronavirus 2019-nCoV, but not amplify other types of coronavirus. The primer sequences and probe sequences designed in this example of the present invention are shown in ...

Embodiment 2

[0060] Example 2 Performance test of the fluorescent RPA detection kit for novel coronavirus 2019-nCoV

[0061] 1. Verification of accuracy

[0062] The gold standard for viral nucleic acid detection is genome sequencing. The test results of the secondary kit are compared with the results of viral genome sequencing to analyze its accuracy. In this example, 4 samples 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 3 below. It can be seen that all 4 positive samples were detected, indicating that the accuracy of the novel coronavirus 2019-nCoV fluorescent RPA detection kit provided by the invention is 100%.

[0063] Table 3 Accuracy analysis of the present invention

[0064] Virus type Number of cases Sequencing positive Fluorescent RPA method Accuracy Novel coronavirus 2019-nCoV 4 4 4 100％

[0065] 2. Specificity Ve...

Embodiment 3

[0078] Embodiment 3 clinical experiment

[0079] The above method was used to detect the alveolar lavage fluid, nasal swab, and pharyngeal swab sputum from patients with a positive diagnosis of the new coronavirus 2019-nCoV. The results are as follows: image 3 As shown, the alveolar lavage fluid, nasal swabs, throat swabs, and sputum from patients with a positive diagnosis of the new coronavirus 2019-nCoV all have amplification curves. At the same time, the amplification curves of the internal reference gene RNase P in these samples were normal, as shown in Figure 4 As shown, it shows that the nucleic acid extraction and amplification process of this experiment is normal, and the positive and negative results are accurate. This proves that this method is suitable for the detection of alveolar lavage fluid, nasal swabs, throat swabs, and sputum of patients suspected of novel coronavirus 2019-nCoV infection.

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Abstract

The invention discloses novel coronavirus 2019-nCoV fluorescent RPA detection primers, a probe, a kit and a method. The kit comprises novel coronavirus 2019-nCoV specific primers shown in SEQ ID NO. 1-2 and a probe shown in SEQ ID NO. 3. According to the invention, the existence of the novel coronavirus 2019-nCoV ORF1ab gene and the internal reference gene RNase P is simultaneously detected by adopting a single-tube double-fluorescence channel, and the existence of the novel coronavirus 2019-nCoV RNA in pulmonary alveolar lavage fluid, nasal swab, pharyngeal swab, sputum and other samples canbe detected. The method is short in detection time and suitable for clinical and bedside virus nucleic acid rapid screening, the kit has extremely high sensitivity and specificity, an internal reference gene is added into the detection system, and quality monitoring is performed on the extraction and amplification process of the sample according to the detection result of the internal reference gene.

Description

technical field [0001] The invention belongs to the field of biotechnology and molecular diagnosis, in particular to a novel coronavirus 2019-nCoV fluorescent RPA detection primer and probe, kit and method Background technique [0002] The World Health Organization named the pathogen of viral pneumonia cases since December 2019 as the 2019 novel coronavirus, or "2019-nCoV". The new coronavirus 2019-nCoV belongs to the Coronaviridae family and the genus betacoronavirus. It has an envelope, and the particles are round or oval, often pleomorphic, with a diameter of 60-140nm. It is the seventh known virus that can infect humans. The remaining 6 coronaviruses are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV. Among them, the first four are more common in the population, less pathogenic, and generally only cause mild respiratory symptoms similar to the common cold; the other two are SARS coronavirus and MERS coronavirus (Middle East Respiratory Syndrome Corona...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2531/119C12Q2522/101
Inventor 唐乃平龚剑肖钢
Owner 湖南润美基因科技有限公司
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