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Primers and method for simultaneously detecting Orf1ab, E and N regions of 2019-nCov virus in one tube

A 2019-ncov and detection method technology, applied in the fields of life sciences and biology, can solve the problems of missed diagnosis, low detection accuracy and false positives of infected people in the incubation period, so as to improve specificity and accuracy, save detection cost and detection time Effect

Pending Publication Date: 2021-03-16
杭州艾迪康医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serum new coronavirus-specific IgM and IgG antibody detection, this method has the advantages of simplicity, rapidity, safety and low cost, but its detection has a certain hysteresis, the detection accuracy of latent infection population is low, and this method is easily affected by blood samples. False positive due to interference from other substances
Nucleic acid detection is the etiological "gold standard" for detection of viral infection. Viral gene sequencing has high specificity, but it is time-consuming and labor-intensive and prone to false negatives leading to missed diagnosis

Method used

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  • Primers and method for simultaneously detecting Orf1ab, E and N regions of 2019-nCov virus in one tube
  • Primers and method for simultaneously detecting Orf1ab, E and N regions of 2019-nCov virus in one tube
  • Primers and method for simultaneously detecting Orf1ab, E and N regions of 2019-nCov virus in one tube

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Real-time fluorescence quantitative PCR kit for detecting 2019-nCov or SARS-Cov-2 virus nucleic acid, mainly including:

[0052] (1) Viral RNA extraction reagent;

[0053] (2) Reagents for reverse transcription and PCR amplification: one-step reverse transcription-fluorescent quantitative reagents;

[0054] (3) Specific primers and Taqman fluorescent probes for amplification, including upstream and downstream primers and probes for amplifying the three regions of Orf1ab, E and N covering the 2019-nCov virus nucleic acid, Orf1ab-F, Orf1ab respectively -R, Orf1ab-Probe; E-F, E-R, E-Probe; N-F, N-R, N-Probe; the upstream and downstream primers and probes used to detect the internal standard gene Rnase P are Rnase P-F, Rnase P-R, and Rnase P-Probe respectively; The base sequence is:

[0055] orflab-F: 5'-TGAGTGAAATGGTCATGTGTGG-3';

[0056] orflab-Pro: 5'-FAM-CAGGTGGAACCTCATCAGGAGATGC-BHQ1-3';

[0057] orflab-R: 5'-ACACTATTAGCATAAGCAGTTGTGG-3';

[0058] E gene-F: 5'-CGG...

Embodiment 2

[0074] The operation process of the inventive method:

[0075] 1. Extract viral RNA from throat swabs (serum, plasma, etc.):

[0076] (1) Use a pipette to add 560 μL of Carrier RNA working solution (the ratio of Lysis Buffer RL to Carrier RNA is 100:1) into a 1.5 mL centrifuge tube.

[0077] (2) Add 140 μL throat swab culture solution or serum, plasma and other specimens to the centrifuge tube, vortex for 15 sec to mix, and incubate at room temperature (15-25°C) for 10 min.

[0078] (3) Add 560 μL of absolute ethanol after centrifugation, and vortex for 15 sec (when the ambient temperature is higher than 25°C, the absolute ethanol needs to be pre-cooled in advance).

[0079] (4) After the instant centrifugation, transfer 630 μL to the adsorption column CR2, centrifuge at 8000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube.

[0080] (5) Pass the remaining liquid in the centrifuge tube through the column again according to ...

Embodiment 3

[0092] Embodiment 3 Sensitivity detection of this kit

[0093] With the plasmid concentration being 1000, 100, 10copies / μL as a template, the experiment was carried out according to the method shown in Example 2, each concentration was repeated 10 times, and a blank control was set at the same time. As a result, it was found that the detection limit of the present invention was 1000 copies / μL.

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Abstract

The invention provides primers, probes and a detection method for nucleic acid detection of 2019-nCov or SARS-Cov-2 virus, including upstream and downstream primers and probes used for amplifying Orf1ab, E and N regions covering 2019-nCov virus nucleic acid, and upstream and downstream primers and probes used for detecting internal standard gene RNase P. The four probes are labeled by different fluorophores, and all detection can be completed in one tube through condition optimization, so that the detection cost and the detection time are greatly saved. The detection reagent and the detectionmethod have the advantages of good detection specificity, high sensitivity and simple operation, and can realize rapid and accurate detection of 2019-nCov or SARS-Cov-2 viruses.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and is a kit for detecting 2019-nCov or SARS-Cov-2 virus nucleic acid. Background technique [0002] Coronaviruses belong to the genus Coronaviridae in the systematic classification. Viruses of the genus Coronavirus are positive-strand single-stranded RNA viruses with a mantle, about 80-120nm in diameter, spherical or oval, and pleomorphic. The virus has an envelope with spikes on the envelope, and the whole virus is like a corona. In addition to humans, coronaviruses can also infect various mammals and birds such as pigs, cattle, and cats. The human diseases caused by coronavirus are mainly respiratory system infection and intestinal infection, and the general symptoms are mild, but there are two kinds that can cause severe respiratory system disease, including severe acute respiratory syndrome coronavirus (Severe Acute Respiratory Syndrome Coronavirus, SARS-CoV) and Middle East ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2600/166C12Q2531/113C12Q2561/101C12Q2545/101
Inventor 牛林梅王淑一
Owner 杭州艾迪康医学检验中心有限公司
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