Detection method and kit for novel coronavirus (SARS-CoV-2)

A detection method and reagent technology, applied in the biological field, can solve problems such as time-consuming, time-consuming, labor-intensive, costly, unsafe, etc.

Pending Publication Date: 2021-09-03
SHANGHAI TOLO BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the content of viral nucleic acid in the tested sample is very low and the Ct value of the test result is close to or even greater than 40, it is difficult to judge whether the sample is "weak positive" or "true negative".
[0006] At present, although the method of deep sequencing can be used for further judgment, this method is time-consuming, laborious and expensive
In addition, although clinical symptoms or treatment effects can also be combined to make a comprehensive judgment, this solution is not only time-consuming, but also unsafe, and the standard of judgment cannot be unified, resulting in inaccurate results

Method used

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  • Detection method and kit for novel coronavirus (SARS-CoV-2)
  • Detection method and kit for novel coronavirus (SARS-CoV-2)
  • Detection method and kit for novel coronavirus (SARS-CoV-2)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0195] 1.1 SENA analysis targeting SARS-CoV-2 ORF1ab

[0196] In this example, in order to screen a suitable guide RNA to target the sequence specifically amplified in SARS-CoV-2 ORF1ab, according to the primers recommended by China CDC (see primer pair 2, SEQ ID No: 20 and 21) and supporting The qRT-PCR amplified Taqman fluorescent probe sequence (5'-FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3', SEQ ID No: 8) determined the qRT-PCR amplification interval of ORF1ab.

[0197] The following crRNA sequences were designed from the amplified sequences:

[0198] sequence (5'-3') SEQ ID No: crRNA guide sequence 1: 5'-UGGCUGUAGUUGUGAUCAACUCC-3' 1 crRNA guide sequence 2: 5'-AUCACAACUACAGCCAUAAC-3' 2 crRNA guide sequence 3: 5'-GCGGAGUUGAUCACAACUACAGC-3' 3 crRNA guide sequence 4: 5'-AAAACACAGUCUGUACCGUCUGC-3' 4

[0199] To prepare crRNA probes, T7-crRNA-F was annealed to synthetic oligonucleotides to prepare transcription templates. Specifi...

Embodiment 2

[0217] In this example, the SARS-CoV-2 RNA standard control was serially diluted with HEK293T total RNA, and the qRT-PCR reaction and SENA sensitization reaction were performed respectively. Negative control and positive control were water and synthetic SARS-CoV-2 ORF1ab fragment respectively; the qRT-PCR amplification primers and probes used were the primers recommended by China CDC (see primer pair 2) and matching Taqman fluorescent probes .

[0218] After qRT-PCR reaction, take 2 μL qRT-PCR reaction solution and add it to 18 μL premixed SENA reaction system (LbaCas12a, crRNA1, FAM-N 12 -BHQ1 and reaction buffer), reacted at 37 degrees for 10 minutes.

[0219] The reaction results of qRT-PCR and SENA are shown in Table 1 below. Among them, the detection sensitivity of qRT-PCR can only detect sample No. 5, while SENA can effectively detect sample No. 8, which is at least 8 times more sensitive than qRT-PCR. Among them, UD indicates that qRT-PCR did not amplify the curve, a...

Embodiment 3

[0223] In this example, the synthetic positive control DNA containing the SARS-CoV-2 N gene fragment was serially diluted with human throat swab extract, and qPCR reaction and SENA sensitization reaction were performed respectively. The qPCR amplification primers used are the primers recommended by China CDC (see primer pair 10) and matching Taqman fluorescent probes. After the qPCR reaction, 2 μL of the qPCR reaction solution was added to the pre-mixed 18 μL SENA reaction system (LbaCas12a, crRNA7, FAM-N 12 -BHQ1 and reaction buffer), reacted at 37 degrees for 10 minutes.

[0224] The results of qPCR and SENA sensitization experiments were performed on the positive control samples after serial dilution, respectively, as shown in Table 2 below. When the target DNA molecules in the qPCR reaction system are less than 32, qPCR cannot obtain a stable and effective amplification Ct value. In contrast, the detection limit of SENA can reach 2 target nucleic acid molecules, that is,...

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Abstract

The invention provides a detection method and a kit for a novel coronavirus (SARS-CoV-2).The detection method comprises the following steps of (a) providing a to-be-verified sample containing a nucleic acid amplification product; (b) mixing the to-be-verified sample with a sensitization reagent or a sensitization buffer solution containing the sensitization reagent so as to form a detection system, wherein the sensitization reagent comprises guide RNA, Cas protein and a sensitization nucleic acid probe; and (c) detecting whether the sensitizing nucleic acid probe in the detection system is cut by the Cas protein or not. According to the characteristics of the method, the method is named as SENA (Specific Enhancement For Nucleic Acid Assays), and is used for the specific sensitization of a PCR (Polymerase Chain Reaction) reaction product of the novel coronavirus.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a detection method and a kit for novel coronavirus. The invention also relates to a method and kit for sensitive detection of nucleic acid molecule amplification products. Background technique [0002] CoV is an enveloped, positive-sense, single-stranded RNA coronavirus that caused an outbreak of acute respiratory viral disease in 2019. On February 11, the International Committee on Taxonomy of Viruses designated the virus name as severe acute respiratory syndrome coronavirus 2, abbreviated as SARS-CoV-2, which stands for "Severe Acute Respiratory Syndrome Coronavirus Type 2". The disease caused by it was named "COVID-19" by the World Health Organization, which means "coronavirus disease 2019". [0003] In the clinical examination of COVID-19, nucleic acid detection of throat swabs, nasal swabs, lower respiratory lavage fluid, stool and other samples is curre...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2561/101C12Q2521/301C12Q2525/161
Inventor 王金赵国屏黄卫人
Owner SHANGHAI TOLO BIOTECH CO LTD
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