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Kit for detecting SARS-CoV-2 novel coronavirus nucleic acid at constant temperature by using enzyme digestion probe

A coronavirus, sars-cov-2 technology, applied in the field of molecular biology, can solve problems such as inability to meet detection

Active Publication Date: 2020-10-20
JIANGSU MACRO&MICRO TEST MED TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the PCR method has high requirements for laboratories and equipment, it cannot meet the detection requirements in some outdoor environments or relatively backward areas, so it is necessary to develop a simple and rapid SARS-CoV-2 detection kit

Method used

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  • Kit for detecting SARS-CoV-2 novel coronavirus nucleic acid at constant temperature by using enzyme digestion probe
  • Kit for detecting SARS-CoV-2 novel coronavirus nucleic acid at constant temperature by using enzyme digestion probe
  • Kit for detecting SARS-CoV-2 novel coronavirus nucleic acid at constant temperature by using enzyme digestion probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Novel coronavirus detection kit (enzyme-cleaved probe constant temperature amplification method) primer probe screening

[0028] 1. Primer probe sequence screening

[0029] a. Primer probe sequence

[0030] Select the novel coronavirus-specific ORF1ab conserved region as the target gene for amplification, design specific primers and probes containing RNA bases (rProbe), where the left and right ends of the RNA bases of rProbe are respectively labeled with FAM fluorescent bases group and BHQ1 quenching group; in the system, a conserved sequence in the recombinant plant is used as an exogenous internal reference, as a quality control of the reagent and the operation itself, to avoid false negatives, and to design specific primers and probes containing RNA bases. Needle (rProbe), wherein the left and right ends of the RNA base of rProbe are labeled with ROX fluorescent group and BHQ2 quencher group respectively. The sequences of selected primer-probe combinatio...

Embodiment 2

[0057] Example 2 Preparation of novel coronavirus detection kit (enzyme-cleaved probe constant temperature amplification method)

[0058] 1. Kit components: nucleic acid reaction solution, detection enzyme solution, positive control substance, negative control substance and internal reference substance, wherein the nucleic acid reaction solution includes primers, probes, ribonuclease RNaseH, betaine, dNTP, MgSO4, buffer ; The detection enzyme solution includes Bst polymerase and AMV reverse transcriptase; the positive control substance is a pseudovirus of the SARS-CoV-2 ORF1ab gene, and the concentration is 10 6 Copies / mL; The internal reference reference substance is the described exogenous internal reference pseudovirus, and the concentration is 10 4 Copies / mL; Negative control substance is RNase-free and DNase-free water.

[0059] 2. Sample Processing

[0060] QIAamp Viral RNA Mini Kit (52904), Viral RNA Extraction Kit (YDP315-R) from Tiangen Biochemical Technology (Beiji...

Embodiment 3

[0074] Example 3 Detection Sensitivity of Novel Coronavirus Detection Kit

[0075] Select 3 SARS-CoV-2 positive samples from different sources, use ddPCR for concentration calibration, and dilute the 3 SARS-CoV-2 positive samples that have been calibrated according to their concentrations with SARS-CoV-2 negative samples to obtain 1000Copies / Samples with concentrations of mL, 500Copies / mL, 250Copies / mL and 100Copies / mL, the samples of each gradient were tested 20 times to determine the detection sensitivity of the kit, and the detection was carried out according to the operation steps of Example 1, and the kit detected 3 samples The results of different concentrations are shown in Table 8 below, and the results of 250Copies / mL of 3 samples were repeated 20 times in Figure 1-3 .

[0076] The results showed that the kit detected 250Copies / mL of 3 samples from different sources and repeated 20 times, all of which could be stably detected, so the detection limit of the kit was ...

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Abstract

The invention provides a kit for detecting SARS-CoV-2 novel coronavirus nucleic acid at a constant temperature by using an enzyme digestion probe. The kit comprises an isothermal amplification primerfor detecting an ORF1ab gene of SARS-CoV-2 novel coronavirus and a corresponding RNA-containing base probe capable of being digested by RNase H, wherein the left end and the right end of an RNA base of the base probe are respectively marked with a reporter fluorophore and a quencher. By utilizing the efficient isothermal amplification primer and the probe, constant-temperature multiple detection can be carried out on the SARS-CoV-2 novel coronavirus, and the kit has high sensitivity and high specificity. The novel coronavirus nucleic acid detection kit disclosed by the invention has high sensitivity and specificity, can be used for detecting throat swabs, alveolar lavage fluid and sputum samples of suspected novel coronavirus pneumonia cases, suspected aggregated case patients and other patients needing novel coronavirus infection diagnosis or differential diagnosis, and has the characteristics of simplicity, convenience, quickness and accuracy.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a kit for constant temperature detection of SARS-CoV-2 novel coronavirus nucleic acid by an enzyme cutting probe. Background technique [0002] Coronaviruses are non-staged single-stranded positive-sense RNA viruses that belong to the subfamily Orthocoronaviridae of the family Coronaviridae of the order Nidoviridae. According to their serotype and genome characteristics, they are divided into four genera: α, β, γ, and δ. There are 7 coronaviruses known to infect humans, including HCoV-OC43, HCoV-229E, HCoV-HKU1, HCoV-NL63, MERSr-CoV, SARSr-CoV and the newly discovered SARS-CoV-2; SARS-CoV-2 It is a new type of coronavirus of the genus Beta. [0003] SARS-CoV-2 is sensitive to ultraviolet rays and heat. 56°C for 30 minutes, ether, 75% ethanol, chlorine-containing disinfectants, peracetic acid, etc. can effectively inactivate coronaviruses. Based on the current epidemio...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2521/327C12Q2563/107C12Q2561/113Y02A50/30
Inventor 冯华华胡小许常沙沙张祺刘利成
Owner JIANGSU MACRO&MICRO TEST MED TECH CO LTD
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