Nucleic acid composition and kit for detecting S gene of novel coronavirus and production method of kit

A nucleic acid composition and coronavirus technology, applied in the field of in vitro nucleic acid detection of viruses, can solve the problems of missed detection, hidden dangers of epidemic prevention and control, false negatives of nucleic acid detection kits, etc., and achieve the effect of improving accuracy and reducing the probability of occurrence

Active Publication Date: 2020-07-03
NANJING LIMING BIO PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, judging from the current actual clinical use, the existing nucleic acid detection kits still have false negatives, which will cause missed detection and bring great hidden dangers to epidemic prevention and control.
The reason for the false negative may be that the primers or probes used in the real-time fluorescent quantitative detection of the virus are not highly specific, resulting in the inability to specifically amplify the target gene, so the virus signal in the sample cannot be detected
In view of this, those skilled in the art still need to continue to develop highly specific nucleic acid detection solutions to improve the false negative problem existing in existing nucleic acid detection kits

Method used

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  • Nucleic acid composition and kit for detecting S gene of novel coronavirus and production method of kit
  • Nucleic acid composition and kit for detecting S gene of novel coronavirus and production method of kit
  • Nucleic acid composition and kit for detecting S gene of novel coronavirus and production method of kit

Examples

Experimental program
Comparison scheme
Effect test

preparation example 1

[0051] A novel coronavirus S gene detection nucleic acid composition:

[0052] Through the Global Influenza Sequence Database (GISAID) (www.gisaid.org), obtain the published whole genome sequence of the new coronavirus and the whole genome sequence of other 6 coronaviruses, and online (http: / / www.ebi.ac) .uk / ) Perform sequence comparison analysis on the gene sequence of 2 new coronaviruses and 6 other coronaviruses, and find the specific conservative sequence S gene on the whole genome sequence of the new coronavirus. The base sequence of this gene fragment is shown in the sequence table. As shown in SEQ ID NO. 4, S gene is used as the indicator gene, and a conservative sequence is selected as the target gene to be amplified on the S gene sequence. The base sequence of the conservative sequence is as SEQ ID NO. 5 in the sequence table. As shown, using real-time TaqMan fluorescent quantitative PCR design software BeaconDesigner7.0, for the target gene on the S gene, design a forwa...

preparation example 2

[0058] Positive control substance:

[0059] The recombinant plasmid DNA of pUC57-T containing the target gene SEQ ID NO.5 was constructed, and the recombinant plasmid DNA was used to transform E. coli DH5α competent cells, which were purchased from Beijing Soleibao Technology Co., Ltd. After the proliferation of Escherichia coli DH5α, the DNA was extracted by alkaline lysis, purified by DNA purification kit, and the A of the extracted DNA was measured with a spectrophotometer. 260 , A 280 And the concentration, the pseudovirus particles were obtained as a positive control.

Embodiment 1

[0061] A novel coronavirus S gene detection kit, which contains liquid forward primers, reverse primers, probes, DNA polymerase, reverse transcriptase, Tris-HCl, cations, dNTPs, RNase inhibitors and positive controls The forward primer, reverse primer and probe are prepared in Preparation Example 1, and the positive control product is prepared in Preparation Example 2. Forward primers, reverse primers, probes and positive controls are packaged separately.

[0062] The DNA polymerase is a hot-start Taq enzyme, and the reverse transcriptase is selected from one of c-MMLV reverse transcriptase or AMV reverse transcriptase, preferably c-MMLV reverse transcriptase. The RNase inhibitor is RNasin. The hot-start Taq enzyme, c-MMLV reverse transcriptase and RNasin can be packaged separately or at least two types can be mixed. In this embodiment, the hot-start Taq enzyme, c-MMLV reverse transcriptase and RNasin are in accordance with the table The final concentration in 2 is mixed as a pa...

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Abstract

The invention discloses a nucleic acid composition and kit for detecting an S gene of a novel coronavirus and a production method of the kit, and relates to the technical field of in-vitro nucleic acid detection of viruses. The nucleic acid composition for detecting the S gene of the novel coronavirus contains a forward primer, a reverse primer and a probe. The detection kit contains the nucleic acid composition, an enzyme mixture and PCR MIX and can be prepared into liquid and powder. The production method of the powdery detection kit comprises a mixing step and a freeze-drying step. The nucleic acid composition and the kit can be used for amplifying a target gene of a specific conserved gene, namely the S gene of the novel coronavirus, are high in detection sensitivity, can be used for quantifying the initial copy number of the viruses and can be widely applied to the nucleic acid detection of the novel coronavirus.

Description

Technical field [0001] The invention relates to the technical field of virus in vitro nucleic acid detection, more specifically, it relates to a novel coronavirus S gene detection nucleic acid composition, a kit and a production method. Background technique [0002] Since December 2019, many cases of pneumonia of unknown cause have been discovered one after another, which has now been confirmed as an acute respiratory infection caused by a new type of coronavirus infection. The epidemic is highly contagious. Clinically, fever, fatigue, and dry cough are the main manifestations. A few patients have symptoms such as nasal congestion, runny nose, and diarrhea. Some patients only have low-grade fever and mild fatigue. There are no symptoms of pneumonia, and those in critical condition. Can cause death. On January 12, 2020, the World Health Organization named the pathogen causing this viral pneumonia case 2019-nCoV, the 2019 new coronavirus. [0003] The new type of coronavirus belong...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2521/101C12Q2521/107
Inventor 张树文葛斌文
Owner NANJING LIMING BIO PROD CO LTD
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