Nucleic acid composition for 2019-nCoV E gene detection as well as kit and production method thereof

A technology of 2019-ncove and nucleic acid composition, which is applied in the field of in vitro nucleic acid detection of viruses, can solve the problems of inability to detect the existence of viruses, amplify target genes, and hidden dangers of epidemic prevention and control, and achieve suitable for large-scale production and good specificity , The effect of shortening the detection time

Inactive Publication Date: 2020-11-13
NANJING LIMING BIO PROD CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Since the outbreak of the epidemic, 2019-nCoV nucleic acid detection kits have emerged one after another at home and abroad. However, judging from the current actual clinical use, the existing nucleic acid detection kits still have false negatives, which will cause missed detection. Epidemic prevention and control brings great hidden dangers
The reason for the false negative may be that the primer sequences used have more or less mutations in the primer region, and the specificity is not high, so that the target gene cannot be specifically amplified, so the presence of the virus in the sample cannot be detected
In view of this, it is still necessary to continue to develop highly specific nucleic acid detection solutions to improve the false negative problems existing in existing nucleic acid detection kits

Method used

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  • Nucleic acid composition for 2019-nCoV E gene detection as well as kit and production method thereof
  • Nucleic acid composition for 2019-nCoV E gene detection as well as kit and production method thereof
  • Nucleic acid composition for 2019-nCoV E gene detection as well as kit and production method thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example 1

[0053] A kind of nucleic acid composition for 2019-nCoV E gene detection

[0054] This application downloaded 3295 whole-genome sequences from the Global Influenza Sequence Database (GISAID), analyzed and compared the sequences with other six coronavirus genome data, and used the real-time TaqMan fluorescent quantitative PCR design software BeaconDesigner7.0, in 2019 - Three sets of primer-probe combinations were designed on the nCoV E gene, and the designed primer-probe combinations completely matched the 3295 whole-genome sequences without mutations. Moreover, the identity of each sequence with the other 6 new coronavirus genes is less than 80%; the sequence identity with other respiratory viruses, influenza virus and respiratory syncytial virus is less than 60%. In the NCBI database, the amplification primers had no matching results for species other than 2019-nCoV virus.

[0055] See Table 1 for the sequence information of the three sets of primer-probe combinations in th...

preparation example 2

[0060] Preparation of positive control substance

[0061] The pUC57-T recombinant plasmid DNA containing the amplified region was constructed according to the 2019-nCoV E gene sequence published on NCBI, and the recombinant plasmid DNA was used to transform Escherichia coli DH5α competent cells (purchased from Beijing Suo Laibao Technology Co., Ltd.). The DNA of Escherichia coli DH5α after proliferation was extracted by alkaline lysis, purified by a DNA purification kit, and the A of the extracted DNA was measured by a spectrophotometer. 260 、A 280 And concentration, obtain the pseudovirion particle as positive control substance, concentration is 10 9 copies / mL.

Embodiment 1

[0063] A test kit for 2019-nCoV E gene detection, which includes any one of the three groups of primer probe combinations of Preparation Example 1, Tris-HCl (pH=8.3), KCl, MgCl 2 , dNTPs, hot start Taq enzyme, c-MMLV enzyme and RNasin, and the positive control substance prepared in Example 2, all of the above reagents are in liquid state.

[0064] When using the above kit for 2019-nCoV virus detection and configuring the reaction system, the final concentration of each primer is 50-250nmol / mL, and the final concentration of each probe is 50-200nmol / mL. Particularly preferably, the final concentration of each primer is 200 nmol / mL, and the final concentration of each probe is 100 nmol / mL. The final concentrations of hot start Taq enzyme, c-MMLV enzyme and RNasin were 10 5 U / L, 4×10 6 U / L, 8×10 5 U / L. dNTPs, Tris-HCl (pH=8.3), KCl and MgCl 2 The final concentrations were 200 μM, 50 mM, 50 mM, 3.5 mM, respectively. The hot start Taq enzyme, c-MMLV enzyme and RNasin in the k...

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Abstract

The invention discloses a nucleic acid composition for 2019-nCoV E gene detection as well as a kit and a production method of the nucleic acid composition, and belongs to the technical field of virusin-vitro nucleic acid detection. The nucleic acid composition for detecting the 2019-nCoV E gene comprises any one of three primer probe combinations, and each primer probe combination comprises a forward primer, a reverse primer and a probe; the invention further provides a detection kit comprising any primer probe combination, the kit further comprises an enzyme mixture and PCR MIX, and the detection kit can be prepared into a liquid state or a powder state. According to the nucleic acid composition and the detection kit, specific amplification can be carried out on the target gene in the 2019-nCoV E gene, the sensitivity is high, the probability of occurrence of false results is reduced, and the detection result is accurate and reliable.

Description

technical field [0001] This application relates to the technical field of virus in vitro nucleic acid detection, in particular to a nucleic acid composition for 2019-nCoV E gene detection, a kit and a production method thereof. Background technique [0002] Since December 2019, a number of cases of pneumonia of unknown cause have been discovered one after another, and it has been confirmed as an acute respiratory infectious disease caused by a new type of coronavirus infection. On January 12, 2020, the World Health Organization named the pathogen causing this case of viral pneumonia as 2019-nCoV, and on February 11, 2020, it was named SARS-CoV-2 by the International Committee on Taxonomy of Viruses. [0003] 2019-nCoV is an RNA virus with an envelope. The particles are round or oval, often pleomorphic, with a diameter of 60-140nm. It is highly infectious and is mainly transmitted through respiratory droplets (sneezing, coughing) and contact Infection, which mainly causes hu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701
Inventor 张树文葛斌文刘光明孙娈燕蒋明军苏晓红李黎明
Owner NANJING LIMING BIO PROD CO LTD
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