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Kit for detecting coronavirus through real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and application thereof

A technology of RT-PCR and detection kits, which is applied in the field of real-time fluorescent RT-PCR detection kits for coronaviruses, can solve the problems of application limitations, lack of cross-reactive antigens, etc., and achieve high specificity and high sensitivity.

Active Publication Date: 2014-01-01
湖北朗德医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] (2) Serological testing: including complement fixation test, hemagglutination inhibition test, indirect immunofluorescence and enzyme-linked immunoassay, etc., but due to the lack of suitable cross-reactive antigens to cover many CoV serotypes, the application of these methods is greatly restricted. limit

Method used

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  • Kit for detecting coronavirus through real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and application thereof
  • Kit for detecting coronavirus through real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and application thereof
  • Kit for detecting coronavirus through real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Development of coronavirus one-step real-time fluorescent quantitative PCR reagent

[0036] 1. The design of primers and probes: by using the DNAman software to carry out sequence comparison analysis on the existing coronavirus nucleic acid sequences in the Genebank database, the conserved fragments of the human coronavirus genome are used as the amplification target site, and the primers and probes are designed The basic principle is to use software to manually design multiple pairs of primers and probes.

[0037] 2. Selection of samples: According to relevant domestic and foreign literature reports, samples such as sputum and nasopharyngeal swabs can be selected.

[0038] 3. Establishment and optimization of the reaction system

[0039] Preparation of samples: 10 samples identified as coronavirus-positive by the virus were used as CoV-positive reference materials, respectively CoV-1, CoV-2, CoV-3, CoV-4, CoV-5, CoV-6, CoV-7 , CoV-8, CoV-9, CoV-10; 10 non-...

Embodiment 2

[0048] Embodiment 2: Human coronavirus one-step fluorescent real-time quantitative RT-PCR detection kit and its use

[0049] 1. Prepare a kit including the following components: RNA extraction solution, RT-PCR amplification reaction solution, negative quality control substance, positive quality control substance, quantitative reference substance, and DEPC-treated water.

[0050] 2. Collection, transportation and storage of specimens

[0051] 2.1 Applicable specimen types: sputum, nasopharyngeal swab, etc.

[0052] 2.2 Specimen collection and pretreatment (pay attention to aseptic operation)

[0053] 2.2.1 Sputum sample collection: Morning sputum is better. Before collecting samples, you should rinse your mouth with clean water or cold boiled water or use a toothbrush (without toothpaste) to clean your mouth and teeth. If you have dentures, you should remove them (in order to reduce the normal oral flora Contaminated specimens), forcefully cough up the sputum in the deep part...

Embodiment 3

[0070] Example 3: Clinical detection of coronavirus one-step fluorescent real-time quantitative RT-PCR detection kit

[0071] The above method was used to detect 18 sputum specimens of patients with suspected coronavirus infection, of which 4 cases were positive for CoV detection, and the virus fluorescence quantitative PCR amplification curve is shown in image 3 , according to the C of these 4 positive results t Value combined with the amplification curve, the Roche LightCycler480 analysis software automatically analyzed the virus concentration of these 4 cases of CoV positive samples, the specific results are shown in Table 1.

[0072] Table 14 Cases of CoV positive specimen virus concentration

[0073] Sample serial number

Ct value

CoV virus concentration (copy number / μl)

sample 1

21.34

2.35×10 2

sample 2

20.98

2.92×10 2

sample 3

22.46

2.48×10 3

Sample 4

21.68

3.48×10 2

[0074]

[00...

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Abstract

The invention relates to a kit for detecting coronavirus through real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and application thereof, belonging to the field of gene detection. The kit disclosed by the invention is very high in sensitivity and specificity. By means of the kit disclosed by the invention, rapid early detection and quantitative analysis of coronavirus in the samples, such as sputum, nasopharyngeal swab and the like, are realized. The kit disclosed by the invention has the advantages of being short in detection period, high in efficiency, strong in detection virus specificity and high in accurate rate and is capable of quantitatively analyzing while quantitatively analyzing virus; the lowest concentration of detected virus is 1.0*10<2> copies / mL; compared with the ordinary PCR and the immunologic detection method, the sensitivity of the kit disclosed by the invention is high; the kit disclosed by the invention is simple for operation, easy for popularizing and good in repeatability of experimental results.

Description

technical field [0001] The invention belongs to the field of gene detection, and relates to a real-time fluorescence RT-PCR detection kit for coronavirus and its application. The kit of the present invention contains a pair of oligonucleotide primers and an oligonucleotide probe obtained by screening. The kit of the present invention has the characteristics of early, rapid, sensitive and specific, and can also be used for quantitative analysis of coronaviruses . Background technique [0002] Coronaviruses belong to the genus Coronavirus of the family Coronaviridae in systematic classification. A variant of coronavirus is the pathogen that causes atypical pneumonia. It was first isolated from chickens in 1937. The diameter of the virus particles is 60-200nm, with an average diameter of 100nm, spherical or oval, and pleomorphic. The virus has an envelope, and there are spines on the envelope. The whole virus is like a corona, and the spines of different coronaviruses are obv...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/686C12Q1/70C12Q2561/113C12Q2563/107C12Q2531/113
Inventor 石康江城名朱世新
Owner 湖北朗德医疗科技有限公司
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