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Primer and probe combination product for detecting SARS-CoV-2

A combined product and primer-probe technology, which is applied in the field of molecular biology, can solve problems such as the lack of widespread promotion and application, the sensitivity and specificity gap of constant temperature amplification technology, and achieve high-efficiency constant temperature nucleic acid amplification with low hardware requirements. , Conducive to the widespread promotion of technology

Pending Publication Date: 2021-06-15
GUANGZHOU PLUSLIFE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing constant temperature amplification technology still has a certain gap compared with qPCR in terms of sensitivity and specificity, so it has not been widely promoted and applied.

Method used

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  • Primer and probe combination product for detecting SARS-CoV-2
  • Primer and probe combination product for detecting SARS-CoV-2
  • Primer and probe combination product for detecting SARS-CoV-2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1 Establishment of a SARS-CoV-2 virus fluorescence detection kit detection method

[0069] Combined with the recommendations in the guidelines and the analysis of the conservation of the SARS-CoV-2 genome, the N gene was selected for primer and probe design. The designed primers and probes are as follows:

[0070] Group A:

[0071] Primer F3: 5'-AACACAAGCTTTCGGCAG-3' (SEQ ID NO: 1)

[0072] Primer B3: 5'-GCGTCAATATGCTTATTCAGC-3' (SEQ ID NO: 2)

[0073] Primer FIP: 5'-TGCGGCCAATGTTTGTAATCAGCCAAGGAAATTTTGGGGAC-3' (SEQ ID NO: 3)

[0074] Primer BIP: 5'-TCAGCGTTCTTCGGAATGTCGCTGTGTAGGTCAACCACG-3' (SEQ ID NO: 4)

[0075] Primer LF: 5'-TTCCTTGTCTGATTAGTTC-3' (SEQ ID NO: 5)

[0076] Primer LB: 5'-TGGCATGGAAGTCACACC-3' (SEQ ID NO: 6)

[0077] Primer probe: 5'-FAM-AATTGCACAATTT[rG]CCCCCAGCGCT-BHQ1-3' (SEQ ID NO: 7)

[0078] Group B:

[0079] Primer F3: 5'-TGGCTACTACCGAAGAGCT-3'

[0080] Primer B3: 5'-TGCAGCATTGTTAGCAGGAT-3'

[0081] Primer FIP: 5'-TCTGGCCCAGT...

Embodiment 2

[0106] Example 2 Sensitivity test of SARS-CoV-2 virus fluorescence method detection kit

[0107] The SARS-CoV-2 genome standard substance was diluted to 5000copies / mL, 500copies / mL, and 50copies / mL respectively according to the N gene, and the sensitivity of the kit was tested.

[0108] The specific operation is as follows:

[0109] Standard substances of different concentrations, as well as negative and positive controls were taken for detection, and each concentration gradient of the standard substance was tested 5 times respectively, and the results were recorded.

[0110] The result is as figure 2 shown. The positive and negative control test results were normal. All concentrations of 5000copies / mL and 500copies / mL are detected, and 50copies / mL is detected with probability. According to the conversion of 10μL sample volume, concentrations above 5copies / reaction can be detected stably, and 0.5copies / reaction is affected by sampling with a probability of detection.

Embodiment 3

[0111] Example 3 SARS-CoV-2 virus fluorescence method detection kit specificity test

[0112] Three cases of SARS-CoV-2, one case of influenza A virus (Influenza A virus, FluA), and one case of respiratory syncytial virus (Respiratory Sycytial Virus, RSV) were collected clinically, a total of 5 cases were verified by fluorescent quantitative PCR as The corresponding virus-positive samples were tested to test the specificity of the kit.

[0113] The specific operation is as follows:

[0114] Take 5 samples and negative and positive controls respectively for detection, and record the results.

[0115] The result is as image 3 shown. Three cases of SARS-CoV-2 were positive, one case of FluA and one case of RSV were negative, and the negative and positive controls were normal. Show the specificity of the detection result of the kit of the present invention.

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Abstract

The invention relates to the field of molecular biology, in particular to a primer and probe combination product for detecting SARS-CoV-2. The primer and probe combination product comprises an outer primer pair composed of F3 and B3, an inner primer pair composed of FIP and BIP, a loop primer and a probe. A kit provided by the primer and probe combination product is used for SARS-CoV-2 virus RNA detection, has the characteristics of simplicity in operation, rapidness and sensitivity, provides an effective technical means for on-site rapid detection and screening of the SARS-CoV-2 virus, and has important significance.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a primer-probe combination product for detecting SARS-CoV-2. Background technique [0002] Severe acute respiratory syndrome coronavirus 2 (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), is a positive-sense single-stranded RNA virus with an envelope, belonging to the Coronaviridae Betacoronavirus genus severe acute respiratory syndrome related coronavirus species. Its gene sequence and SARS virus and MERS virus belong to the same lineage but different clades. It is the seventh known coronavirus that can infect humans. The hosts of the virus include mammals and avian animals, which cause novel coronavirus disease (COVID-19). The SARS-CoV-2 virus can invade the human body through the human upper respiratory tract, and achieve infection through the ACE2 expressed on the surface of various cells; the main infected organs include lungs, heart, kidneys and other major ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6848C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6848
Inventor 萧卓冯耀恒陈翀
Owner GUANGZHOU PLUSLIFE TECH CO LTD
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