Combined detection kit and detection method for influenza A virus h8n7 and h13n6

A technology of influenza A virus and H8N7 is applied in the field of detection methods and detection kits of influenza A virus H13N6 to achieve the effects of promoting PCR amplification system, accurate typing detection and improving PCR detection efficiency

Active Publication Date: 2021-11-02
SHANGHAI BIOGERM MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the type detection method for influenza A virus H13N6 and H8N7 on the fluorescent quantitative PCR technology platform. Therefore, it is extremely necessary for those skilled in the art to use this platform to develop a highly sensitive, highly specific and highly accurate Multiple one-step real-time fluorescent quantitative RT-PCR detection method and kit to realize multiple joint detection and distinguish influenza A virus H13N6 and H8N7 subtypes

Method used

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  • Combined detection kit and detection method for influenza A virus h8n7 and h13n6
  • Combined detection kit and detection method for influenza A virus h8n7 and h13n6
  • Combined detection kit and detection method for influenza A virus h8n7 and h13n6

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1—Combined Detection Kit for Influenza A Virus H8N7 and H13N6

[0091] In this example, a kit for the dual combined detection of influenza A virus H8N7 and H13N6 by real-time fluorescent PCR, including: virus nucleic acid rapid extraction reagent, PCR amplification reagent, PCR enzyme mixture, H8N7 detection reagent, H13N6 detection reagent, positive control substance , Negative control substance. in:

[0092] (1) The quick extraction reagent of viral nucleic acid comprises lysis reagent, and lysis reagent comprises: 500mM Tris-HCl (pH10.0), 10%v / v TritonX100, 4M guanidine isothiocyanate, 100mM KCl, 0.2M EDTA (pH8 .0).

[0093] (2) The PCR amplification reagent includes a PCR amplification buffer; comprising: 2×PCRbuffer, 50mM MgCl 2 , 100 mM Tris-HCl (pH 10.0), 0.5 mM dNTPs, 5% g / ml BSA.

[0094] (3) The PCR enzyme mixture includes 0.5U / μl reverse transcriptase, 5U / μl DNA polymerase, 5×RNase inhibitor;

[0095] (4) The H8N7 detection reagent includes H8N7-...

Embodiment 2

[0100] Embodiment 2——A joint detection method of influenza A virus H8N7 and H13N6

[0101]The real-time fluorescent PCR detection method of influenza A virus H8N7 and H13N6 of this embodiment comprises the following experimental steps:

[0102] (1) Main reagents and instruments: the kit reagents in Example 1 were used; the fluorescent quantitative PCR instrument was ABI7500.

[0103] (2) Specimen preparation: Positive samples are inactivated virus after titration of human influenza A virus H8N7 and inactivated virus of human infection of influenza A virus H13N6 after titration, and then diluted with DEPC water in different times, and negative control samples are healthy people saliva swab.

[0104] (3) RNA extraction: Mix equal volumes of the viral nucleic acid rapid extraction reagent and the sample to be tested, let stand at room temperature for 5-10 minutes, and then directly use the lysed mixture for PCR detection. (4) Amplification of the target gene:

[0105] a. Desig...

Embodiment 3

[0144] Embodiment 3——The detection kit of influenza A virus H13N6

[0145]In this embodiment, the kit for detecting influenza A virus H13N6 by real-time fluorescent PCR includes: virus nucleic acid rapid extraction reagent, PCR amplification reagent, PCR enzyme mixture, H13N6 detection reagent, positive control substance, and negative control substance. in:

[0146] (1) The viral nucleic acid rapid extraction reagent includes a lysis reagent, specifically comprising: 500mM Tris-HCl (pH10.0), 10% v / v TritonX100, 4M guanidine isothiocyanate, 100mM KCl, 0.2M EDTA (pH8. 0).

[0147] (2) The PCR amplification reagent includes a PCR amplification buffer; comprising: 2×PCRbuffer, 50mM MgCl 2 , 100 mM Tris-HCl (pH 10.0), 0.5 mM dNTPs, 5% g / ml BSA.

[0148] (3) The PCR enzyme mixture includes 0.5U / μl reverse transcriptase, 5U / μl DNA polymerase, 5×RNase inhibitor;

[0149] (4) The H13N6 detection reagent includes H13N6 specific primers and probes; H13N6-F1, H13N6-F2, H13N6-R1, H13N6...

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Abstract

The invention discloses a combined detection kit for influenza A virus H8N7 and H13N6, the kit includes virus nucleic acid rapid extraction reagent, PCR amplification reagent, PCR enzyme mixture, H8N7 detection reagent, H13N6 detection reagent, positive control substance, Negative control substance; H8N7 detection reagent contains H8N7 amplification primers and probes; H13N6 detection reagent contains H13N6 amplification primers and probes. The viral nucleic acid rapid extraction reagent formula adopted in the present invention can be used for rapid extraction of viral RNA, which greatly shortens the extraction time, improves the extraction efficiency, and has a promoting effect on the subsequent PCR amplification system, improving the PCR detection efficiency; and the present invention is aimed at Type A The specific amplification primer probes for influenza virus H8N7 subtype and H13N6 subtype are the first and uniquely designed, with good specificity and high sensitivity, which can realize efficient and accurate typing detection of influenza A virus H8N7 subtype and H13N6 subtype . The invention also provides a detection method for influenza A virus H8N7 and H13N6, and a detection kit and detection method for influenza A virus H13N6.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a RT-PCR amplification technology, in particular to a multiple combined detection method and detection kit for influenza A virus H8N7 and H13N6, and a detection method and detection kit for influenza A virus H13N6 Reagent test kit. Background technique [0002] Influenza viruses are divided into three types: A, B, and C. Influenza A virus is the most common and most likely to mutate. Influenza pandemics are caused by the emergence of new subtypes or the reappearance of old subtypes of influenza A viruses. Influenza A virus has two specific envelope proteins, namely Hemagglutinin (HA) and Neuraminidase (Neuraminidase, NA), which are mainly divided into 16 H subtypes according to the antigenicity of HA and NA proteins. type (H1-H16) and nine N subtypes (N1-N9). [0003] Studies in recent years have found that there is a certain balance between the HA and NA genes that has a great relat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12N15/10C12Q1/686C12N15/11C12R1/93
CPCC12N15/1003C12Q1/686C12Q1/701C12Q2537/143C12Q2563/107C12Q2527/125
Inventor 蒋小琴朱兆奎赵百慧
Owner SHANGHAI BIOGERM MEDICAL TECH CO LTD
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