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One-tube method with multiplex detection for human Influenza A and B and new Influenza A H1N1 virus and kit

A type of influenza B, influenza virus technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc. High specificity, low cost, simple and convenient operation

Active Publication Date: 2013-06-05
广东省南山医学发展基金会
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the 20th century, there were four influenza pandemics in the world, all of which were caused by mutated influenza A viruses, causing significant human and property losses
[0006] However, the currently developed influenza virus typing reagents are basically single-plex fluorescent quantitative PCR detection reagents, which cannot detect these three influenza viruses at the same time; if they want to detect them simultaneously, they need to be carried out in three tubes respectively, which is cumbersome and time-consuming. laborious

Method used

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  • One-tube method with multiplex detection for human Influenza A and B and new Influenza A H1N1 virus and kit
  • One-tube method with multiplex detection for human Influenza A and B and new Influenza A H1N1 virus and kit
  • One-tube method with multiplex detection for human Influenza A and B and new Influenza A H1N1 virus and kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: the specific experiment of primer, probe

[0045]Using bioinformatics and biological software for designing primers and probes, we designed primers and probes for the conserved regions of influenza A and B viruses on the sequence data of influenza A, B and new influenza A H1N1 viruses, and designed primers and probes for the new influenza A and H1N1 viruses. Primer probes were designed for the conserved region of the H1 gene of the H1N1 influenza virus, and the probes were labeled with TAMRA, FAM, and JOE respectively. The detailed sequences are shown in Table 1, and Table 1 shows the sequences of primers and probes for human influenza A, influenza B and new influenza A H1N1 viruses.

[0046] Table 1

[0047]

[0048] In order to verify the specificity of the designed primers and probes, a single-plex fluorescent PCR reaction was designed first, and the primers and probes were evaluated, that is, the three sets of primers and corresponding probes of In...

Embodiment 2

[0052] Embodiment 2: Specificity experiment of M-ABH1 multiple detection reagent

[0053] Multiple detection reagents are prepared using the RT-PCR buffer of TAKARA'PrimeScript One-Step RT-PCR Kit (Ver.2)' (TAKARA'PrimeScriptOneStepRT-PCRKitVer.2'), in which the amount of primer used is 7-15pmol, The amount of probe used is 0.5-5 pmol. Prepare the InfA / InfB / A(H1N1) primers and each probe in Table 1 into a tube of reaction solution (M-ABH1). The total reaction volume is 25 μl / reaction, the volume of prepared reagents is 18 μl / reaction, 2 μl is reserved for adding enzyme mixture; 5 μl is reserved for adding template. Use single positive template (common influenza A; influenza B or new H1N1); double positive template (common influenza A-influenza B; common influenza A-new H1N1; influenza B-new H1N1); triple positive The template (common influenza A-influenza B-new influenza A H1N1) was tested for multiple detection reagents, and single-plex fluorescent PCR reagents were used fo...

Embodiment 3

[0057] Embodiment 3: Sensitivity experiment of M-ABH1 multiple detection reagent

[0058] In order to further test the sensitivity of the reagents, a 10-fold gradient dilution was performed on human influenza A (H3N2 type), influenza B and new influenza A H1N1 influenza virus positive samples, which were recorded as dilutions 1 to 6, respectively representing infA-H3N2 , infB, and A(H1N1) viruses were diluted 10 to 106 times, respectively, compared with the single-fold detection with multiple influenza virus detection reagents, and the virus culture detection was carried out at the same time. The experimental results of quantitative PCR are shown in Table 4. The identifiable range of virus culture is at dilution 4. When the dilution is 5, the culture method is negative, and the sensitivity is far lower than that of single or multiplex influenza quantitative PCR detection. Table 4 is a data table of comparative experimental results of multiplex and singlex parainfluenza quanti...

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Abstract

The invention provides a one-tube method with multiplex fluorescent PCR detection for human Influenza A and B and new Influenza A H1N1 virus. The method adopts the primers of which the sequence is shown in SEQ ID NO: 1-6, and also adopts the probes of which the sequence is shown in SEQ ID NO: 7-9. The invention also provides a kit for one-tube method with complex fluorescent PCR detection for human Influenza A and B and new Influenza A H1N1 virus. The kit comprises the primers and the probes. The invention adopts InfA / InfB / A (H1N1) specific primers and Taqman probes, and uses FAM / JOE / TAMRA multiplex fluorescein labels to realize the multiplex detection for the human Influenza A and B and new Influenza A H1N1 virus. The invention has the advantages of high specificity, high sensitivity, high speed, simple and convenient operation, low cost and the like, can be used as a multiple-detection reagent for scientific research and clinical application.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to a multi-test method and kit for human influenza A, influenza B and new influenza A H1N1 viruses in one tube. Background technique [0002] Human influenza virus is an RNA virus that causes influenza in humans. In taxonomy, influenza virus belongs to the Orthomyxoviridae family. It can cause acute upper respiratory tract infection and spread rapidly through the air. It often occurs around the world. Periodic pandemics. [0003] Human influenza viruses are divided into A, B and C influenza viruses (also known as A, B and C influenza viruses) according to the antigenicity of their nucleoproteins. Among the three influenza viruses infecting humans, influenza A virus has a strong variability, followed by influenza B, and the antigenicity of influenza C virus is very stable. Therefore, it is generally the first two that cause large-scale epidemics. [0004] Influ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 周荣刘文宽高文娟苏晓波
Owner 广东省南山医学发展基金会
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