Method for simultaneously detecting influenza A virus, influenza B virus and influenza C virus and kit

A technology of influenza virus and detection reagents, applied in the field of biotechnology applications, can solve the problems of long time-consuming, difficult detection of trace nucleic acid and accurate quantification, and cumbersome operation

Inactive Publication Date: 2014-09-10
王全意
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the classic method of detecting influenza virus at home and abroad is to isolate and cultivate the virus, but its operation is cumbersome and time-consuming; electron microscopy, immunohistochemistry, ELISA, conventional PCR, etc. have their own outstanding advantages, but it is difficult to detect trace amounts of nucleic acid and Accurate quantification, the real-time fluorescent quantitative PCR technology (Real-time fluorescent quantitative PCR) developed at the end of the 20th century is a method of adding fluorescent groups to the PCR reaction system and using the accumulation of fluorescent signals to monitor the entire PCR process in real time. Qualitative and quantitative, and has the characteristics of high sensitivity, stronger specificity and reliability, ability to experiment with multiple reactions, high degree of automation, no pollution, real-time and accuracy, etc.

Method used

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  • Method for simultaneously detecting influenza A virus, influenza B virus and influenza C virus and kit
  • Method for simultaneously detecting influenza A virus, influenza B virus and influenza C virus and kit
  • Method for simultaneously detecting influenza A virus, influenza B virus and influenza C virus and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1. Design and synthesis of various types of M genes, NS genes and NP gene-specific primers and probes of type A, type B and type C influenza viruses

[0034] 1.1 Selection of corresponding target genes M, NS and NP of representative strains of influenza A, B and C and determination of conserved regions

[0035] The sequences used in this study were selected from NCBI GenBank (http: / / www.ncbi.nlm.nih.gov) and the influenza virus database of Los Amos National Laboratory (http: / / www.flu.lanl.gov) Among them, the main basis for selection is: (a) strains with a relatively recent age; (b) strains with full-length sequences and main sequences; (c) vaccine strains recommended by WHO; (d) selection of strains with Representative strains. The information of the corresponding M gene, NS gene and NP gene of selected representative strains of influenza A, B and C are shown in Tables 1-1, 1-2 and 1-3.

[0036] In this study, the corresponding M genes, NS genes and NP gen...

Embodiment 2

[0047] Example 2. Establishment of one-step multiplex fluorescent RT-PCR detection system for influenza A, B and C viruses

[0048] 2.1 According to Invitrogen's nucleic acid extraction kit ( Viral RNA Mini Kit) to extract sample nucleic acid.

[0049] 2.2 Preparation of one-step multiplex fluorescent RT-PCR detection system for influenza A, B and C viruses The AgPath-IDTM One-step RT-PCR Kit from Ambion Company was used for the preparation of reagents, and the system volume was 50 μl:

[0050] 2×RT-PCR buffer 25μl

[0051] 25×RT-PCR Enzyme 2μl

[0052] Detection Enhancer 3μl

[0053] Add 0.5 μl of primers shown in SEQ NO: 1, 2, 4, 5, 7, 8, 10, and 11 at the same time for primers and probes, and the final concentration of primers is 200 nM; add primers such as SEQ NO: 3, 6, 9, 0.5 μl each of the probe primers indicated in 12, the final concentration is 100 nM

[0054] Template 6 μl (add 2 μl for each of the three positive templates, and add each template after 10-1 fold ...

Embodiment 3

[0062] Example 3. Specificity identification of type A, type B and type C influenza virus one-step multiplex fluorescent RT-PCR detection method

[0063] The one-step multiplex fluorescent RT-PCR reaction system established in Example 2 is used for influenza A virus, influenza B virus, influenza C virus, parainfluenza virus, respiratory syncytial virus, enterovirus, adenovirus, Boca virus, respectively. The positive nucleic acid of the virus was detected, and the results showed that only influenza A, B, and C had corresponding specific fluorescence amplification curves in the FAM, JOE, and ROX detection channels respectively, and there was no cross-reaction, while the other 5 In addition to the amplification curve of the internal quality control CY5 channel of the strain virus, the other three channels have no amplification curve, indicating that the method has strong specificity (see Figure 1).

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Abstract

The invention belongs to the field of biotechnology application and relates to a multiple fluorescence RT-PCR method with internal quality control for simultaneously detecting influenza A virus, influenza B virus and influenza C virus, and a kit. Specific primers and probes are designed for conserved sequences of an M gene, an NS gene and an NP gene of representative strains of the influenza A virus, the influenza B virus and the influenza C virus. The one-step multiple fluorescence RT-PCR method with internal quality control is established. The method is simple and convenient in operation, overcomes tediousness of single detection in single hole of conventional fluorescence RT-PCR detection, simplifies operation processes, and saves the testing cost. The method and the kit provide strong technical support for virus on-site detection, hygienic evaluation of food and water, clinical diagnosis, and the like by utilization of high specificity, high sensitivity, high efficiency and high stability of the method and the kit.

Description

technical field [0001] The invention relates to the application field of biotechnology, especially for the simultaneous detection and identification of type A, type B and type C influenza viruses. Background technique [0002] Influenza A, B, and C viruses are the main pathogens that cause human influenza. They belong to the Orthomyxoviridae family and are single-strand negative-strand, segmented RNA viruses. Influenza A virus is also highly contagious to a variety of birds and mammals, and can also cause annual seasonal epidemics and repeated pandemics, causing serious harm to the world's economy and public health. [0003] The eight genomes of influenza A virus encode at least 10 proteins, including two surface glycoproteins (HA and NA), six internal proteins (NP, PB1, PB2, PA, M1, and M2) and two nonstructural proteins ( NS1 and NS2). According to the different antigenicity of HA and NA, influenza A virus can be divided into 16 HA subtypes (H1-H16) and 9 NA subtypes (N1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/70C12Q1/686C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 王全意杨鹏崔淑娟张莉彭晓旻张代涛吴双胜
Owner 王全意
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