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H1N1/H5N1 type avian influenza virus detection kit and application thereof

A detection kit, avian influenza virus technology, applied in the direction of DNA/RNA fragmentation, microbial determination/inspection, recombinant DNA technology, etc. , easy cross-contamination and other problems, to achieve the effect of shortening the detection time, easy operation, and avoiding mutual interference

Inactive Publication Date: 2013-09-25
浙江国际旅行卫生保健中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2007, someone used two sets of multiplex PCR to carry out molecular diagnosis on 12 kinds of respiratory viruses, but these two methods rely on gel electrophoresis for result analysis, and they have their own shortcomings such as poor sensitivity and easy cross-contamination
In 2009, multi-tube multiplex fluorescent quantitative PCR was used to detect new H1N1 influenza virus and seasonal influenza virus. In 2010, single-tube multiplex fluorescent quantitative PCR began to be used for influenza virus subtype typing, but H1N1 / H5N1 influenza was not carried out Establishment of Single Tube Multiplex Fluorescence Quantitative PCR Method for Viruses

Method used

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  • H1N1/H5N1 type avian influenza virus detection kit and application thereof
  • H1N1/H5N1 type avian influenza virus detection kit and application thereof
  • H1N1/H5N1 type avian influenza virus detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1H1

[0060] Design and synthesis of embodiment 1 H1N1 / H5N1 double fluorescent PCR detection kit detection primer probe

[0061] The FAM fluorophore is labeled at the 5' of the H5N1 probe, and the HEX fluorophore is labeled at the 5' of the H1N1 probe. Synthesize primers and probes for H1N1 and H5N1.

[0062] H1N1

[0063] H1N1 upstream primer: 5'-CACCAGTCCACGATTGCAAT-3' (SEQ ID NO: 1)

[0064] H1N1 downstream primer: 5'-ATGGGAGGCTGGTGTTTATAGC-3' (SEQ ID NO: 2)

[0065] H1N1 probe: 5'HEX-CAACTTGTCAGACACCCAA-BHQ1-3' (SEQ ID NO: 3)

[0066] H5N1

[0067] H5N1 upstream primer: 5'-GGAATGGTAGATGGTTGGTATGG-3' (SEQ ID NO: 4)

[0068] H5N1 downstream primer: 5'-CTTTTGAGTGGATTCTTTGTCTGC-3' (SEQ ID NO: 5)

[0069] H5N1 probe: 5'FAM-ACCACCATAGCAACGAGCAGGGGA-BHQ1-3' (SEQ ID NO: 6)

Embodiment 2

[0070] The preparation of embodiment 2 kit dual fluorescence PCR detection mixed solution

[0071] H1N1 upstream primer 0.5 μl / test; H1N1 downstream primer 0.5 μl / test; H1N1 probe 0.1 μl / test; H5N1 upstream primer 0.5 μl / test; H5N1 downstream primer 0.5 μl / test; H5N1 probe 0.1 μl / test; RT -PCR MIX 12.5 μl / test; process water 4.3 μl / test, mix well to obtain dual fluorescent PCR detection mixture.

Embodiment 3H1

[0072] Sensitivity Analysis of Example 3 H1N1 / H5N1 Type Avian Influenza Virus Double Fluorescent PCR Detection Kit

[0073] 1. Experimental method

[0074] 1) Sample preparation:

[0075] The construction method of the H1N1 plasmid: PCR method is used to amplify the nucleic acid of the H1N1 virus sample to obtain the nucleic acid fragment containing the target amplification sequence of H1N1 (sequence shown in SEQ ID NO: 7). The primer sequences are shown in SEQ ID NO: 1-2 Show. After the amplified fragment was purified, it was cloned into the pMD8-T vector by TA, identified by sequencing, and the recombinant vector with correct sequencing result was transformed into DH5α and amplified to obtain the H1N1 plasmid.

[0076] The construction method of the H5N1 plasmid: PCR method is used to amplify the nucleic acid of the H5N1 virus sample to obtain a nucleic acid fragment containing the target amplification sequence of H5N1 (sequence shown in SEQ ID NO: 8). The primer sequences...

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Abstract

The invention relates to the field of biological detection technology, and in particular relates to a primer, a probe and a kit for synchronously detecting avian influenza viruses H1N1 and H5N1 at a single tube. The nucleotide sequences of the primer and the probe for the double fluorescent PCR (polymerase chain reaction) detection of the H1N1 / H5N1 type avian influenza virus are represented by SEQ ID NO: 1-6; the kit for the double fluorescent PCR detection of the H1N1 / H5N1 type avian influenza virus comprises double fluorescent PCR detection mixed solution which contains the primer and the probe. The H1N1 type avian influenza virus and the H5N1 type avian influenza virus can be quickly detected by using the kit, the primer and the probe provided by the invention; the kit has the advantages of simple and convenient operation, high sensitivity, good specificity and the like; by adopting the kit, suspect cases can be found and be confirmed in time, the monitoring level to the disease is improved accordingly.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a primer, a probe and a kit for H1N1 / H5N1 double fluorescent PCR detection. Background technique [0002] Influenza viruses belong to the Orthomyxoviridae family and are RNA viruses, including three types: A, B, and C. Influenza A virus can be divided into 15 H subtypes (H1-H15) and 9 N subtypes according to the different antigenic properties of the virus surface hemagglutinin (H) and neuraminidase (Neuramindase, N). (N1-N9). Among them, only H1N1, H2N2, and H3N2 mainly infect humans, and the natural hosts of many other subtypes are various birds and animals. In general, bird flu viruses do not infect animals other than birds and pigs. However, since 1997, individual cases of human infection with highly pathogenic H5N1 subtype influenza virus have also occurred from time to time. [0003] Type A is the most important type of influenza virus variation, and influen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 吴忠华吕沁风郑伟罗鹏朱旭平朱勤玮刘燕潘旭光
Owner 浙江国际旅行卫生保健中心
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