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Primer, kit and detection method for detecting avian influenza H7N9 virus by using RT-LAMP method

A RT-LAMP, avian influenza virus technology, applied in the biological field, can solve the problems of unsuitability for on-site detection, low sensitivity, expensive, etc., and achieve the effects of convenient and intuitive result identification, reliable results, and simple use.

Inactive Publication Date: 2013-09-25
中国人民解放军南京军区军事医学研究所
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

The existing immunocolloidal gold detection method has low sensitivity and is difficult to detect clinical cases (Baas C, et al. Euro surveillance, 2013)
Real-time fluorescent quantitative PCR (rRT-PCR) based on the HA gene and NA gene of H7N9 avian influenza virus (rRT-PCR, the method recommended by the World Health Organization and the Chinese Center for Disease Control and Prevention, see http: / / www.who.int / influenza / human_animal_interface / influenza_h7n9 / en / ), has the advantages of sensitivity and specificity, but requires expensive equipment, professional technology, and cumbersome operations, and is not suitable for on-site testing or grassroots laboratories with poor conditions

Method used

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  • Primer, kit and detection method for detecting avian influenza H7N9 virus by using RT-LAMP method
  • Primer, kit and detection method for detecting avian influenza H7N9 virus by using RT-LAMP method
  • Primer, kit and detection method for detecting avian influenza H7N9 virus by using RT-LAMP method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: H7, N9 primer set

[0055] First, retrieve the HA gene sequence and NA gene sequence of the H7N9 avian influenza virus in the EpiFlu data center of GISAID (Global Influenza Avian Influenza Shared Database, http: / / www.gisaid.org / ), and perform sequence comparison by ClustalX software (respectively compared with H1, H3, H5, H7, H9 subtype influenza virus sequences, and N1, N2, N7, N9 subtype influenza virus sequences) to obtain the specific conserved sequences of the two genes; then, through LAMP primers The design software (Primer Explorer software, version4.0) designed LAMP primers for the above-mentioned specific conserved sequences, manually selected and corrected according to professional experience, and then screened among the 10 synthetic primer combinations based on multiple rounds of experimental tests The H7 and N9 primer sets that can be applied to the same reaction system were obtained.

[0056] The H7 primer set includes outer primer pair H7-F3...

Embodiment 2

[0058] Embodiment 2: the kit that detects H7N9 avian influenza virus with RT-LAMP method

[0059] The kit of this example includes the H7 primer set and the N9 primer set of Example 1. Among them, the primer mixture composed of the H7 primer set is placed in tube I, the molar ratio of the outer primer pair H7-F3 and H7-B3, the inner primer pair H7-FIP and H7-BIP, and the loop primer H7-LB It is 1:(5-10):(2-6), preferably 1:8:4. The primer mixture composed of the N9 primer set was placed in tube II, and the molar ratio of the outer primer pair N9-F3 and N9-B3, the inner primer pair N9-FIP and N9-BIP, and the loop primer N9-LB was 1:( 5-10):(2-6), preferably 1:8:4.

[0060] For example: in tube I, each 2.5 μl primer mixture contains 4 pmol H7-F3, 4 pmol H7-B3, 32 pmol H7-FIP, 32 pmol H7-BIP, and 16 pmol H7-LB; in II tube, each 2.5 μl primer mixture contains 4 pmol N9-F3, 4 pmol N9-B3, 32 pmol N9-FIP, 32 pmol N9-BIP, and 16 pmol N9-LB.

[0061] The test kit of this embodiment...

Embodiment 3

[0067] Embodiment 3: Non-diagnostic purpose detects the rapid detection method of H7N9 avian influenza virus with RT-LAMP method

[0068] The detection method of this embodiment uses the kit of Embodiment 2 for detection.

[0069] The detection method of this embodiment comprises the following steps:

[0070] Step 1: Add 2× reaction solution of the kit, primer mixture of tube I or tube II, enzyme solution, chromogenic solution, and ultrapure water of the same volume to transparent reaction tubes A and B respectively, and then mix to obtain the volume The same reaction solution A and B; the primer mixture of tube I is added to reaction tube A, and the primer mixture of tube II is added to reaction tube B; the kit 2× reaction solution, primer mixture of tube I or tube II, The volume ratio of enzyme solution, chromogenic solution and ultrapure water is (8-12): (1.5-3.5): (0.5-1.5): (0.3-0.9): (3.1-4.7);

[0071] For example, reaction solution A can be obtained as follows:

[0...

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Abstract

The invention relates to a primer, a kit and a detection method for detecting an avian influenza H7N9 virus by using an RT-LAMP method. The primer comprises an H7 primer group and an N9 primer group and can effectively detect the avian influenza H7N9 virus. The kit comprises the above-mentioned primer and can be used for detecting the avian influenza H7N9 virus. The detection method employs the above-mentioned kit and can intuitively determine results according to the color of a reaction solution, being rapid and accurate. According to the invention, the results are reliable; cost is low; the characteristics of easiness, rapidness and sensitivity are obtained; the primer, the kit and the detection method are especially suitable for on-site detection and applicable to most hospitals and laboratories of epidemic prevention departments lacking real-time fluorescence quantification PCR instruments in China.

Description

technical field [0001] The invention relates to a primer, a kit, a detection method and a kit inspection method for detecting H7N9 avian influenza virus by RT-LAMP method, and belongs to the field of biotechnology. Background technique [0002] Avian influenza virus belongs to Orthomyxoviridae Influenza A virus genus. Avian influenza A virus particles are pleomorphic, with a spherical diameter of 80-120nm and an envelope; its genome is segmented single-stranded negative-strand RNA. According to the different antigenicities of outer membrane hemagglutinin (H) and neuraminidase (N) proteins, it can be divided into 16 H subtypes (H1-H16) and 9 N subtypes (N1-N9). In addition to infecting poultry, avian influenza A virus can also infect pigs, horses, mink and marine mammals. Since 1959, subtype viruses such as H5, H7 and H9 have crossed species barriers to infect humans more than 10 times. There are currently eight avian influenza viruses that have been confirmed to infect hu...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 王长军张锦海李丙军胡丹吕恒
Owner 中国人民解放军南京军区军事医学研究所
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