RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) kit for detecting avian influenza virus subtype H7N9

A RT-LAMP, avian influenza virus technology, applied in DNA/RNA fragments, recombinant DNA technology, microbial determination/inspection, etc. The high cost of needle kits can achieve the effect of low primer-dimer probability, easy to popularize and apply on a large scale, and shorten the detection time.

Inactive Publication Date: 2014-04-23
SOUTH CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the market price is high. If it is used for H7N9 screening in live poultry markets or poultry farms, the cost of fluorescent probe kits is...

Method used

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  • RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) kit for detecting avian influenza virus subtype H7N9
  • RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) kit for detecting avian influenza virus subtype H7N9
  • RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) kit for detecting avian influenza virus subtype H7N9

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Example 1 Design of RT-LAMP primers for detecting avian influenza H7N9 subtype

[0054] RT-LAMP primer design of the present invention: the hemagglutinin gene (HA gene, GenBank accession number is KC853766.1) of avian influenza H7N9 (AIV-H7N9) is used as the target gene, and the nerve gene of avian influenza H7N9 (AIV-H7N9) The aminoacidase gene (NA gene, GenBank accession number KC853765.1) was used as the target gene, and the online design software Primer Explorer version 4 (http: / / primerexplorer.jp / e) was used to design LAMP primers.

[0055] This embodiment provides the process of screening the best primers, and selects several pairs of alternative primers designed with software to screen, and the alternative primers are as follows:

[0056] Table 1 Primer sequences used in the screening process of HA primers

[0057]

[0058]

[0059] For the screening results of HA primers, see Figure 1-3 , showing that the peak times of HA-1 primer and HA-3 primer ...

Embodiment 2

[0066] Example 2 Construction of Standard Positive Plasmid for Detecting Avian Influenza H7N9 Subtype

[0067] The T vector clone containing the avian influenza H7N9 (AIV-H7N9) hemagglutinin (HA) gene fragment is prepared by using the synthetic HA gene fragment as a template and using the external primers in Table 3 (SEQ ID NO: 1 and SEQ ID NO: 2) Amplify the synthetic HA gene fragment, the length of the obtained gene fragment is 265bp, the sequence is shown in SEQ ID NO: 13, the amplified fragment is recovered, and connected to the T vector by conventional methods, that is Standard positive plasmid containing HA gene.

[0068] The T vector clone containing the avian influenza H7N9 (AIV-H7N9) neuraminidase gene fragment is prepared by using the synthetic NA gene fragment as a template and using the external primers in Table 3 (SEQ ID NO: 7 and SEQ ID NO:8) Amplify the synthesized NA gene fragment, the length of the obtained gene fragment is 373bp, the sequence is shown in SEQ...

Embodiment 3

[0069] Example 3 Construction of RT-LAMP kit for detecting avian influenza H7N9 subtype

[0070] The avian influenza H7N9 subtype RT-LAMP detection kit of the present invention includes the RT-LAMP primer combination of Example 1, RT-LAMP reaction solution, Bst DNA polymerase, AMV reverse transcriptase, positive control (obtained in Example 2) 2 positive plasmids) and a negative control. RT-LAMP reaction solution: containing 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 Aqueous solution, the volume ratio of the three is 8:5:2. The negative control was DEPC treated water.

[0071] The method for detecting avian influenza H7N9 (AIV-H7N9) virus using the kit of the present invention comprises the following steps:

[0072](1) Extraction of RNA from the sample to be tested: the sample RNA was extracted using a virus RNA extraction kit; the avian influenza H7N9 subtype virus used was the G1 strain, the isolation site was Hangzhou, the host was human, and it was isolated b...

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Abstract

The invention discloses an RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) kit for detecting avian influenza virus subtype H7N9. The kit comprises six pairs of primers, the nucleotide sequences are shown in SEQ ID No.1-12 respectively. The kit can rapidly, specifically and sensitively detect the avian influenza virus subtype H7N9, a turbidity meter is utilized for judging the reaction in real time, or SYBR Green I is injected after the reaction is finished, and a detection result is judged by color reaction; the lowest detection limit of a primer for detecting HA gene is 48fg/mul of H7N9 RNA; and the lowest detection limit of a primer for detecting NA gene is 4.8pg/mul of H7N9 RNA. The kit is convenient and simple to use, low in cost and good in specificity, is very suitable for disease surveillance, on-site emergency and clinical specimen detection, and is suitable for large-range popularization and application, and the reaction result is easy to observe.

Description

technical field [0001] The invention relates to the field of virus detection, in particular to a reverse transcription-loop-mediated isothermal amplification technique (RT-LAMP) specific primer combination and a detection kit for detecting the H7N9 subtype of avian influenza virus. Background technique [0002] In the past, H7 subtype avian influenza viruses such as H7N2, H7N3 and H7N7 have occurred in the world. Most of the H7 avian influenza is transmitted among birds, not among humans, and the symptoms are generally relatively weak. The earliest H7N9 virus was recorded in 1988, from the US National Center for Biotechnology Information, infecting animal turkeys. In late March 2013, human infection with Influenza A H7N9 virus and cases began in Shanghai and were successively discovered in cities along the Yangtze River Delta in China. This is the first time the virus has infected humans in the world. According to statistics, before the emergence of the H7N9 virus, a total...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/70C12Q2531/119
Inventor 廖明李红梅亓文宝黄丽红李华楠石磊唐大运
Owner SOUTH CHINA AGRI UNIV
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