H7N9 Avian Influenza Virus Duplex Fluorescent Quantitative RT-PCR Detection Kit

A bird flu virus and detection kit technology, applied in fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of lack of correction function of RNA polymerase, high frequency of gene mutation of bird flu virus, etc. Achieve good specificity and high amplification

Active Publication Date: 2016-01-27
GUANGXI VETERINARY RES INST
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] Because the genome of avian influenza virus replicates in segments, it relies on RNA polymerase to complete the replication, and RNA polymerase lacks the correction function, so the frequency of gene mutation of avian influenza virus is extremely high

Method used

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  • H7N9 Avian Influenza Virus Duplex Fluorescent Quantitative RT-PCR Detection Kit
  • H7N9 Avian Influenza Virus Duplex Fluorescent Quantitative RT-PCR Detection Kit
  • H7N9 Avian Influenza Virus Duplex Fluorescent Quantitative RT-PCR Detection Kit

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Embodiment 1

[0019] The design of embodiment 1 primer and probe and the preparation of kit

[0020] Avian influenza virus (H2N3, H4N5, H7N2, H8N4 and H10N3) RNA was donated by the University of Hong Kong; avian influenza virus (H1N7, H5N1, H11N9) RNA was donated by the University of Pennsylvania; H7N9 avian influenza virus positive RNA was donated by Guangxi Zhuang Autonomous Region Epidemic Control Center gift. Avian influenza virus (H3N2, H6N6, H9N2), Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and infectious laryngotracheitis virus (ILTV) were isolated and preserved by the Veterinary Research Institute of Guangxi Zhuang Autonomous Region.

[0021] 1. Design of primers and probes

[0022] According to the conserved sequence of H7 subtype avian influenza virus HA gene and N9 subtype avian influenza virus NA gene conservative sequence in the gene bank, the conserved sequence of H7 subtype avian influenza virus HA gene was determined (genbank: KC853766.1 No. 1054-1157 ...

Embodiment 2

[0043] Application of Example 2 Primers and Kits in Double Fluorescence RT-PCR Detection of H7N9 Avian Influenza Virus

[0044] 1. The specificity of primers and probes in the detection of H7N9 avian influenza virus duplex fluorescent RT-PCR

[0045] Using the above cDNA as a template, add 2 μL of the template to the sample tubes containing 11.4 μL of the above reaction solution, make up with ultrapure water to form a 20 μL reaction system, put the above sample tubes into the LightCycler 2.0 PCR instrument of Roche Company, and set the following conditions Reaction: pre-denaturation at 94°C for 30s; then a two-step reaction: denaturation at 94°C for 5s, annealing at 60°C for 20s, 40 cycles, collecting fluorescence signals after each cycle, and finally ending the reaction at 40°C. After the reaction, judge the result according to the amplification curve.

[0046] For the results of the FAM channel (under 530nm excitation light), see figure 1 , shows the amplification curve of...

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Abstract

The invention discloses a double fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit for an H7N9 avian influenza virus. PCR primers and fluorescence TaqMan probes which aim at the conserved sequences of an H7 subtype avian influenza virus HA gene and an N9 subtype avian influenza virus NA gene are designed respectively by the inventor and form a primer group and a probe group which specially aim at H7 subtype and H9 subtype avian influenza viruses and are combined to be the double fluorescence quantitative RT-PCR detection kit. The double fluorescence quantitative RT-PCR detection kit can be used for determining single H7 subtype or N9 subtype avian influenza virus infection, and also can be used for determining whether H7N9 avian influenza virus infection exists or not through one-pipe detection without mutual interference, so that the sensitivity and specificity of detection are further improved. According to the detection kit and a detection method thereof, the high amplification performance and excellent specificity of a PCR technology and quick sensitivity of a fluorescence detection technology are utilized completely, and the detection kit can judge the H7N9 avian influenza virus infection according to an amplification curve when reaction is finished.

Description

technical field [0001] The invention belongs to the technical field of RT-PCR detection kits, in particular to a dual fluorescence quantitative RT-PCR detection kit for H7N9 avian influenza virus. Background technique [0002] Avian Influenza Virus (Avian Influenza Virus, AIV) belongs to Orthomyxoviridae Influenza A virus genus, single-stranded negative-sense RNA, polymorphic enveloped virus. The genome of avian influenza virus is divided into 8 segments, encoding HA, NA, NP, NS, MP, PA, PB1 and PB2 proteins respectively, and the filaments on the virus envelope have hemagglutinin (HA) and neuramin Acidase (NA) activity. These two genes are the specific antigens of the virus. According to the different antigenicity of HA and NA proteins, they can be divided into 16 H subtypes (H1-H16) and 9 N subtypes (N1-N9). [0003] Because the genome of avian influenza virus is segmentally replicated, the replication is completed by relying on RNA polymerase, and RNA polymerase lacks co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 谢芝勋罗思思刘加波庞耀珊邓显文谢志勤谢丽基范晴
Owner GUANGXI VETERINARY RES INST
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