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Fluorescence quantification RT-PCR (reverse transcription-polymerase chain reaction) detection kit of Eurasian avian-like type H1N1 swine influenza virus and application thereof

A swine flu virus and detection kit technology, applied in the biological field, can solve the problems of threats to human life and health, limited research content, and economic losses in the pig industry, and achieve the advantages of easy operation and application at the grassroots level, simple operation, and easy promotion Effect

Active Publication Date: 2015-03-25
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

At the same time, recombinant viruses produced by gene rearrangement of (EA) H1N1 and pH1N1 / 2009 viruses also appeared in pig herds in my country. The prevalence of these viruses has caused huge economic losses to the development of my country's pig industry, and it has also affected human health. Life and health have caused a serious threat. Therefore, the establishment of a detection kit for the Eurasian avian (EA) H1N1 subtype swine influenza virus can not only meet the needs of swine influenza virus monitoring, but also can be used for human influenza. Pandemic early warning offers an effective detection method
At present, there are also a large number of research reports on SIV diagnosis, but the research content is limited to general gene detection and subtype detection. According to the SI epidemiological survey in my country, there are (EA) H1N1, pH1N1 / 2009, (CS ) H1N1 and (Hu) H1N1 and other H1 subtype SIVs of different lineages, among which (EA) H1N1 and pH1N1 / 2009 are dominant strains, but there is currently no real-time fluorescent quantitative PCR diagnostic method that can completely distinguish (EA) H1N1 and pH1N1 / 2009

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  • Fluorescence quantification RT-PCR (reverse transcription-polymerase chain reaction) detection kit of Eurasian avian-like type H1N1 swine influenza virus and application thereof
  • Fluorescence quantification RT-PCR (reverse transcription-polymerase chain reaction) detection kit of Eurasian avian-like type H1N1 swine influenza virus and application thereof
  • Fluorescence quantification RT-PCR (reverse transcription-polymerase chain reaction) detection kit of Eurasian avian-like type H1N1 swine influenza virus and application thereof

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Effect test

Embodiment 1

[0028] Embodiment 1, fluorescent quantitative RT-PCR primer pair and fluorescent probe design

[0029] The HA genes of Eurasian avian H1N1 swine influenza virus, H1N1 (2009) swine influenza virus, classic H1N1 swine influenza virus and human H1N1 swine influenza virus from all over the world were downloaded from the NCBI gene bank in the United States. It carried out a homology comparison, and designed primers and probes for the detection of Eurasian avian H1N1 swine influenza virus with strong specificity in the HA gene region of all influenza virus genomes. The sequences are as follows:

[0030] Upstream primer: AlikeH1F 5'-CTCAGCAAGTCATACACAA-3'(SEQ ID NO.1)

[0031]Downstream primer: AlikeH1R 5'-CTGGTAGAGGGTTTGTTG-3' (SEQ ID NO.2)

[0032] Specific probe: AlikeH1MGB probe 5'-F-CACCACCCTCCGACCGACAG-Q-3' (SEQ ID NO.3).

Embodiment 2

[0033] Embodiment 2, the specific detection of fluorescent quantitative RT-PCR primer pair

[0034] 1. Total RNA extraction

[0035] 1: (EA) H1N1; 2: (CS) H1N1; 3: pH1N1 / 2009, (Hu) H1N1, H1N2, H3N2, H5N1, H9N2, PRRS virus. The extraction of viral RNA can be carried out according to conventional methods, or can be carried out by using the German QIAGEN company (RNeasy Mini Kit, 74104), or other kits can be purchased separately; extract according to the instructions of the kit, and obtain viral RNA for the next step experiment.

[0036] 2. Fluorescent quantitative RT-PCR amplification detection primer pair and probe specificity

[0037] Use step 1 to extract the RNA of the sample to be tested as a template, and use the one-step method to mix the following substances to prepare a reaction system for PCR reaction: specific primers and fluorescent probes, PCR buffer, deoxynucleoside triphosphate mixture and DNA polymerase, reverse transcriptase, RNase inhibitor; the reaction prod...

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Abstract

The invention discloses a fluorescence quantification RT-PCR (reverse transcription-polymerase chain reaction) detection kit of a Eurasian avian-like type H1N1 swine influenza virus and an application thereof. By virtue of multiple alignments of sequences, and aiming at conserved gene segments of the Eurasian avian-like type H1N1 swine influenza virus, an A(H1N1) (2009) swine influenza virus, a classical type H1N1 swine influenza virus and a human-derived H1N1 swine influenza virus, a primer and a probe with strong specificity for detecting the Eurasian avian-like type H1N1 swine influenza virus are designed and are used for performing real-time fluorescence RT-PCR detection. Experiment results show that by using the kit provided by the invention to detect the Eurasian avian-like type H1N1 swine influenza virus, the specificity is high, and the detection sensitivity to virus RNA (ribonucleic acid) can reach 4.6*10<-7>ng / reaction, so that tissue samples of nasal swabs, lungs, tracheas and the like of a to-be-detected swine herd can be detected, and chick embryo allantoic fluid can also be detected. The kit is simple to operate, easy to popularize and convenient in basic-level operation and application, and can become a useful detection tool for epidemic disease diagnosis of the Eurasian avian-like type H1N1 swine influenza virus and epidemiologic investigations.

Description

technical field [0001] The invention relates to a real-time fluorescent detection method and kit for identifying Eurasian avian H1N1 swine influenza virus. The invention belongs to the field of biotechnology. Background technique [0002] Swine influenza (SI) is an acute respiratory infectious disease of pigs caused by type A influenza virus. Swine influenza virus (SIV) circulating around the world includes 3 main subtypes: H1N1, H3N2 and H1N2 subtypes, which also includes different genotypes: classic H1N1 [Classical swine H1N1 influenza A virus, (CS ) H1N1], Human H1N1 influenza A virus [Human H1N1 influenza A virus, (hu) H1N1], Eurasian avian-like swine H1N1 [Eurasian avian-like swine H1N1, (EA) H1N1], Influenza A H1N1 virus [pandemic ( H1N1)2009, pH1N1 / 2009], human H3N2, gene rearranged H3N2 and multi-genotype H1N2 subtypes, etc. Since the discovery of SIV, a stable genetic lineage has been established in pig herds in my country. Swine influenza virus can cause respir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/70C12Q1/701C12Q2531/113C12Q2561/101C12Q2561/113
Inventor 杨焕良陈艳陈化兰乔传玲
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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