Real-time fluorescence RT-PCR detection kit for H1N1 type A swine influenza virus and application of detection kit
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A swine flu virus and detection kit technology, applied in the biological field, can solve the problems of distinguishing between pH1N1 and limited research content, etc., and achieve the effects of easy promotion, simple operation, and convenient grassroots operation and application
Inactive Publication Date: 2015-03-25
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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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, (C
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Embodiment 1
[0033] Embodiment 1, PCR primer pair and fluorescent probe design
[0034] The HA genes of H1N1 (2009) swine influenza virus, Eurasian avian H1N1 swine influenza virus, classic H1N1 swine influenza virus and human H1N1 swine influenza virus were downloaded from the NCBI gene bank in the United States. The homology comparison was carried out, and primers and probes for detecting pH1N1 swine influenza virus with strong specificity were designed in the HA gene region of all influenza virus genomes. The sequences are as follows:
[0035] Upstream primer: PanH1F 5'-AAATCTAGTGGTACCGAGATATGCA-3', (SEQ ID NO.1)
[0036] Downstream primer: PanH1R 5'-GGGAGGCTGGTGTTTATAGCAC-3', (SEQ ID NO.2)
[0037] Specific probe: PanH1MGB probe 5'-F-CAATGGAAAGAAATGCTGG-Q-3' (SEQ ID NO.3).
Embodiment 2
[0038] Embodiment 2, the specific detection of PCR primer pair
[0039] 1. Total RNA extraction
[0040] 1: pH1N1 / 2009; 2: (CS) H1N1; 3: (EA) H1N1, (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.
[0041] 2. Fluorescent quantitative RT-PCR amplification detection primer pair and probe specificity
[0042] 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 product is placed in a quantitat...
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Abstract
The invention discloses a fluorescent quantitative RT-PCR detection kit for an H1N1 type A swine influenza virus, and an application of the detection kit. Through multiple sequence alignment, a primer and a probe with high specificity for detecting the H1N1 type A swine influenza virus is designed aiming at conservative gene segments of the H1N1 (2009) type A swine influenza virus, a Eurasian avian-like H1N1 swine influenza virus, a classical type H1N1 swine influenza virus and a human-derived H1N1 swine influenza virus, and is applied to real-time fluorescence RT-PCR detection. An experiment result proves that the specific PCR primer and TaqMan fluorescence probe disclosed by the invention are high in specificity when being used for detecting the H1N1 type A swine influenza virus; the sensitivity can reach 2.6*10<-5>ng; tissue samples such as nasal swabs, lungs and tracheas of to-be-detected swinery can be detected; the chick embryo allantoic fluid can also be detected; the detection kit is simple to operate and easy to popularize; basic operation and application are facilitated; and the detection kit can become a useful detection tool for diagnosis of H1N1 type A swine influenza virus diseases and epidemiological survey.
Description
technical field [0001] The invention relates to a real-time fluorescence RT-PCR detection method and kit for identifying swine influenza A (H1N1 (2009)) 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 H1N1influenza 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. Receptors for both avian influenza virus, sialic acid a-2,3-galactoside (SA a 2,3Gal) and human influenza virus receptor sialic acid a-2,6...
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