Establishment And Application Of A Real-time PCR To Detect Swine Influenza Viruses

Swine influenza(SI)is an acute respiratory infectious disease caused by Swine influenza virus(SIV),and all types of pigs can become infected.Its clinical symptoms are sudden onset,persistent high fever,depression,decreased intake or disuse,different degrees of respiratory symptoms,accompanied by tears and nasal fluid mucus.SI the mortality rate is not high,the end of the course can quickly recover,but the pig infection not only cause the loss of its growth speed,production performance and economic benefit are low,and easy to make the illness aggravating secondary other diseases and even cause death.Since there is no vaccine for swine influenza virus in China,the detection and genetic evolution of the disease is of great significance.Belong to influenza virus is sticky virus,single strand RNA virus negative chain,consists of eight independent genes encoded respectively its structural protein(PB2,PB1,PA,HA,NP,NA,M1,M2)and non structural protein(NS1,NS2).The influenza virus nucleoprotein is by NP gene encoding,as specific antigen of influenza virus,can stimulate the host organism in the production of specific neutralizing antibodies,and NP fragment extremely conservative,mutation rate is low,so the study of influenza virus NP could help swine flu immune prevention and control and the development of diagnostic reagents and vaccines.By using NP gene as the target primer design,this experiment cloned the NP gene fragment,constructed the positive plasmid and optimized the fluorescence quantitative PCR amplification condition.A fluorescence quantitative detection method which can detect 100 copies,specificity and repeatability is established.This method was used to detect 712 samples of various kinds of diseases collected from parts of shandong,and to carry out normal PCR detection.The results showed that the fluorescence quantitative PCR detected 21 samples positive,the positive rate was 2.9%,and the routine PCR was used to detect the above-mentioned diseases,and 10 samples were detected,and the positive rate was 1.4%.The total test positive sample SPF chicken embryo was isolated and 1 strain of influenza virus with blood coagulation activity was isolated.Through the study of the whole genome sequencing of isolates and the homology comparison,the results showed that the strain of eight gene segments are similar to those of Europe and Asia,H1N1 class birds,showed that the isolates from Europe and Asia,class type avian H1N1 swine source spectrum.The genetic evolution of the strain was studied from themolecular level,and the epidemiological data of swine influenza in shandong province were further enriched.