Establishment of real-time fluorescence quantitative RT-PCR detection method for swine influenza virus
In order to establish an efficient,sensitive and specific method to accurately detect swine influenza virus(SIV),specific primers and a TaqMan probe were designed according to the con-served sequence of SIV viral matrix protein(M)gene registered in GenBank,and the recombinant plasmid was constructed as an absolute quantitative template sensitivity and repeatability.The re-sults showed that the established method had no cross reaction with classical swine fever virus,porcine reproductive and respiratory syndrome virus,porcine epidemic diarrhea virus,transmissible gastroenteritis virus,porcine circovirus type 2,porcine pseudorabies virus,porcine parvovirus and porcine Japanese encephalitis virus,and had strong specificity.The minimum detection limit was 1.0 copies/μL,10 times more sensitive than ordinary RT-PCR methods.The coefficient of variation of intra group and inter group repeatability tests were below 2.2％and 1.9％,respectively,indica-ting good repeatability.The method was applied to detect 220 clinical suspected swine flu samples,and the detection rates of fluorescence quantitative RT-PCR and conventional RT-PCR were 2.7％and 1.8％,respectively,with a positive coincidence rate of 100％.The results indicate that the es-tablished TaqMan fluorescence quantitative RT-PCR method has strong specificity,high sensitivi-ty,and good repeatability,and can quickly detect various SIVs in samples,thereby better diagno-sing and monitoring swine influenza.
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