Establishment and preliminary application of a real-time PCR method for detecting N1 subtype swine influenza virus
In order to establish a sensitive and accurate SYBR Green real-time PCR method for the detection of N1 subtype SIV,specific primers were designed with the conserved region of the N1 subtype SIV NA gene as target gene,and plasmid standards were constructed.A real-time PCR assay was developed by optimizing the reaction conditions and evaluating its specificity,sensitivity and reproducibility.This method was compared with conventional PCR method and applied to the detection of clinical samples.The results showed that the standard curve of the real-time PCR method was y=-3.329 7x+36.881 with an R2 value of 0.999 5,and the amplification efficiency was 99.6％.The results showed that there was a linear relationship between the number of plasmid copies and the Ct value.This method performed well with high specificity.There was no crossreaction with PCV2,CSFV,PRV and PRRSV.The minimum detection amounts of plasmid standards was 1.03 × 101 copies/μ L,which was 100 times higher than that of the con-ventional PCR.The results of the repeatability experiment showed that the coefficient of variation between inter-groups and intra-groups were both less than 3.0％.The detection rate of the proposed method for clinical samples was in high concordance with the chicken embryo isolation assay.This method has the advantages of high sensitivity and strong specificity,and can be applied to the detec-tion and identification of N1 subtype of SIV in clinical samples.
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