Feline Calicivirus Genetic Phylogenetic Analysis And Establishment Of SYBR Green ? RT-qPCR Detection Method

Feline Calicivirus(FCV)is a single-strand RNA virus as well as a positive chain without segments,belongs to the genus Vesivirus of Calicivirudae.FCV is highly infectious in felines,especially in kittens,which mainly cause lesions of upper respiratory tract and oral cavity.It was reported that the genome sequences of different subtypes of PCV showed diversity,so this paper mainly analyzed the genetic evolution diversity of the genetic tree of the entire genome sequence of FCV,and based on this,establishes a SYBR Green I real-time quantitative PCR detection method.The results are as follows:The primers were designed based on the conserved regions of the FCV whole genome sequence of different regions published on the NCBI.PCR tests were performed to detect the swabs of 32 cats' s mouth,nose,and eye,that were collected from Hefei,Anhui.The results showed that the positive rate of FCV was 31.25 %,indicating that the FCV infection rate was really high.The genetic evolution tree was analyzed by MEGA 7.0software confirmed the diversity of the FCV genome.Four pairs of primers were designed baesd the characteristics of the entire genome sequence of FCV strains in each region,and the FCV whole genome of the positive sample in Hefei regions were sequenced and analyzed.Its entire genome is 7687 bp,including 3open reading frames(ORFs).Its specific conserved segments are only displayed in the head and tail segment of ORF1 and the front segment of ORF2.This further confirms the wide diversity of FCV genome.According to the characteristics of the conserved segments of the entire genome of FCV,the real-time fluorescent quantitative PCR primers were designed and the RT-q PCR detection method based on SYBR Green I was established.In addition to the reaction conditions and system were optimized,sensitivity and repeatability tests were conducted,and clinical samples were preliminary detected.The results showed that in addition to FCV,feline panleukopenia virus(FPV),feline coronavirus(FCo V),feline herpes virus(FHV)and other common feline diseases were tested negative,indicating good specificity by this method.The detection sensitivity of the SYBR Green I RT-q PCR established in this study was 42.4 copies/?L,that shows the sensitivity of this method is approximately 100-fold higher than that of conventional RT-PCR methods.The coefficient of variation within the group is less than 1 % and the coefficient of variation between the groups is small,indicating that the detection method is reproducible.Thirty-two clinical samples fromregions of Anhui were tested with the detection method of SYBR Green I RT-q PCR,and40.63 % tested positive.The figure was higher than regular RT-PCR,indicating that the method established in this study can be used for detection of clinical samples.The establishment of this method provides technical support for the rapid and efficient detection of FCV clinical samples.