Genetic Evolution Analysis Of Feline Panleukopenia Virus In Hefei Area And Establishment Of SYBR Green?Real-time PCR Detection

Feline panleukopenia virus belongs to the Caivore protoparvovirus 1,mainly causing feline leukopenia,fever,vomiting,diarrhea and other clinical manifestations.The disease is widespread throughout the world.FPLV is found in many provinces and cities in my country,but there are still few reports on FPLV molecular biology information.In addition,there are frequent clinical cases of immunization failure,and it is not ruled out that the mutation of the virus causes the base change of the epidemic strain to produce the effect.Therefore,this study launched a series of studies to understand the genetic evolution of FPLV in Hefei,Anhui,and to establish a new detection method for FPLV.In this study,the suspected FPLV material was first collected from a number of animal hospitals in Hefei,using the FPLV standard strain(registration number:M38246)that has been registered in Gen Bank as a template for FPLV identification and genome-wide primer design.The 8 FPLV genome sequences of Hefei region were cloned by PCR,recombinant plasmid construction and other techniques,and the resulting genome sequence was analyzed by genetic evolution using biological information software such as Megalign.In addition,VP2 gene was selected from 10FPLV strains registered in Gen Bank,and fluorescent quantitative primers were designed to establish a SYBR Green I PCR detection method for detecting FPLV.Comparative analysis with the reference strains published at home and abroad by Gen Bank found that the overall gene similarity was 92.5%-99.2%.The NS1 gene has different degrees of mutations in nucleotides and amino acids;the similarity of the VP2 gene is 97.9%-99.9%,and the amino acid encoding position 91 is completely mutated from alanine(Ala)to serine(Ser).The genetic evolution tree shows that the analysis of the entire FPLV gene sequence and the VP2 gene in the Hefei region have evolved in separate branches,and the VP2 gene is far from the vaccine strain.In the NS1 gene analysis,AN-HF 02 and the MEV strain belong to the same branch.The genetic distance to MEV is closer.In addition,the FPLV SYBR Green?PCR detection method was established,with a minimum detection of 8.4×10~1copies/?L.The correlation coefficient R~2=0.998,the amplification efficiency Eff=95.4%,and the Tm=83.0?.The repeatability is good,the stability is high,and FPLV can be specifically detected in the test,which is suitable for the first-line clinical.In summary,this study systematically analyzed the genetic evolution of FPLV inHefei,Anhui.The functional site analysis found that FPLV AN-HF 04 strain had 6amino acid point mutations and FPLV AN-HF 06 strain had 1 amino acid.A point mutation occurred.It is worth noting that all the 91 positions of the FPLV VP2 protein of the eight strains of Hefei were changed from A?S,suggesting that there is a regional epidemic of FPLV in Hefei,and it is constantly evolving.The results of this study provide a reference for its epidemiological research and provide direction for genetic engineering vaccine research.In addition,this study also established the FPLV fluorescence quantitative detection method,which provides some help for FPLV from prevention to clinical diagnosis.