Identification Of Classical Swine Fever Virus Wild Strains And The Establishment Of HRM Diagnosis Method For Distinguishing From Vaccine Strains

13samples isolated from3districts, including Zhanjiang and Qingyuan from Guangdongprovince and Taiyuan from Shanxi Province, were detected positive for CSFV. As NS5B region ofCSFV is the best conserved region for genotyping, a pair of primers which amplification size of965bp were designed. Genetic similarity analysis showed that different isolates from same regionhad high similarity, up to99.7%. The highest genetic similarity between isolates andreference strains ranged from84.3%(genotype type3, L49347) to95.8%(genotype2,HQ148063). The similarity analysis of amino acid sequence deriving from the genetic sequenceshowed that it had the highest similarity between Guangdong Zhanjiang isolates (99.7%). Theamino acids sequence similarity derived from the genetic sequence showed it had a high percentage(95.7%) similarity between CSFV field isolates and a genotype2reference strain, and a lowsimilarity (84.0%) with genotype3reference strains. According to genetic evolution analysis,isolates of CSFV belong to genotype2.With the utilization of advantage of PCR-high resolution melting curve analysistechnology, a rapid method to differentiate the wild-type and vaccine strains of classicalswine fever virus (CFSV) was developed. Through the alignment of sequences of29wild-type strains and9vaccine strains of CSFV published on NCBI(National Center ofBiotechnology Information) database, a pair of generic primers were designed at3′UTRarea. Analyzing13CSFV suspected clinical samples and3CSFV vaccine strains, theresult showd that wild-type strains and vaccine strains have their own unique meltingcurves. Both strains have two melting peak named Tm1and Tm2at the meltingtemperature range from70to90℃. The Tm2of vaccine strain is higher than that ofwild-type strain so that the Tm value between Tm1and Tm2is significantly increased(P<0.01). Therefore the differences of Tm value can be used to distinguish vaccine strainand wild strain of CFSV. Specific test showed that the primers can’t amplify the BVDV,PEDV, TGEV, PRRSV, PRV and PCV positive samples. Sensitivity test showed thatPCR-HRM curve can detect the minimum concentration low to83.4copies/μL.This paper compared several pathogens of animal diseases (Classical swine fevervirus/Eimeria species/Avain leukemia virus) the effects of different fragments amplified,different fluorescent dyes (SYBR green/LCgreen/SYTO9) and different instruments(LightScanner96(Idaho)/LightCycler480(Roche)/Rotor-GeneQ(Qiagen)).The experimentalresults showed that HRM results were mainly affected by different fragments amplifiedand fluorescent dyes with different properties; nucleic acids with multiple melting rangehad higher specificity than SNP and were more suitable for differentiation; difference ofTm on multiple melting range influenced HRM results; Saturated fluorescent dyes (LCGreen and SYTO9) had higher resolution than unsaturated dye (SYBR Green Ⅰ);concentration of fluorescent dye had a greater influence on the value of Tm; There was nosignificant difference on the HRM results by using different PCR-HRM analyzers.