Establishment And Preliminary Application Of SYBR Green ? Real-time PCR For Detection Of CDV Wild-type Strain

Aim:Canine distemper(CD)is an acute and highly contagious disease caused by canine distemper virus(CDV),resulting in acute intestinal infectious diseases in dogs,Fur animals and carnivorous wild animals.The most variated H gene have brought great difficulties to the prevention and control of the CDV disease.It is significant to grasp the molecular characteristics and rapid diagnosis for controlling CDV.This study aimed to develop a SYBR Green I real-time PCR for detection of CDV wild-type strain,and provide new technical support for the differential diagnosis of CDV.To verify the accuracy of this method in clinical application,23 clinical CDV strains were analyzed for genetic variation,and the genomic information data of CDV wild strains were also enriched.Test 1 Establishment and application of SYBR Green I real-time PCR for detection of CDV wild-type strainMethod:According to the CDV H gene difference region of vaccine strain(CDV3:DQ778941;Onderstepoort:EU143737;Snyder Hill:AF259552;Convac:Z35493)and wild-type strain(Sichuan06:JX406739)in GenBank,the SYBR Green I real-time PCR assay was established basing on a pair of specific primers targeting CDV wild-type strains.The recombinant plasmid was constructed using CDV wild-type strain cDNA as a template,and its OD260 value and OD280 value were measured and converted to copy numbers.After optimizing the optimal reaction system and procedure,the standard curve and the dissolution curve by using 10 fold diluted recombinant plasmid,then the specificity,sensitivity as well as repeatability of the assay were evaluated.The established real-time PCR assay was applied to 95 CD clinical samples and compared with conventional PCR.Result:The copy number of recombinant plasmid was 6.22×109 copies/?L;The optimized primers concentration was 0.4?0.6 ?M,and the optimized annealing temperature was 57.6?61.6 ?;The real-time PCR had a broad linear range(R2=0.987,E=102.7%);The dissolution curve showed a single peak(Tm=82.50 ?);The assay could efficiently distinguish wild-type CDV isolateing from the vaccine strains,and no cross-reactions with canine adenovirus,canine parvovirus,caine coronavirus,and canine parainfluenza virus;The sensitivity of the real-time PCR assay could reach 6.22×101 plasmid copies/?L of initial templates,which was 100 times more sensitive than the conventional PCR;The variability of the intra-assay and inter-assay were evaluated with coefficients of variation(CV)less than 1%;A total of 95 CD clinical samples were tested by the real-time PCR and conventional PCR method,with the wild-type strains detection rate in 93.68%(89/95)and 82.11%(78/95),respectively.Test 2 Genetic variation analysis of CDV epidemic strains in SichuanMethod:Totally 23 samples were randomly selected from positive and negative samples of test,and their H gene was amplified,cloned and sequenced,then compared with the 38 H gene sequences published at home and abroad using the analysis of DNAMAN 7.0?MEGA6 and NetNGlyc 1.0 software.And the reliability of the test 1 was established.Result:The 21 field strains belonged to the Asia-I genotype by genetic variation analysis except the C11 and C53 strains.Their nucleotides(amino acid)homology was 97.1%?99.9%(97.7%-99.8%),with 8-9 N-glycosylation sites.The C24 strain had the Y549H mutation;The C11 strain belonged to the Asia-IV genotype,and its nucleotides(amino acid)homology with other strains was 92.9%?93.6%(93.9%?95.4%),with eight N-glycosylation sites and the G530D,K213T,L272M,N427Y,M235V,A298D,I506T,A588S substitutions;The C53 strain belonged to the America-I genotype,and its nucleotides(amino acid)homology with other strains was 90.6%?91.4%(90.2%?91.7%),with seven N-glycosylation sites,and which is closest to the CDV3 vaccine strain;The genetic variation analysis results were consistent with the results of the SYBR Green I real-time PCR assay for the detection of CDV wild strains.