Study On AS-PCR Nucleic Acid Strip Of Canine Distemper Virus

CDV is a non-segmented,non-overlapping single-stranded negative-strand RNA virus which belongs to Paramyxoviridae,Paramyxoviridae subfamily,and Measles virus.It can invade immune system,blood system,respiratory system,digestive system,and even nervous system,leading to systemic pathological changes.At present,in order to prevent canine distemper,vaccines are used for immunization.However,canine distemper vaccine is mainly attenuated vaccine for now.Due to the widespread application of attenuated vaccines,it is very difficult to identify natural infections and vaccine immunizations of animals.Therefore,it is imperative to establish a detection method that can distinguish CDV wild strains and vaccine strains.Currently,there are many CDV detection methods,which includes virus isolation culture,animal inoculation test,serological diagnosis method,RT PCR and real-time fluorescent quantitative RT PCR and etc.Virus isolation and animal testing have disadvantages such as long diagnostic cycle,cumbersome operation and low detection rate.As the main conventional serological test methods on the market,colloidal gold immunochromatographic test strip technique is widely used because of its simple and rapid operation.But it is difficult to identify CDV wild-type infection and vaccine immunity.At present,the only method can identify CDV wild-type infection and vaccine immunity is real-time fluorescent quantitative RT PCR,which has high requirements for experimental instruments and is not suitable for clinical detection.Therefore,this study uses the AS-PCR colloidal gold method to distinguish CDV wild strains and vaccine strains.In this study,2 kinds of genome-wide sequencing of canine distemper wild strains and clinically widely used canine distemper virus vaccine strains isolated from Wuhan was conducted,and the six protein genes of canine distemper virus were compared from amino acid level and base level.Combining the whole genome sequence of the known vaccine strain CDV/R-20/8 strain,the six protein genes of 6 wild strains and 3 vaccine strains were compared and analyzed from the amino acid level and the base level,and F protein gene and H protein gene has a maximum difference of 10%.At the same time,it combines the functions of two proteins of F protein and H protein in the process of CDV infection,and determines that H gene is a target gene designed by AS-PCR primers.Then typed 6 wild-type strains and 3 vaccine strain,and it was found that the canine distemper virus popular in Wuhan was Asia-I type and Vaccine-1 and Vaccine-3(CDV/R-20/8 strain)are American-I and Vaccine-2 is American-II.The CDV-H gene already on NCBI was typed,and a total of 173 CDV-H genes of the same type as Asia-I were screened out,and 179 strains were screened(6 strained wild strains + 173 strains of AsiaI).The CDV-H gene sequence of those strains and the H gene sequence of the three vaccine strains were analyzed.It was found that wild strain and vaccine strain had 9 stable base mutation sites on the H gene,and combined with the primer mismatch principle.71 pairs of AS-PCR primers were tested to find that AS-PCR primers capable of distinguishing CDV wild strains and vaccine strains were G at the 469 th base of the H gene(Asia-I)?A(vaccine strain),the upstream specific primer DI2F-157 designed for the mutation site of V(Asia-I)?I(vaccine strain)at amino acid 157 and the downstream universal primer DIR-15 R designed for the conserved region of H gene.Combine 179 strains of Asia-I type CDV-H gene statistical base difference sites,optimizing the primers,and finally determining the upstream specific primer of AS-PCR: 5'-TTAAAATGATTAATGACACTATGTG-3',downstream of AS-PCR Primer: 5'-CCTGGCAAGGCAAGAA-3',and clinically validated.Clinical validation results show clinically determined the positive test rate for dogs with canine distemper is 70%,and the wild strain and vaccine strain can be distinguished.The upstream specific primers of AS-PCR were labeled with biotin,the general primers for AS-PCR were labeled with FAM,and the optimum pH and optimum concentration of streptavidin gold were determined to complete the streptavidin colloidal gold labeling.The Anti-Fluorescein antibody was streaked into a detection line,and the biotinylated BSA was streaked as a quality control line,and then assembled according to the structure of the nucleic acid test strip.The nucleic acid test strip adopts the doubleanti-sandwich method,the positive sample of the CDV wild strain is the double-line color of the detection line and the quality control line,and the negative sample of the CDV vaccine strain is the single-line color development of the quality control line.The clinical verification of the nucleic acid test strips revealed that the nucleic acid test strips can clearly distinguish between CDV wild strains and vaccine strains.The method is simpler,faster and more accurate to detect the natural infection of canine distemper,in order to eliminate the clinical interference caused by vaccine immunization.