Development And Application Of Real-Time PCR Methods For Three Feline Susceptible Viruses

In recent years,with the progress of urbanization in China,more and more people keep felines,and the health of felines have also attracted widespread attention.As one of feline's "killers",infectious diseases have always threatened feline's health.In this study,through the analysis of the cases of animal hospitals in Nanjing,the most common pathogens of feline infectious diseases were feline leukopenia virus(FPV),feline calicivirus(FCV)and feline herpesvirus I(FHV-1).This study conducted an epidemiological survey of these three viruses in Nanjing,and analyzed the effects of variety,gender,age,and season on the incidence of the three viruses.At the same time,the Real-time PCR methods were developed to detect these three viruses,and the specificity,sensitivity and reproducibility of the methods were evaluated,and finally used for the detection of clinical samples.The Real-time PCR methods established in this study can quickly and accurately diagnose the diseases,greatly improve the efficiency of diagnosis and treatment,and have important significance for the clinical detection and prevention of feline diseases.Experiment 1.Epidemiological investigation of three feline virusesA statistical analysis was conducted on 2,214 feline cases of animal hospitals in nanjing from February 2018 to February 2020.The results showed that there were 402 cases of feline infectious diseases,of which FPV accounted for 9.95%,FCV 35.32%and FHV-1 11.94%.Further statistical analysis found that pure breed felines are more susceptible to disease than pastoral felines.The influence of gender on incidence rate is not significant.Kittens less than one year old are more susceptible than adult felines.Among felines with FPV,FCV,and FPV,felines aged 2-3 months account for 50%,36.23%,and 46.67%,respectively,which were the highest incidence groups.The incidence of FPV is highest in autumn,and the incidence of FCV and FHV-1 has no significant correlation with seasonExperiment 2.Establishment of uniplex Real-time PCR method for detection of three feline virusesThis experiment established three uniplex Real-time PCR methods for three common feline viruses.First,we download all subtypes of these three viruses in GenBank and find the conserved regions of these genes by sequence alignment.Three pairs of specific primers and probes were designed in the conserved regions according to certain principles and then three uniplex Real-time PCR methods were established.The reaction system was 20 ?L,in which the primer concentration was 0.4 ?M and the probe concentration was 0.1 ?M.The reaction procedure was as follows:pre-denaturation at 95? for 2min,denaturation at 95? for 10s,annealing at 54? for 30s,and 40 cycles.The specificity,sensitivity and repeatability of the methods were evaluated and the results showed that these methods could only amplify specific templates and did not cross-react with other common animal pathogens.The detection limit of these three viruses was 5 ×101 copies/?L,which is 10-100 times more sensitive than conventional PCR.The coefficient of variation of the intra-and inter-assay was less than 2%,indicating good repeatability.Experiment 3.Establishment of triplex Real-time PCR method for detection of three feline virusesBased on the uniplex Real-time PCR methods,the primer concentration,probe concentration and annealing temperature were optimized,and a triplex Real-time PCR method was established to detect these three viruses simultaneously.The optimized primer concentration was 0.45?M,probe concentration was 0.05?M,and annealing temperature was 56?.The sensitivity of the method was 5 ×101 copies/?L,intra-day relative standard deviation and inter-day relative standard deviation were lower than 3%,which represents good repeatability.In order to verify the practicability of the method,we established a co-infection model.The results showed that the method can flexibly detect random combinations of different concentrations of viruses.In the detection of clinical samples,the positive rate of the triple Real-time PCR method was 70.83%,which was higher than that of the commercial kit 62.5%.All positive samples were also confirmed to be positive by sequencing,indicating that the accuracy of the method is higher than that of the commercial kit.