Establishment And Application Of A Triplex Taqman Quantitative RT-PCR For Simultaneous Detection Of Feline Panleukopenia Virus, Feline Calicivirus And Feline Herpesvirus 1

In recent years,with people’s living standards continuously improving,more and more people choose to raise pets.Because cats love clean,quiet and other characteristics are very in line with the actual needs of urban families,more and more families raise cats,and even one family raises several pet cats.The number of cats is increasing year by year.Nowadays,stray cats can be seen everywhere in cities with a wide range of activities.The increase of cat population density is more conducive to the occurrence of infectious diseases.With high incidence and rapid spread of Feline panleukopenia virus（FPV）,Feline calicivirus（FCV）and Feline herpesvirus 1（FHV-1）,And can occur throughout the year,serious harm to feline health.Therefore,this study established a single TaqMan fluorescence quantitative PCR for three viruses respectively,and then established a triplex TaqMan quantitative RT-PCR on this basis for the rapid diagnosis of FPV,FCV and FHV-1 in clinical practice.The details are as follows:1.Establishment of FPV,FCV and FHV-1 single TaqMan fluorescence quantitative PCR methodIn this study,primers and probes were designed according to the sequences of FPV VP2 gene,FCV ORF2 gene and FHV-1 TK gene,and recombinant plasmids p MD18-T-FPV,p MD18-T-FCV and p MD18-T-FHV-1 were successfully constructed as standards.The results showed that the three TaqMan quantitative PCR methods had no cross-reaction with other common cat pathogens.The reaction system and conditions were optimized,and their sensitivity and repeatability were explored.In terms of sensitivity.The minimum detection limit of the standard plasmid was 1×10¹copies/μL,about 100 times more sensitive than the ordinary PCR.Moreover,the minimum detection limit of the three TaqMan fluorescence quantitative PCR methods for different strains of FPV,FCV and FHV-1 was 10^0.5 TCID₅₀/m L,and the R² of the standard curve drawn according to the results were all greater than 0.99,which could be used for quantitative analysis of the virus.2.Establishment and application of FPV,FCV and FHV-1 triplex TaqMan quantitative RT-PCR methodIn order to simultaneously detect FPV,FCV and FHV-1,a triplex TaqMan real-time quantitative PCR method was established.Specificity test results showed that this method could only detect FPV,FCV and FHV-1,and there was no cross-reaction with other common feline pathogens.Sensitivity test showed that the minimum detection limit of standard plasmid was 1.0×10¹ copies/μL.The minimum detection limit of virus was about 10^0.5 TCID₅₀/m L.The results of the repeatability test showed that the coefficient of variation（CV）between the two groups was lower than 3%.In order to simulate the clinical real mixed infection,the three viruses were mixed in different proportions for detection.The method could accurately detect and the results were stable.On this basis,309 clinical samples were tested by the established triplex TaqMan quantitative RT-PCR method,single TaqMan quantitative PCR method and conventional PCR methods.The results showed that the triplex TaqMan quantitative RT-PCR method was consistent with the single TaqMan quantitative PCR detection results,and the total coincidence rate was 100%.The positive rates of FPV,FCV and FHV-1 detected by triplex TaqMan quantitative RT-PCR were 43.04%,23.95%and 19.09%,respectively,which were higher than those detected by conventional PCR.In conclusion,this study successfully established the triplex TaqMan quantitative RT-PCR detection method,and the test proved that it has good specificity,sensitivity and repeatability,which can be applied to clinical detection,providing good technical support for the rapid detection of FPV,FCV and FHV-1.