Establishment And Preliminary Application Of Molecular Identification Method Of Distinction Of Variants Of CPV And Discrimination Between CPV And FPV

Canine parvovirus(CPV)and Feline parvovirus(FPV)have close relationship and both belong to Carnivore protoparvovirus 1,genus Protoparvovirus within the family Parvoviridae.These two parvoviruses caused an intense infectious disease in animals of families Canidae,Felidae,Mustelidae,Procyonidae and Viverridae.These diseases have similar sets of signs and symptoms.Furthermore,CPV has more than one variant,and as the new variant types,CPV-2a,CPV-2b,CPV-2c have also penetrated the feline host-range,co-infected with CPV and FPV in cat may occur.In short,clinical signs are not enough to differentiate.There is an urgent need for the development of a simple,rapid and efficient detection method.Two set of primers and five probes were designed by Beacon designer 7 software,which based on the alignment of VP2 gene sequences of four types of CPV from GenBank.A set of primers and probes,CPV-305F/CPV-305R and CPV-2-305P(for CPV-2)/CPV-2a-305P(for CPV-2a,2b and 2c),was able to differentiate CPV-2 and its variants(CPV-2a,2b and 2c).Another set of primers and probes,CPV-426f/CPV-426R and CPV-2-426P(for CPV-2 and 2a)/CPV-2b-426P(for CPV-2b)/CPV-2c-426P(for CPV-2c),was able to differentiate CPV-2a(2),CPV-2b and CPV-2c.With these primers and probes,the multiplex TaqMan real-time PCR assay detected effectively and differentiated CPV-2,2a,2b,and 2c by two separate real-time PCRs.The detection limit of the assay was 10~1 genome copies/μl for CPV-2,CPV-2a,and CPV-2b,and 10~2 copies/μl for CPV-2c.No cross reactivity was observed with canine distemper virus,canine adenovirus,and canine coronavirus.The multiplex real-time PCR had 100%agreement with DNA sequencing.We provide a sensitive assay that simultaneously detects and differentiate four antigenic types of CPV and the method was also used for quantification of CPVs viral genome.In this study,we developed a High resolution melting(HRM)analysis assay to distinguish FPV and CPV.The assay can screen single nucleotide polymorphism(SNP)and changes in base compositions.A pair set of primers was designed for qPCR amplification.And the amplification product contains the SNP(A4408C)of FPV and CPV.Subsequently,data was auto-analysed and plotted using Applied Biosystems?High Resolution Melt Software v3.1.Results showed that the assay could simultaneously detect and differentiate FPV and CPV in infected animal fecal samples.No cross-reactions were observed with canine adenovirus,canine coronavirus and canine distemper virus.The detection limit of the assay was 4.2 genome copies/μl for CPV and FPV.A total of 80 clinical samples were subjected to HRM assay,PCR-sequence assay and virus isolation.The coincidence rate of the assays and other methods was 92.5%and 85%,respectively.In general,we develop a sensitive method which can simultaneously detect and distinguish CPV and FPV.