Diagnosing Canine Parvovirus And Canine Distemper Viral Without Nucleic Acid Extraction And Genetic Evolution Analysis Of Canine Parvovirus In Dogs

Canine parvoviruses(CPV)and canine distemper virus(CDV),both globally important viral diseases in dogs,cause serious and often fatal infections.To rapidly detect CPV or CDV without nuclear acid extraction,we established a direct amplification TaqMan real-time PCR/RT-PCR method(DARPM)with high sensitivity and specificity for CDV and CPV detection in clinical samples.We compared this new method against real-time PCR/reverse transcriptase(RT)-PCR and it showed no cross-reactivity with other pathogens,including type 1 canine adenovirus,type 2 canine adenovirus,rabies virus,and canine par influenza virus.Sensitivity testing showed the minimum detection limits of the real-time PCR/RT-PCR were 7.44×101 copies·?L-1(CPV)and 4.20×101 copies·?L-1(CDV).DARPM detection of CPV and CDV with DNA and cultured viruses showed a minimum detectable CPV copy number of 1.53×101 copies·?L-1,while the minimum detectable amount from the virus culture supernatant was 6.70×101 copies·?L-1.The minimum detectable copy numbers for CDV cDNA and the virus culture supernatant were 9.56×101 copies·?L-1 and 7.77×101 copies·?L-1,respectively.To validate the accuracy of the method,112 clinical samples suspected of harboring CPV and 97 clinical samples suspected of harboring CDV were tested.DARPM showed a 100%compliance rate with ordinary PCR and colloidal gold rapid detection methodology,while the coincidence rate for DARPM and the same method with DNA added was also 100%.Therefore,our new method(DARPM)detects CPV and CDV without the need for pre-PCR nuclear acid extraction.Our results show that DARPM is a specific,sensitive,fast and powerful method for detecting CPV and CDV in clinical samples.We investigated infection by canine parvovirus and genetic variation of the VP2 gene.We collected feces samples of 50 diarrheal dogs in Sichuan Province,China.Analyses DARPM,polymerase chain reaction(PCRs),agarose gel electrophoresis,and amplification of the complete sequence of canine parvovirus were done.We observed 19 DARPM-positive samples.Sequencing analyses of 15 PCR-positive samples based on amplification of the complete VP2 gene showed all to be CPV-2a,and to be polymerized with Sichuan isolates.These results suggest that the common epidemic strain in Sichuan Province is CPV-2a,and may originate from the same strain.Compared with reference strains,there were no significant variations in canine parvovirus in Sichuan Province.