犬细小病毒2型TaqMan探针实时荧光定量PCR检测方法的建立及应用OA

[Objective]This study aimed to establish a rapid and highly efficient Real-time quantitative PCR method for detecting Canine parvovirus type 2(CPV2),in order to meet the urgent needs for early diagnosis and epidemiological investigation of CPV2.[Method]By comparing the VP2 gene sequences of different subtypes of CPV2,the conserved regions were obtained.Based on this,specific primers and TaqMan probes were designed,and the pMD18-T-CPV2a plasmid standard was constructed.The reaction conditions of Real-time quantitative PCR were optimized through response surface experimental design and a standard curve was plotted.To further evaluate the sensitivity,specificity and repeatability of the detection method established in this study,and thereby establish a specific TaqMan probe Real-time quantitative PCR method for detecting CPV2.Using the established method,59 clinical samples of canine anal swabs were tested to evaluate the feasibility of its application in clinical samples.[Result]The standard curve of the CPV2 TaqMan probe-based Real-time quantitative PCR method established in this study was y=―3.132x+44.500(R2=0.999),with an amplification efficiency of 108.59%,demonstrating excellent linear relationship and amplification performance.The Real-time quantitative PCR method established in this study showed no cross-reaction with Canine distemper virus(CDV),Canine adenovirus(CAV),and other common canine viruses'nucleic acids and Feline parvovirus(FPV)nucleic acid,and had strong specificity.The minimum detection limit of this method was 10 copies/μL,which was 1 000 times higher than that of the conventional PCR method,and had high sensitivity.The coefficient of variation for both intra-batch and inter-batch repeatability tests was less than 1.5%,indicating good repeatability and stability.Using the method established in this study,59 clinical samples of canine anal swabs were tested.The results showed that the positive detection rate of clinical samples detected by Real-time quantitative PCR was 62.71%,which was higher than that of the conventional PCR method(50.85%).[Conclusion]This study successfully established a TaqMan probe-based Real-time quantitative PCR detection method for CPV2.This method had a good linear relationship,high sensitivity,strong specificity,and good repeatability.It could quickly,efficiently and specifically detect CPV2 in clinical samples.The results of this study provided an efficient detection technique for the clinical diagnosis and epidemiological investigation of CPV2.