猪细小病毒PCR检测方法的建立与应用

Development of PCR Method for Determination of Porcine Parvovirus

目的建立猪细小病毒(PPV)PCR检测方法。方法设计一对针对PPV的特异引物,经方阵滴定法确定PCR检测的最佳条件,以多种病毒DNA为模板进行PCR扩增,并对扩增产物进行斑点杂交鉴定。提取已知滴度的病毒培养物DNA为模板,稀释后进行PCR扩增,测定PCR方法的敏感性。采用此方法检测了180份猪各种组织样品的DNA及PPV-猪组织混合感染PK-15细胞模型。结果仅PPV野毒株和北京分离株AV7909能够扩增出一条531bp的片段,测序结果证明为PPV的特异片段。该方法所能检测的最小病毒滴度值为0.398TCID50,可以检测出PPV-猪组织混合感染PK-15细胞模型中0.500 TCID50的感染量。180份组织样品均未检出PPV。结论该方法具有良好的特异性和敏感性。

Objective To develop a PCR method for the determination of porcine parvovirus （PPV）. Methods Design a pair of primers specific for PPV,and optimize the condition for PCR by block titration. Amplify gene fragments by PCR using the DNAs extracted from wild and AV7909 strains of PPV as well as other virus strains, such as canine parvovirus （ CPV）, rat parvovirus （RPV） and minute virus of mice（MVM） ,as templates respectively. Identify the PCR products by dot hybridization. The developed PCR method was tested for sensitivity by amplification of the DNA of virus culture with known titers, and used for the determination of 180 various porcine tissue specimens as well as PK-15 cell model with mixed infection of PPV and porcine tissues. Results The gene fragment at a length of 531 bp was amplified from both wild PPV strain and AV7909 strain and proved as PPV-specific by sequencing. However, no PPV gene was amplified from other virus strains. The minimum detectable titer of PPV by the developed PCR method was 0. 398 TCID50. The infective titer as low as 0. 5 TCID50 was detected in the PK-15 cell model with mixed infection was detected,which further proved the sensitivity of the developed PCR method. By the method, no PPV was detected in 180 porcine tissue specimens. Conclusion The developed PCR method showed good specificity and sensitivity.