猪细小病毒荧光定量PCR检测方法的建立及初步应用

Development and preliminary application of fluorescent quantitative PCR method for porcine parvovirus

目的 建立猪细小病毒（porcine parvovirus,PPV）荧光定量PCR检测方法,并进行验证及初步应用。方法 根据GenBank中登录的PPV NADL-2株序列,针对VP2区设计引物,以提取的PPV核酸为模板,进行荧光定量PCR扩增;优化反应体系及反应条件,并进行专属性、重复性和敏感性验证。用建立的荧光定量PCR法检测辛酸沉淀及L、P、S公司生产的纳米膜过滤器去除制品中PPV的效果。结果 优化后的荧光定量PCR反应体系：Eva Green预混液7.5μl,上下游引物各0.5μl,模板DNA 1μl,补加dH2O至15μl;反应条件：95℃3 min,95℃10 s和60℃10 s,共40个循环;模板浓度的对数值与循环数的相关性较好（R2=0.999）,扩增效率为102.5%。该方法可特异性扩增PPV,而对伪狂犬病病毒（pseudorabies virus,PRV）、牛细小病毒（bovine parvovirus,BPV）、鼠细小病毒（minute virus of mice,MVM）及猪肾细胞PK-15无扩增反应;试验内CV为0.79%～2.81%,试验间CV为1.23%～2.21%,均〈5%,最低检测限为102copies/μl。辛酸沉淀可使样品中PPV浓度下降5 Logs;不同公司生产的20 nm膜滤器过滤量及过滤效果均有明显差异,其中S公司生产的滤器效果最好,可使样品中的PPV滴度下降4 Logs。结论 已成功建立了PPV荧光定量PCR检测方法,为快速、准确的检测病毒去除工艺对PPV的去除效果奠定了基础。

Objective To develop, verify and preliminarily apply a fluorescent quantitative PCR （QPCR） method for porcine parvovirus （PPV）. Methods Primers were designed according to VP2 region of PPV NADL-2 strain in GenBank for PCR amplification using the extracted PPV nucleic acid as a template. The reaction system and condition were optimized, and the developed method was verified for specificity, reproducibility and sensitivity. The efficaeies of removal of PPV in products by caprylie acid precipitation and by nanofihers manufactured by manufacturers 1,, P and S were determined by the developed method. Results The optimized QPCR system consisted of pre-mixture of Eva Green, 0. 5 p,1 of upstream and downstream primers and 1 μl of template DNA, supplemented with dH20 to a total volume of 15 μl. The reaction condition was optimized as follows： 95 ℃ 3 min, 95℃C 10 s and 60℃ 10 s, 40 cycles in total. The log of template concentration showed good relationship to the cycle number （R^2 = 0. 999）. The amplification efficacy was 102. 5%. PPV was amplified specifically by the developed method, while no pseudorabies virus （PRV）, bovine parvovirus（BPV）, minute virus of mice （MVM） or porcine kidney cell PK-15 were amplified. The CVs of intra- and inter-assays were 0. 79% - 2. 81% and 1. 23% - 2. 21% respectively, both of which were less than 5%, while the mininmm detection limit wsa 10^2 copies/μl. Capryfic acid precipitation decreased the PPV concentration in test samples by 5 Logs. Both the filtration volume and efficacy of 20 nm membrane filter manufactured by various manufacturers showed significant difference. However, the filter manufacturer manufactured by manufacturer S showed satisfactory filtration efficacy, which decreased the PPV titer in test samples by 4 Logs. Conclusion The fluorescent QPCR method for PPV was developed successfully, which laid a foundation of rapid and accurate evaluation of removal efficacy of PPV by virus removal procedure.