PPV SYBR GreenⅠ实时荧光定量PCR检测方法的建立及应用

【目的】建立一种能够快速、灵敏地检测猪细小病毒（PPV）的SYBR GreenⅠ实时荧光定量PCR方法。【方法】根据GenBank中的PPV VP2基因序列，设计并合成1对引物。通过常规PCR，扩增猪细小病毒VP2基因，并将其纯化的PCR产物克隆入pGEM-T Easy 载体中，构建重组质粒。对扩增程序中的荧光染料浓度、引物浓度和Mg²⁺浓度条件进行优化，建立最佳的荧光定量 PCR 反应体系和标准曲线。以阳性重组质粒为模板，建立SYBR GreenⅠ荧光定量PCR方法，对其灵敏性、特异性和重复性进行检验。应用建立的SYBR GreenⅠ荧光定量PCR方法，对临床60份疑似PPV病料进行检测，同时与血凝试验(HA)和常规PCR方法的检测效果进行比较。【结果】 在25 μL扩增体系中，Mg²⁺终浓度为4.5 mol/L、SYBR Green染料浓度为20 μmol/L、引物浓度为25 μmol/L时，本底反应最小、循环阈值最低、扩增效率最高。所建立的SYBR GreenⅠ实时荧光定量PCR方法能够特异、定量地检测猪细小病毒，灵敏度达20 TCID₅₀/mL；在临床样品检测中，其检出率比常规PCR方法高13.3%。【结论】 成功建立了PPV SYBR GreenⅠ实时荧光定量PCR检测方法，为临床猪细小病毒的早期诊断及定量分析病毒的感染程度奠定了基础。

Development and application of a SYBR GreenⅠ real-time fluorescent quantitative PCR method for detection of PPV

【Objective】 The study developed a SYBR GreenⅠ real-time fluorescent quantitative PCR which can detect porcine parvovirus(PPV) quickly and specially.【Method】 According to protein 2(VP2) nucleotide sequences of porcine parvovirus(PPV) published in GenBank,a pair of primer was designed.The VP2 gene was amplified with traditional PCR.The PCR product was cloned into pGEM-T Easy vector and sequenced.A real-time fluorescent quantitative PCR was established by optimizing the SYBR GreenⅠ’s concentration,primers concentration and Mg2+ concentration.The positive recombinant plasmid was used as quantitative template to generate standard curve and melt curve.Sensitivity assay,reproducibility of the assay and specificity assay were determined.Clinical 60 suspected PPV disease materials were detected by established SYBR GreenⅠ real-time fluorescent quantitative PCR method and compared with HA and the result of the conventional PCR testing methods.【Result】 The background response and Ct is minimum,the amplification efficiency is maximum when the Mg2+ concentration is 4.5 mol/L,SYBR GreenⅠ concentration is 20 μmol/L,primer concentration is 25 μmol/L in 25 μL amplification system.The real-time fluorescent quantitative PCR method can specificly and quantitatively detect porcine parvovirus.The detection limit of real-time PCR for PPV was 20 TCID50/mL.The detection rate is 13.3% higher than the conventional PCR in the clinical.【Conclusion】 A SYBR GreenⅠ real-time fluorescent quantitative PCR to detect VP2 gene of PPV was developed on the basis of early and rapid detection and analysis of the infect degree of PPV quantitatively.