SYBR Green荧光PCR检测美洲钩虫方法的建立

Developing SYBR green fluorescent quantitative PCR for detecting Necator americanus

目的建立荧光定量PCR检测美洲钩虫的方法。方法提取虫体基因组DNA、根据GenBank中美洲钩虫ITS-2序列设计特异引物,PCR扩增ITS-2序列、克隆、测序和比对。常规PCR检验引物特异性。将ITS-2序列扩增产物回收、纯化后经T克隆转入大肠埃希菌DH5α,提取重组质粒,鉴定后作为标准品模板建立荧光定量PCR标准曲线,并做灵敏性和重复性试验。结果构建的荧光定量PCR标准曲线循环阈值与模板浓度呈良好的线性关系,溶解曲线的吸收峰单一,实验重复性良好。结论成功构建了SYBR Green Ⅰ荧光定量PCR检测美洲钩虫的方法,可用于快速、准确、定量检测美洲钩虫。

The objective was to establish a sensitive and specific SYBR Green I real-time quantitative PCR method for the detection of Necator americanus.In this study,Necator americanus were obtained from the stool and identified chiefly based on the morphological characteristics.According to the ITS-2 sequences from GenBank,the specific primers were designed.Specificity assay was determined by a series of conventional PCR,and the ITS-2 sequence was amplified and cloned into T vector which was subsequently transformed into E.coli DH5α.Following by extraction and identification,the positive recombinant plasmid was used as quantitative template to generate standard curve and melt curve.Sensitivity assay and reproducibility assay were determined.The standard curves established by recombinant plasmid showed fine linear relationship between threshold cycle（Ct）and template concentration.Melt curves were specific and the correlation coefficient was above 0.993.The real-time quantitative PCR was more reproductive and specific than traditional PCR.Besides as the real-time quantitative PCR detection had the advantage of fast testing,it only needed 3 hours from the sample treatment to result report.A SYBR Green I fluorescent quantitative PCR for detecting Necator americanus was developed successfully.It is apparent that real-time PCR for the detection of Necator americanus is rapid,sensitive and specific.