梨小食心虫实时荧光PCR检测技术的建立与验证

Establishment and Validation of Real-Time Fluorescence Quantitative PCR for Identification of Grapholitha molesta

梨小食心虫(Grapholitha molesta)为我国常见蛀果性害虫,与其他梨果害虫的组织形态非常相似,仅凭形态学特征难以区分,需建立实时荧光PCR检测方法在基因水平上进行鉴定。通过样品核酸提取与质控,设计并验证荧光探针特异性,进一步通过优化反应条件和反应体系,结合特异性分析、灵敏性比对和盲样测试开发了梨小食心虫实时荧光定量PCR检测方法。结果表明:方法特异性强,重复性好(重复间变异系数为0.055~0.359),可靠性高(标准曲线中的R2值为0.991),检出限低(最低检出限3 pg/μL),盲样测试结果与形态学鉴定结果一致,能满足日常检验检疫需求。该操作方法简单易行,检测时限短(约3 h),通量高,特别适合于大批量的抽样检查,可应用于梨小食心虫的检疫工作。

Grapholitha molesta is a common internal fruit feeders from orchard in China,and is very similar to other pear pests in the external morphology.It is difficult to distinguish only by morphological characteristics,a real-time fluorescent PCR detection method should be established for identification at the gene level.The specificity of fluores⁃cent probe was designed and verified through sample nucleic acid extraction and quality control,and a real-time fluo⁃rescent quantitative PCR detection method for Grapholitha molesta was developed by further optimizing reaction condi⁃tions and reaction systems,combined with specificity analysis,sensitivity comparison and blind sample testing.The re⁃sults showed that the method had strong specificity,good repeatability(the coefficient of variation between repeats was 0.055~0.359),high reliability(R2 value of standard curve was 0.991),and low detection limit(the minimum detection limit was 3 pg/μL),the results of blind sample test were consistent with morphological identification results,which could meet the needs of daily inspection and quarantine.This method is simple and easy to operate,with short detec⁃tion time limit(about 3 h)and high flux,which is especially suitable for sampling inspection in large quantities,and can be applied to the quarantine of Grapholitha molesta.