保鲜与加工StorageandProcess2023，23（0）：00-00加工研究梨小食心虫实时荧光PCR检测技术的建立与验证王凤军，周晓红（浙江经贸职业技术学院，浙江杭州310018）摘要：梨小食心虫（Grapholithamolesta）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3pg/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3h），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。关键词：梨小食心虫；实时荧光定量PCR；快速鉴定EstablishmentandValidationofReal-TimeFluorescenceQuantitativePCRforIdentificationofGrapholithamolestaWANGFeng-jun,ZHOUXiao-hong（ZhejiangInstituteofEconomicandTrade,Hangzhou310018,China）Abstract:GrapholithamolestaisacommoninternalfruitfeedersfromorchardinChina,andisverysimilartootherpearpestsintheexternalmorphology.Itisdifficulttodistinguishonlybymorphologicalcharacteristics,areal-timefluorescentPCRdetectionmethodshouldbeestablishedforidentificationatthegenelevel.Thespecificityoffluores⁃centprobewasdesignedandverifiedthroughsamplenucleicacidextractionandqualitycontrol,andareal-timefluo⁃rescentquantitativePCRdetectionmethodforGrapholithamolestawasdevelopedbyfurtheroptimizingreactioncondi⁃tionsandreactionsystems,combinedwithspecificityanalysis,sensitivitycomparisonandblindsampletesting.There⁃sultsshowedthatthemethodhadstrongspecificity,goodrepeatability(thecoefficientofvariationbetweenrepeatswas0.055~0.359),highreliability(R2valueofstandardcurvewas0.991),andlowdetectionlimit(theminimumdetectionlimitwas3pg/µL),theresultsofblindsampletestwereconsistentwithmorphologicalidentificationresults,whichcouldmeettheneedsofdailyinspectionandquarantine.Thismethodissimpl...
