PMA-qPCR联用快速检测苹果中扩展青霉活菌  

Rapid Detection of Viable Penicillium expansum in Apple by PMA-qPCR

作　　者：阳瑾 丁宇 于吕健 范盈盈 刘峰娟 赵雪祺 焦子伟 王成 YANG Jin;DING Yu;YU Lüjian;FAN Yingying;LIU Fengjuan;ZHAO Xueqi;JIAO Ziwei;WANG Cheng(College of Bio-and Geo-Sciences,Yili Normal University,Ili 835000,China;Institute of Quality Standards&Testing Technology for Agro-Products,Xinjiang Academy of Agricultural Sciences,Key Laboratory of Functional Nutrition and Health of Characteristic Agricultural Products in Desert Oasis Ecological Region(Co-construction by Ministry and Province),Ministry of Agriculture and Rural Affairs,Laboratory of Quality and Safety Risk Assessment for Agro-Products(Urumqi),Ministry of Agriculture and Rural Affairs,Key Laboratory of Agro-products Quality and Safety of Xinjiang,Urumqi 830091,China;College of Food Science and Pharmacy,Xinjiang Agricultural University,Urumqi 830052,China)

机构地区：[1]伊犁师范大学生物与地理科学学院,新疆伊犁835000 [2]新疆农业科学院农业质量标准与检测技术研究所,农业农村部荒漠绿洲生态区特色农产品功能营养与健康重点实验室(部省共建),农业农村部农产品质量安全风险评估实验室(乌鲁木齐),新疆农产品质量安全实验室,新疆乌鲁木齐830091 [3]新疆农业大学食品科学与药学学院,新疆乌鲁木齐830052

出　　处：《食品工业科技》2023年第16期331-338,共8页Science and Technology of Food Industry

基　　金：国家重点研发计划(2019YFC1604502);伊犁师范大学高层次人才项目(YLSDXSDTR22001)。

摘　　要：目的:为建立一种叠氮溴化丙锭(Propidium Monoazide,PMA)与实时定量聚合酶链式反应(Quantitative real-time Polymerase Chain Reaction,qPCR)联用的快速检测扩展青霉活菌方法。方法:通过优化PMA处理浓度、黑暗孵育及曝光时间,筛选扩展青霉特异性引物,结合qPCR技术,建立一种基于PMA-qPCR联用快速检测扩展青霉活菌的方法,构建定量标准曲线,应用于人工污染的苹果样品中活菌的检测,与平板菌落计数比较评估该方法的可靠性。结果:PMA处理浓度10μg/mL、黑暗孵育5 min、曝光10 min为最佳PMA处理条件。4种引物中Pexp-patF对扩展青霉具有极强的特异性,可作为引物用于PMA-qPCR检测。构建的定量标准曲线的R2=0.9948,最低检测限为102.6 CFU/mL,方法检测结果与平板菌落计数无明显差异,并发现在苹果的未腐烂部分中可检测出扩展青霉活菌。结论:研究建立的PMA-qPCR技术可应用于苹果中扩展青霉活菌的检测,为扩展青霉精准防控提供一定技术支撑。Objective:To establish a rapid detection method for viable Penicillium expansum by propidium monoazide(PMA)combined with quantitative real-time polymerase chain reaction(qPCR).Methods:PMA-qPCR detection method for viable P.expansum was established,including the optimization of the treatment concentration,dark incubation and exposure time of PMA,the screening of specific primers of P.expansum,and the conduction of qPCR.In addition,the standard curve was constructed,which was applied to the detection of artificially contaminated apples samples.The reliability of this method was also evaluated by comparing with the plate counting method.Results:The optimal PMA treatment conditions were:10μg/mL for PMA concentration,5 min for the dark incubation and 10 min for the exposure time.Among the 4 pairs of primers,Pexp-patF showed strong specificity for P.expansum,which could be used as a optimal primer for PMA-qPCR detection.The correlation coefficient of the established quantitative standard curve was 0.9948,and the detection limit of the method was 102.6 CFU/mL.There was no obvious difference between the detection result of this method and the plate counting method,and the viable P.expansum could be detected in the non-rotted part of apples.Conclusion:The established PMA-qPCR technique could be applied to the detection of P.expansum in apples,which would provide technical support for the prevention and control of P.expansum.

关 键 词：扩展青霉 叠氮溴化丙锭 实时定量聚合酶链式反应 苹果 活菌 

分 类 号：TS207.4[轻工技术与工程—食品科学]