扁桃拟茎点霉TaqMan荧光定量PCR检测方法的建立与应用  

Development and application of TaqMan fluorescence quantitative PCR for detection of Phomopsis amygdali

作　　者：王律[1,2] 张华[3] 赵玉强 褚姝频[5] 吴翠萍[6] 田艳丽[1,2] 胡白石[1,2] WANG Lv ZHANG Hua ZHAO Yu-qiang CHU Shu-pin WU Cui-ping TIAN Yan-li HU Bai-shi(College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China z Key Laboratory of Integrated Management of Crop D~seases and Pests （ Nanj~ng Agricultural Universi- ty）, Ministry of Education, Nanjing 210095, China a Wuxi Entry-Exit Inspection and Quarantine Bureau, Wuxi 214101, Chi- n Shanghai Agricultural Technology Extension and Service Center, Shanghai 201103, China Jiangsu Plant Protection Sta- tion, Nanjing 210013, China Jiangsu Entry-Exit Inspection and Quarantine Bureau, Nanjing 210001, China)

机构地区：[1]南京农业大学植物保护学院,南京210095 [2]农作物生物灾害综合治理教育部重点实验室,南京210095 [3]无锡出入境检验检疫局,无锡214101 [4]上海农业技术推广服务中心,上海201103 [5]江苏省植物保护站,南京210013 [6]江苏省出入境检验检疫局,南京210001

出　　处：《植物病理学报》2017年第1期26-34,共9页Acta Phytopathologica Sinica

基　　金：无锡市科技发展基金(CSE11N1307)

摘　　要：扁桃拟茎点霉(Phomopsis amygdali)引起的桃缢缩溃疡病是桃生产上重要的病害之一。带菌苗木及接穗是病害向无病区传播的主要途径。严格的检疫措施是保证无病区健康生产的关键。准确、灵敏、快速的检测方法是严格执行检疫措施及研究病害发生规律的有力工具。将扁桃拟茎点霉组蛋白H3(histone H3)基因与其近缘种比对发现,该基因特异性强,适合作为分子检测的靶标。针对histone H3基因为靶标,设计了特异性引物和Taq Man探针,建立了扁桃拟茎点霉的荧光定量PCR检测方法。结果发现,该方法只能特异识别扁桃拟茎点霉。检测灵敏度可达4×10~1copies·uL^(-1),比常规PCR检测方法高1 000倍;孢子检测最低限达2个孢子,比常规PCR检测方法高10倍,且仅需1 h即可完成检测。该方法用于田间样品检测,扁桃拟茎点霉的阳性检出率达86%,高于分离培养法和常规PCR检出结果。本研究建立的基于Taq Man探针的荧光定量PCR检测体系可用于田间对扁桃拟茎点霉的快速检测。The peach constriction canker,caused by Phomopsis amygdali is one of the most important diseases of peach trees.Because the disease transmission is mainly through infected seedlings and scions,strict quarantine measures are the key to prevent the pathogen into the disease-free area.The accurate,sensitive and fast detection method is an effective tool for quarantine and study on the occurrence of disease.In comparison of histone H3 gene of P.amygdali with that of the closely related species,histone H3 was a suitable target for molecular detection.Based on the histone H3 gene,we designed the specific primers and TaqMan probe,and the TaqMan Fluorescence Quantitative PCR Method（FQ-PCR） was established for the detection of P.amygdali.Results showed that the developed FQ-PCR was specific for P.amygdali detection;and this method was able to detect as little as 4x10 copies of recombinant plasmid DNA.Its sensitivity was 1 000 times as high as routine PCR.The detectable limits of P.amygdali were 2 conidia,which were 10 times higher than that of routine PCR;the whole detection process could be finished in one hour.Compared to culture and routine PCR,the positive detection rates of suspicious samples by FQ-PCR were the highest,at 86%.In conclusion,the TaqMan Fluorescence Quantitative PCR developed in this study was simple,fast,sensitive,and specific,and can be used to detect P.amygdali in high-speed from infected peach trees in the field.

关 键 词：扁桃拟茎点霉 HISTONE H3 TAQMAN探针 荧光定量PCR 

分 类 号：S858.23[农业科学—临床兽医学]