基于16S rDNA基因的谷斑皮蠹PCR检测技术  

Study on molecular detection technology of Trogoderma granarium based on 16S rDNA

作　　者：张祥林[1] 李京[1,2] 罗明[2] 李亚伟[1] 王罛 张小菊[1] 

机构地区：[1]新疆出入境检验检疫局,新疆乌鲁木齐830063 [2]新疆农业大学农学院,新疆乌鲁木齐830052

出　　处：《生物安全学报》2017年第1期75-79,共5页Journal of biosafety

基　　金：国家质检公益性行业科研专项(201310091)

摘　　要：【目的】谷斑皮蠹是一种重要的检疫性害虫,在新疆周边多个国家分布,口岸检疫人员多次从进境货物中截获谷斑皮蠹,该虫对新疆的农业生产极具威胁。【方法】以谷斑皮蠹的16S rDNA基因为靶序列,用昆虫通用引物对4种供试皮蠹进行PCR扩增,将扩增产物进行克隆和测序,用生物软件设计检测谷斑皮蠹的特异性引物与探针。【结果】设计的特异性引物(TG-SNP-F/TG-SNP-R)及所建立的常规PCR方法能有效检测出谷斑皮蠹,其扩增产物的片段大小为250 bp,灵敏度为3 ng·μL^(-1)。设计的特异性引物(TG-F/TG-R)和探针(TG-probe),以及所建立的实时荧光PCR方法,对谷斑皮蠹的检测特异性强,灵敏度达0.8 fg·μL^(-1)。【结论】建立的常规PCR方法和实时荧光PCR检测方法能够对谷斑皮蠹进行准确鉴定,为口岸检疫人员检测进境货物中携带的谷斑皮蠹提供技术支持。【 Aim】 The Trogoderma granarium is an important quarantine pest,found in many counties in Xinjiang. It w’as intercep-ted many times by port quarantine officers.【 Method】 The study w’as carried out using the 71. granarium/s 16S rDNA gene as target fragments. The universal insect primer was used amplify DNA from the four Trogoderma spp.. T. granarium ＇s specific primers and probes were designed by using biological software.【 Result】 The designed primers TG- SNP- F/TG- SNP-R could specifically detect the T. granarium by conventional PCR. The amplified fragment was 250 bp, and its sensitivity was 3 ng ·μL^-1. Real-time PCR methods of detecting the T. granarium had strong specificity,with sensitivity of 0.8 fg ·μL^-1【 Conclusion】 T. granarium was accu-rately identified by using the conventional as well as real-time PCR,and provides technical support for port quarantine officers to de-tect T. granarium in imported goods.

关 键 词：谷斑皮蠹 16S RDNA 常规PCR 实时荧光PCR 

分 类 号：S41-30[农业科学—植物保护]