不同DNA抽提方法对普通PCR和实时荧光定量PCR方法检测家蚕微孢子虫的影响  

Influence of DNA extraction methods on detection of Nosema bombycis by traditional PCR and real-time PCR methods

作　　者：何永强 吴姗 鲁兴萌[2] 邱海洪[2] 帅江冰 张晓峰 王素华 徐国群 李光才[2] 董强 

机构地区：[1]浙江出入境检验检疫局技术中心,杭州310012 [2]浙江大学动物科学学院,杭州310029

出　　处：《昆虫学报》2011年第11期1329-1334,共6页Acta Entomologica Sinica

基　　金：浙江省重大科技专项(2010C12005-2);国家质量监督检验检疫总局科研项目(2007IK017);浙江出入境检验检疫局科技项目(ZK200610)

摘　　要：为了优选快速、灵敏、特异的家蚕微孢子虫Nosema bombycis分子检测方法和DNA抽提方法,本文通过对家蚕微孢子虫TaqMan探针荧光定量PCR检测方法和SYBRGreen荧光定量PCR检测方法的建立以及反应体系优化,并与普通PCR方法进行比较;再采用4种不同DNA抽提方法分别对PCR和实时荧光定量PCR方法检测家蚕微孢子虫悬浮液的效果评价。结果显示:不经过DNA抽提,直接将家蚕微孢子虫发芽液进行PCR反应的效果优于其他方法,检测灵敏度由高到低依次为直接法、酚/氯仿抽提法、动物组织DNA试剂盒抽提法和植物组织DNA试剂盒抽提法;TaqMan探针法检测家蚕微孢子虫发芽液的灵敏度和SYBRGreen法相近,达到微孢子102个/mL,两者均优于普通PCR方法。实验表明,直接采用发芽液结合荧光定量PCR方法检测家蚕微孢子虫最为简便、快速、灵敏。该研究结果将有助于提高家蚕微粒子病监控技术和检疫能力,对家蚕微粒子病的检疫和防治具有积极意义。To search out a rapid,sensitive and specific method for detection of Nosema bombycis and DNA extraction,both TaqMan probe real-time PCR method and SYBR Green real-time PCR method were established and optimized compared with traditional PCR method.In addition,four DNA extraction methods have been evaluated for the efficiency for traditional PCR and real-time PCR.The results showed that the germination solution of N.bombycis could be directly used for real-time PCR and traditional PCR,without the DNA extraction procedure.Moreover,the direct method had much higher sensitivity than other extraction methods in the real-time PCR and traditional PCR detection.The sensitivity of the four methods from high to low was in the following order：direct method,phenol-chloroform extraction method,animal tissue DNA extraction kit and plant tissue DNA extraction kit method.TaqMan probe method and SYBR Green method had similar sensitivity in the detection,up to 102 Nb/mL in detecting the germination solution of N.bombycis,and both had higher sensitivity than traditional PCR.Real-time PCR technique combining with the DNA-extraction-skip skill makes a simple,sensitive and specific method for detection of N.bombycis.This study would be helpful for quarantine and control of pebrine.

关 键 词：家蚕微孢子虫 DNA抽提方法 PCR 荧光定量PCR 家蚕微粒子病 检疫 

分 类 号：Q966[生物学—昆虫学]