昆虫学报2011，Vol.54Issue(11)：1329-1334,6.
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
摘要
Abstract
lo search out a rapid, sensitive and specific method tor 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Key words
Nosema bombycis/ DNA extraction/ PCR/ real-time PCR/ pebrine/ quarantine分类
生物科学引用本文复制引用
何永强,董强,吴姗,鲁兴萌,邱海洪,帅江冰,张晓峰,王素华,徐国群,李光才..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[J].昆虫学报,2011,54(11):1329-1334,6.基金项目
浙江省重大科技专项(2010C12005-2) （2010C12005-2）
国家质量监督检验检疫总局科研项目(20071K017) （20071K017）
浙江出入境检验检疫局科技项目(ZK200610) （ZK200610）
