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

Abstract: 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.

Key words: Nosema bombycis, DNA extraction, PCR, real-time PCR, pebrine, quarantine