作　　者： ; ; ;

机构地区： 华南农业大学

出　　处： 《中国农业科学》 2004年第12期1925-1931,共7页

摘　　要： 家蚕微粒子病病原为桑蚕微孢子虫(Nosema bombycis Nageli, N.b)。本文根据文献报道,选用了一对根据N.b孢子及其相近种属微孢子虫的SSU-rRNA保守区段设计的有效引物VIF/530R,从模拟感染不同浓度N.b孢子(3×101-7/ml)的蚕蛾、蚕卵的角度,较系统地研究了PCR诊断技术的可检测灵敏度和对模拟感染家蚕微粒子病的诊断效果,为PCR分子诊断家蚕微粒子病的实用化研究提供参考。在本实验的DNA制备方法和PCR反应条件下,分别对由不同浓度N.b纯孢子抽提的DNA模板和不同浓度N.b纯孢子(3×101～7ml-1)分别与蚕卵、蚕蛾混合抽提的DNA模板进行PCR扩增检测。结果表明:(1)对N.b纯孢子DNA模板,PCR可检测灵敏度为3×104N.bml-1(弱检测信号可到3102～3N.bml-1),对模拟染毒N.b孢子的蚕蛾DNA模板,PCR可检测灵敏度为3×105N.bml-1,两者分别已超过或达到目前生产上用显微镜的检毒水平;(2)对模拟染毒N.b孢子的蚕卵DNA模板,VIF/530R引物不能有效地检出蚕卵中染毒的N.b孢子DNA信号,推测在蚕卵DNA抽提物里可能含有抑制PCR扩增的未明因素,而蚕蛾DNA抽提物里则无此抑制因素。 The pathogen of Pebrine disease in silkworm is Nosema bombycis Nageli (N.b). Based on published data, one effective primer set VIF/530R, which matched conserved regions of microsporidian SSU-rDNA from N.b and the other microsporidium species were selected. In order to consult the practicality of PCR diagnosis to Pebrine disease, the sensitivity and effectivity of PCR assay for different concentration of N.b(3×101-7ml-1)mixed with moths and Bombyx eggs to simulate natural N.b infection were studied, respectively. Under this paper抯 DNA extraction methods and PCR conditions, the template DNA from purified N.b spores and from spores diluted in tissues from Bombyx mori eggs and moths to simulate natural N.b infection were amplified. The results are as follows:(1)the sensitivity of PCR diagnosis was 3?04 N.b穖l-1 for purified spores (the slight bands were up to 3102-3N.bml-1), and 3?05 N. b穖l-1 for spores mixed with tissue from Bombyx moths, respectively. All of them are therefore excessive or equal to the sensitivity of microscopical diagnosis currently used as criteria to quarantine silk moths in the field in China.(2)For the DNA template from spores diluted in tissues from Bombyx eggs simulated naturally N.b infection, there are no target bands with the primer set VIF/530R, it suggested that there exist inhibitory substances from the eggs interfere with PCR amplification, but not exist in the extraction of moths.

关 键 词： 蚕卵 蚕蛾 微孢子虫 家蚕微粒子 模板 引物 感染 染毒 分子诊断技术

领　　域： [农业科学] [农业科学] [农业科学] [农业科学]