核酸分子杂交技术鉴别和检测家蚕微孢子虫的研究

IDENTIFICATION AND DETECTION OF NOSEMA BOMBYCIS NAEGELI BY NUCLEIC ACID HYBRIDIZATION

为了将家蚕微孢子虫 (N .b .)从蚕业生产中流行的 10多种病原性微孢子虫中鉴定出来 ,并对其发病程度进行检测 ,用PCR技术合成了一段 317bp的DNA片段 ,将其克隆到大肠杆菌E .coliDH5α中 ,并大量制备该片段 ,用地高辛 (DIG)标记成探针 ,经点杂交和Southern杂交检测 ,发现该片段为N .b .所特有。该探针为N .b的特异性检测探针 ,灵敏度可达 1ngDNA水平。对蚕业生产上的带毒蚕蛾和蚕卵的模拟检测证明 ,该DIG探针可将N .b发病初期的带毒蚕蛾和蚕卵检出 。

Nosema Bombycis Nageli(N.b.)is the most serious pathogenic spore in sericulture.In order to identify.b.from other related species spores,we amplified a DNA fragment by PCR,Which is about 317 bp.The fragment was cloned into a T-Easy Vector and transferred into Ecoli DH5α,in which it can be largely produced.The fragment is labeled with DIG and made to be a probe.The results of dot blotting and southern blotting show that the probe is peculiar to the N.b.spore and can distinguish N.b.from other spores by nucleic acid hybridization method,the detective sensitivty has reached to 1 ng DNA level.The DIG labeled probe is also used to detect the N.b.DNA isolated from the silkworm eggs and moths,imitating the condition of sericulture,the results show that the probe can detect N.b.spore when N.b.is in its initial stage.