16S rRNA基因多变区序列对不同种属钩端螺旋体的PCR扩增

PCR Amplification of the Leptospiral DNAs from Different Genus and Species with the Variable Sequonces of 16S rRNA Gene

从问号状钩体16S rRNA基因多变区V2和V4序列中设计了一对引物:PⅠ 5’GGGAAC CTA ATA CTG GAT GG;PⅡ 5’ACA TAG TTT CAA GTG GAG GC,并对不同种属钩体DNA进行了扩增。在55℃退火条件下,问号状钩体各群型具有相同大小的扩增产物(473bp)和KpnⅠ酶谱,双曲钩体有约280bp的扩增带,但不被KpnⅠ酶消化,而细丝体属钩体和其它对照DNA未见扩增。以^(32)P标记的lai型Lai株(强毒)扩增产物作探针行Southern杂交发现,问号状钩体各株扩增带均可被杂交,而双曲钩体扩增物无杂交。研究表明,该对引物可用于钩体的分类和检测。

We designed a pair ofprimers from the variable regions (V2 andV4) of 16S rRNA gene of Leptospira inter-rogans, i. e. PⅠ: 5'GGG AAC CTA ATACTG GAT GG; PⅡ: 5'ACA TAG TTT CAAGTG GAG GC, and amplified the lepto-spiral DNAs from different genus andspecies. When denaturing with 55℃. allDNAs of L. interrogans had the sameproducts not only in length but also withKpn Ⅰ-digested pattern. The DNA of L.biflexa could be amplified with a c. a. 280bp-band but not digested by Kpn Ⅰ,whilethe DNAs of Leptonema and other controlbacteria had no amplification. In addition,the products of L. interrogans spp. couldbe hybridized with the PCR product of L.interrogans serovar lai strain Lai labelledwith ^(32)P, while the product of L. biflexahad no hybridization. It proved that the 16S rRNA gene primers is useful for theclassification and detection of leptospires.