兔多杀性巴氏杆菌环介导等温扩增检测方法的建立

Establishment of LAMP for detection of rabbit Pasteurella multocida

为建立一种快速、准确地检测兔多杀性巴氏杆菌(Pm)的环介导等温扩增(LAMP)方法,本研究利用设计的4条特异性引物,在链置换DNA聚合酶的作用下,优化反应条件,并进行特异性和敏感性试验。结果表明,建立的LAMP扩增反应可以在70 min内完成;具有良好的特异性;敏感性为普通PCR的10倍,最低可以检测量为16 cfu的DNA;采用建立的LAMP方法对27份经PCR检测Pm阳性的样品进行检测,结果均呈阳性。该方法的建立为Pm在临床上的快速检测提供了一种新的技术手段。

In order to establish a rapid method for detection of rabbit Pasteurella multocida, a loop-mediated isothermal amplification （LAMP） was developed with four specific primers designed according to the Kmtl gene of P.multocida. Under the optimization of the reaction conditions, the results showed that amplification was completed within 1 hour at a consistence of 63 ~C in water bath. The assay was specific to amplify the DNA from P.multocida, but no cross-amplification from other bacteria DNA. The minimum detection of the assay was 16 cfu of the DNA for P.multocida which was 10 times sensitive than ordinary PCR. In addition, the positive results of 27 suspected P.multocida samples detected by LAMP was consistent with results of ordinary PCR. The establishment of LAMP provided a promising and alternative method for rapid detection of P.multocida.