猪胸膜肺炎放线杆菌环介导等温扩增检测方法的建立与应用

本文建立了敏感、特异的胸膜肺炎放线杆菌环介导等温扩增检测方法。以1型胸膜肺炎放线杆菌ApxIV基因片段为靶序列设计了一组引物，并建立了LAMP反应体系，在63℃等温条件下反应45min，最低能检测到102个拷贝的目的基因，敏感性是PCR方法的10倍。通过对1～10型胸膜肺炎放线杆菌、多杀巴氏杆菌、链球菌、支原体等18种病原微生物的检测，证明该法具有很强的种特异性。另外，通过对8头实验感染猪和127份临床发病猪的鼻拭子的检测发现，该法对鼻拭子中胸膜肺炎放线杆菌的检出率为100%，优于PCR方法。

Detection of Actinobacillus pleuropneumoniae by Newly Developed Loop-mediated Isothermal Amplification Method

The loop-mediated isothermal amplification (LAMP) assay with high sensitivity and specificity for the detection of Actinobacillus pleuropneumoniae was established. Based on the ApxⅣ gene of Actinobacillus pleuropneumoniae serotype 1, a set of six primers were designed. The detection limit of the LAMP assay completed within 45 min at 63 ℃ was 10~2 copies of the target sequence, which was 10 times more sensitive than that of the PCR assay. High species-specificity of the LAMP method were confirmed by the assay of 18 pathogens such as A. pleuropneumoniae serotypes 1～10, Pasteurella multocida, Streptococcus suis and Mycoplasma hyopneumoniae. In addition, all the nasal swab samples from 8 experimental pigs infected by Actinobacillus pleuropnemnoniae and127 clinical cases were detected by LAMP assay, which deteleion rate was 100%, and the sensitivity is preferable to the PCR assay.