嗜水气单胞菌与温和气单胞菌的LAMP检测方法的建立

Loop-mediated isothermal amplification for detection of Aeromonas hydrophila and A.sobria

利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)分别建立嗜水气单胞菌(Aeromonas hydrophila,AH)与温和气单胞菌(A.sobria,AS)的快速检测方法。针对嗜水气单胞菌pilin基因、温和气单胞菌zipA基因设计特异性LAMP引物。在恒温条件下利用实时浊度仪对2组引物进行特异性和灵敏度试验,并以琼脂糖凝胶电泳和核酸染料颜色变化对扩增结果进行判定。结果显示,LAMP实时浊度法能够特异地检测嗜水气单胞菌和温和气单胞菌,最低检出限分别为46 fg·mL-1和320 fg·mL-1,是普通PCR方法的104倍和102倍;并能应用于已知临床样品检测。该研究建立的嗜水气单胞菌与温和气单胞菌LAMP快速检测方法具有高效、特异、灵敏的特点。

The study established a loop-mediated isothermal amplification （LAMP） technology for Aeromonas hydrophila and A. sobria. We designed the primers based on the sequences of the pilin gene of A. hydrophila and the zipA gene of A. sobria, respectively, and conducted specificity and sensitivity tests by real-time turbidimeter under isothermal conditions, which were detected by agarose gel e- lectrophoresis test and change of SYBR Green I colour. The results show that the LAMP method was effective for rapid detection of A. hydrophila and A. sobria with limit of detections of 46 fg·mL^-1 and 320 fg·mL^-1, 104 and 102 times more sensitive than the conven- tional PCR, respectively. In conclusion, the LAMP detective method for A. hydrophila and A. sobria which we established was specif- ic, sensitive, effective and rapid.