桑源阴沟肠杆菌LAMP检测方法的建立和应用

Establishment and Application of Loop-Mediated Isothermal Amplification Method for Detecting Enterobacter cloacae Complex

桑源阴沟肠杆菌是引起桑树发生枯萎症状的重要病原之一。对桑源阴沟肠杆菌全基因组序列进行分析,得到桑源阴沟肠杆菌特异基因序列,并以该特异基因序列为靶序列,设计筛选合适的环介导等温扩增(loop-mediated isothermal amplification,LAMP)引物组,通过荧光扩增曲线法、PCR产物琼脂糖凝胶电泳法及PCR产物SYBR Green Ⅰ染色法3种判定方法优化反应体系,进而建立桑源阴沟肠杆菌LAMP检测技术和产品。采用Yt4引物组,在60℃、外内引物浓度比为1∶10的最优反应条件下,桑源阴沟肠杆菌阳性质粒检测灵敏度为25×10^6拷贝/μL。在特异性实验中,桑源阴沟肠杆菌菌株的阳性反应能产生S型荧光扩增曲线,电泳法可得到梯形条带,染色法反应液变为绿色,而在其他供试菌株及阴性对照中均未发生这些现象。应用建立的LAMP检测法和普通PCR方法分别对来自4个不同地区的11个桑树枯萎症状样品进行同步平行检测,2种方法的检测结果一致,但LAMP检测法更简单、快捷和灵敏。建立的桑源阴沟肠杆菌LAMP检测方法,为桑源阴沟肠杆菌的快速检测及桑树枯萎病的诊断提供了新技术。

Enterobacter cloacae complex is one of the main pathogens causing wilting symptoms of mulberry tree. In this study,the whole genome sequence of E. cloacae complex was analyzed to obtain its specific gene sequence,which was used as the target sequence to design and screen appropriate LAMP(loop-mediated isothermal amplification)primers. LAMP detection technique and products for detecting E. cloacae complex were obtained by optimizing the reaction system with 3 methods including LAMP real-time fluorescence amplification curve detection,agarose gel electrophoresis and SYBR Green Ⅰ chromogenic method. Under optimal reaction conditions when concentration ratio of outer and inner Yt4 primers was 1 ∶ 10 at 60 ℃,detection sensitivity of positive plasmid was 2 5×10^6 copy·μL^-1. Furthermore,specificity test proved that S-type fluorescent amplification curve could be produced by positive reaction of E.cloacae complex,the band ladder could also be observed by agarose gel electrophoresis method,and the reacted solution turned green by chromogenic method. These reactions did not occur in other tested strains and in the negative control group. Finally,simultaneous and parallel detection of E. cloacae complex from 11 wilting mulberry tree samples of 4 different areas were carried out by LAMP and PCR. Though similar results were acquired,LAMP detection method was simpler,faster and more sensitive than another detection method. This study resulted in a LAMP method for rapid detection of E. cloacae complex and diagnosis of mulberry enterobacterial wilt caused by E. cloacae complex.