香蕉枯萎和细菌性软腐病菌的多重PCR检测

Detection of banana Fusarium wilt and bacterial soft rot by multiplex PCR amplification

香蕉枯萎病菌Fusarium oxysporum f.sp.cubense和细菌性软腐病菌Dickeya zeae的复合侵染为害给香蕉产业发展带来严重挑战,有必要建立相关病害的多重聚合酶链式反应(multiplex polymerase chain reaction, multiplex PCR)检测技术。本文基于尖孢镰刀菌古巴专化型1号生理小种(F.oxysporum f.sp.cubense race 1,FOC1)基因组contig 438区间(35 631-37 693 bp)(GenBank:AMGP01000438.1)和4号生理小种(F.oxysporum f.sp.cubense race 4,FOC4)基因组contig 195区间(4 028-6 126 bp)(GenBank:AMGQ01000195.1)存在160 bp插入序列差异设计特异扩增引物FOC-F/-R,同时以香蕉细菌性软腐病菌D.zeae的促旋酶B亚单位基因(the subunit B of gyrase gene)(GenBank:JQ284039)序列设计特异扩增引物gyrB-F/-R。多重PCR检测结果显示:本技术可在一次PCR扩增反应内同时检测香蕉枯萎病菌1号、4号生理小种和细菌性软腐病菌;多重PCR的灵敏度结果表明:检测香蕉枯萎病菌的DNA浓度最低限为0.1 ng/μL,细菌性软腐病菌的灵敏度为103cfu/mL;检测结果稳定可靠。因此,本研究建立的多重PCR检测方法可有效应用于检测香蕉发病组织中的香蕉枯萎病菌和细菌性软腐病菌,也可用于香蕉种苗和田间土壤带病菌的监测,为香蕉种植保驾护航。

The diseases caused by Fusarium oxysporum f.sp.cubense and Dickeya zeae have brought serious challenges to the development of banana industry,and it is necessary to develop a detection method by multiplex polymerase chain reaction(multiplex PCR)technology for them.The specific amplification primers for the molecular multiplex PCR system were designed as followed:FOC-F/-R targeted the sequences of the contig 438(35631-37693 bp)(GenBank:AMGP01000438.1)in the DNA of F.oxysporum f.sp.cubense race 1(FOC1)and the contig 195(4028-6126 bp)(GenBank:AMGQ01000195.1)in the DNA of F.oxysporum f.sp.cubense race 4(FOC4),and there was a difference of 160 bp between FOC1 and FOC4.The primers gyrB-F/-R were designed based on the gene sequence(GenBank:JQ284039)in D.zeae.The results of multiplex PCR showed that this technology could simultaneously detect FOC1,FOC4 and D.zeae in one PCR amplification reaction.Moreover,the minimum limit of DNA concentration for detection of F.oxysporum f.sp.cubense was 0.1 ng/μL,and the sensitivity for detecting D.zeae was 10^(3) cfu/mL.The detection results were stable and reliable.Therefore,the multiplex PCR detection method can be effectively used to detect F.oxysporum f.sp.cubense and D.zeae in infected banana plant tissues,and can also be used to monitor banana seedling-and soil-borne pathogens in the field,which provides a useful tool for safe planting of banana.