钉螺体内细菌群落结构PCR-DGGE分析方法的建立

Examination of Bacterial Microbial Community Structure from Oncomelania hupensis by PCR-DGGE

建立了通过PCR-DGGE研究钉螺体内细菌群落结构的方法,并采用此技术分析了钉螺体内细菌群落结构。钉螺样本采自湖北省荆州市,样本带回实验室经破壳解剖后取清洗的钉螺内脏放入无菌离心管后经酚-氯仿法抽提微生物DNA。使用获得的微生物DNA为模板,选择细菌原核通用引物F357-GC和R518进行PCR扩增得到大约250bp的目的片段,然后采用变性梯度凝胶电泳结合DNA测序技术对获得的目的片段进行分析。结果显示此技术能够区分幼螺和成螺体内细菌群落结构差异,并发现在成螺体内有大量的Pseudomonas属和Acidobacteria属细菌出现。

The present study established the method of investigating the visceral bacterial community of Oncomelania hupensis by polymerase chain reaction-denaturing gradient gel electrophoresis（PCR-DGGE）and analyzed the visceral bacterial community of young and adult O.hupensis.The viscera of O.hupensis were collected into centrifuge tubes after removing their shells.Subsequently,lyses solutions were added to every tube and incubated.After centrifugation,the liquid was transferred into a new sterile tube followed by a phenol/chloroform/isoamyl alcohol extraction procedure.The bacterial DNA was amplified using primers F357-GC and R518.The aim fragments（approximate 250 bp,containing 40 bp GC）amplified from PCR were analyzed applying DGGE,and a DGGE profile was analyzed by clustering.DNA bands excised from DGGE gel were sequenced and the closest sequences blasted to confirm the corresponding species in GenBank.The visceral bacterial community diversity between young and adult snails could be displayed by PCR-DGGE,and a large amount of Pseudomonas and Acidobacteria appeared in adult snails viscera.