采用PCR-DGGE(PCR-denaturing gradient gel electrophoresis)技术对一个典型的凡纳滨对虾(Litopeneaus vannamei)海水养殖系统细菌群落进行分子分析。结果表明,沿岸水、蓄水池、养殖池水具有较高的细菌种类多样性,而蓄水池进水、对虾粪样、肠壁定植细菌样以及排水渠污水的细菌多样性程度低。每种环境群落的优势种明显。3个养殖池水样(Y1、Y2、Y3)、2个沿岸水样(W1、W2)、2个粪样(F1、F2)、蓄水池水样(B1、B2)及2个肠壁定植细菌样(G1、G2)各自具有高度群落相似性。BLAST结果表明,12个条带克隆序列所代表优势种很可能来源于以下几个属:柔发菌属(Flexithrix)、黏纤维菌属(Cytophaga)、Dyella属、聚球菌属(Synechococcus)、Chlorarachnion属、支原菌属(Mycoplasma)、草螺菌属(Herbaspirillum)、河氏菌属(Hahella)、Ruegeria属。本研究表明,PCR-DGGE技术可以用于海水对虾养殖系统的细菌群落结构分析。对于海水对虾养殖系统来说,一些序列所代表的主要细菌种类极有可能是很少被注意到或研究过的,具有潜在的研究价值。
Using PCR-denaturing gradient gel electrophoresis （DGGE） targeting the RNA polymerase beta subunit （rpoB） gene, a simultaneous detection method for Vibrio species was established, rpoB gene-based PCR-DGGE was carried out with eight Vibrio reference strains （each from different species）, mixed sample （including these Vibrio reference strains）, two non Vibrio strains, four environmental Vibrio strains, and three unidentified environmental strains. For comparison, 16S rRNA gene-based PCR-DGGE of the eight Vibrio reference strains was performed with universal primers. In addition, three unidentified strains were identified by 16S rRNA and gyrB gene sequencing and API20E system in order to confirm the accuracy of rpoB gene-based PCR-DGGE detection. Results revealed that rpoB-based PCR-DGGE could well discriminate eight Vibrio reference strains and could not discriminate different strains within the same species. The bands derived from two non Vibrio strains could not match with any bands in reference marker. Meanwhile, 16S rRNA gene-based DGGE failed to distinguish these reference strains. Further-more, four out of eight Vibrio species exhibited heterogenous bands in 16S rRNA gene-based DGGE. Sequencing and API 20E identification of unidentified strains coincided with the detection by rpoB gene-based PCR-DGGE. The results demonstrated that rpoB-based PCR-DGGE provided a rapid and efficient method for simultaneous detection of muhiple Vibrio species, which can avoid the limitations inherent in 16S rRNA gene-based PCR-DGGE.
