水体中盐单胞菌属(Halomonas)细菌实时荧光定量PCR检测方法的建立及应用  

Establishment and application of a real-time PCR method for Halomonas in water

作　　者：张艳[1] 李秋芬[1] 成钰[1] 费聿涛 姜娓娓[1] 

机构地区：[1]中国水产科学研究院黄海水产研究所,青岛266071

出　　处：《应用与环境生物学报》2016年第1期140-145,共6页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(31170113);海洋公益性行业科研专项经费项目(201305043);中央级公益性科研院所基本科研业务费专项资金(20603022013019)资助~~

摘　　要：通过制备标准曲线模板,针对一株盐单胞属细菌X3（经鉴定为Halomonas alkaliphila）的16S r DNA序列设计和筛选特异性引物,建立水体中盐单胞菌属细菌的实时荧光定量PCR检测方法,并将该法用于检测莱州人工鱼礁海域Halomonas属细菌数量.结果表明,引物X3-3F和X3-3R能够对Halomonas属的细菌进行特异性检测,对细菌浓度为6.75×10^-1-6.75×10^7 CFU/m L检测重复性和稳定性较好,最低检出浓度为67.5 CFU/m L.运用建立的方法对莱州人工鱼礁海域水体样品的检测结果表明,在未投礁海域2011年5月和7月水体样品中Halomonas细菌数量分别为8.71×10^3 CFU/m L和2.37×10^3 CFU/m L,而投礁区5月份水样也检出了该属细菌,但荧光信号较弱,7月份水样未检出该属细菌.本研究可为Halomonas属细菌的快速检测及其在海水养殖水质净化方面的推广应用提供有力的技术支撑.This research aimed to develop a real-time quantitative PCR method for Halomonas in water by designing and selecting specific primers based on the gene sequence from 16 S rDNA of Halomonas sp.X_3.The new method was successfully employed in the analysis of bacteria in real environmental samples from the artificial reefs area in Laizhou Bay.The key factors of the method were investigated in detail,including the standard curve,specificity of primers,and the sensitivity,the reproducibility and the practicality of the method.The results showed that the highest specificity could be realized for Halomonas by employing X3-3F and X3-3R primers.Using the DNA extracted from X_3 liquid cultivate as initial template,a standard curve was established.There was a good linear relationship between the concentration of X3 and the fluorescent response over the range of 6.75 × 10^1- 6.75 × 10^7 CFU/mL,with a detection limit of 67.5CFU/mL.According to the readouts,there were 8.71 × 10^3 and 2.37 × 10^3 CFU/mL bacteria in genus Halomonas in the seawater samples collected from the control points in May and July 2011.There were weak fluorescent signals in May and negative signals in July in the samples collected at in the artificial reefs area.The development of the real time quantitative PCR method lays a solid technical basis for rapid detection and water purification in aquaculture of Halomonas.

关 键 词：异养硝化-好氧反硝化细菌 HALOMONAS sp. 实时荧光定量PCR 人工鱼礁 

分 类 号：X832[环境科学与工程—环境工程] X172