荧光原位杂交法在检测硫酸盐还原菌中的应用

Application of fluorescence in situ hybridization (FISH) to detection of sulfate-reducing bacteria

目的探讨荧光原位杂交法在检测淡水湖泊沉积物硫酸盐还原菌检测中的应用。方法建立荧光原位杂交方法并应用该法进行洱海沉积物硫酸盐还原菌含量分析,所用寡核酸苷酸探针为依据硫酸盐还原菌16SrRNA特异性区域而设计的SRB385。结果荧光显微镜下标本中可见发特异性红色荧光的硫酸盐还原菌菌体。经荧光原为杂交分析,洱海沉积物前18cm硫酸盐还原菌含量为(0.08～8.50)×103个/g,平均为(1.91±2.51)×103个/g。硫酸盐还原菌含量在空间分布上表现为悬浮层、5和8cm深度有较小谷值,3、6和11cm深度有较高的峰值,其中11cm深度形成的峰较宽。结论荧光原位杂交法具有快速准确、检测信号强、杂交特异性高的特点,可以进行淡水湖泊沉积物硫酸盐还原菌含量的检测。洱海沉积物硫酸盐还原菌含量丰富且不同深度的沉积物中硫酸盐还原菌含量分布是不均一的。

[Objective] To evaluate the application of FISH to detection of Sulfate-reducing bacteria in the fresh lake sediments. [Methods] FISH （fluorescence in situ hybridization） was established with the oligonucleotide probe （SRB385） designed according to the specific 16SrRNA sequence of SRB and utilized to analyze SRB quantity in Lake Erhai sediments. [Results] SRB with clear red fluorescence were observed by fluorescence microscope. The average quantity of SRB in the upper 18 cm of Lake Erhai sediments was （1.91±＋2.51）×10^3 number/g ranged from 0.08×10^3 number/g to 8.5×10^3 number/g. SRB quantity vertical distribution showed that the lowest peak value was in 0, 5 and 8 cm depth and the highest peak value was in 3, 6 and 11 cm depth. Meanwhile, there was a wide peak around 11 cm depth. [Conclusion] FISH is a sensitive,simple and correct method which can be used to determine SRB in the fresh lake sediments. The quantity of SRB in Lake Erhai sediments is large and SRB quantity distribution is uniform in different depths.