深部热水硫酸盐还原菌微滴数字PCR检测技术的建立与应用

Development and application of a droplet digital PCR technique for detection of sulfate-reducing bacteria in deep geothermal water

摘要:

【背景】地下深部存在一个生物圈，深部沉积岩、玄武岩、花岗岩和变质岩等岩性环境的微生物群落已被调查，而地下深部碳酸盐岩岩溶-裂隙热储层微生物群落特征仍然不清。硫酸盐还原菌(sulfate-reducing bacteria，SRB)是地下深部频繁检出的微生物。【目的】建立快速准确定量深部热水硫酸盐还原菌的微滴数字PCR (droplet digital PCR，ddPCR)技术。【方法】以SRB的功能基因dsrB为检测目标，优化SRB ddPCR技术的退火温度，考察其线性范围、敏感性、重复性和特异性，并将该技术用于实际样品的检测。【结果】SRB ddPCR技术的最佳退火温度为54 °C，检测的线性范围为1.1×100?1.1×105 copies/μL-DNA，相关系数R2为0.996，检出限为1 copy/μL-DNA，重复性的相对标准差优于9%，对3种非SRB人工构建的质粒均没有扩增，显示该技术具有很好的线性关系、敏感性、重复性和特异性。利用该技术对冀中地热区深部热水、浅层水和土壤样品进行了检测，平均含量分别为(4.0±8.4)×103 copies/mL、(1.6±3.5)×102 copies/mL和(1.5±1.2)×103 copies/g-dw。与浅层水和土壤相比，深部热水富含SRB菌。【结论】为了提高地下深部生物圈认识和合理开发利用深部热水，建立了一种快速、灵敏、准确的SRB ddPCR检测技术，同时为其他指示菌检测技术的建立提供了参考。

关键词:硫酸盐还原菌，ddPCR，dsrB，深部热水

Abstract:

[Background] The subsurface harbors a deep biosphere. It has been investigated that microbial communities inhabit deep environments of sedimentary rocks, basalts, granites and metamorphic rocks. However, microbial characteristics of deep carbonate rock karst-fracture geothermal reservoir is still unclear. Sulfate-reducing bacteria (SRB) are frequently detected in the deep subsurface. [Objective] To develop a droplet digital PCR (ddPCR) technique for rapid and accurate quanti?cation of sulfate-reducing bacteria in deep geothermal water. [Methods] Functional gene dsrB of SRB was used as detection target to optimize annealing temperature of SRB ddPCR technique. Its linear range, sensitivity, repeatability and specificity were examined. Field samples were tested using the technique. [Results] The optimized annealing temperature of SRB ddPCR technique was 54 °C. The linear range of the technique was 1.1×100?1.1×105 copies/μL-DNA with the correlation coefficient (R2) of 0.996. The sensitivity was 1 copy/μL-DNA. All of relative standard deviations (RSD) in the repeatability tests were better than 9%. In addition, no amplification was observed in the templates of 3 non-SRB artificial plasmids. Overall, the technique showed good linear relationship, sensitivity, repeatability and specificity. The technique was applied to quantify SRB in deep geothermal water, shallow water and soil samples collected from the geothermal area located in the middle Hebei Province to obtain an average of (4.0±8.4)×103 copies/mL, (1.6±3.5)×102 copies/mL and (1.5±1.2)×103 copies/g-dw, respectively, showing deep geothermal water was rich in SRB compared with shallow water and soil. [Conclusion] The SRB ddPCR technique was developed to rapidly, accurately and sensitively detect SRB in deep geothermal water for improvement of deep biosphere understanding, scientific development and management of deep geothermal water. At the same time, this technique could contribute to detection of other indicator bacteria in deep geothermal water.

Key words:Sulfate-reducing bacteria, ddPCR, dsrB, Deep geothermal water