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

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.