Supplementary Material for: Detection of Dehalococcoides spp. by Peptide Nucleic Acid Fluorescent in situ Hybridization

Chlorinated solvents including tetrachloroethene (perchloroethene and trichloroethene), are widely used industrial solvents. Improper use and disposal of these chemicals has led to a widespread contamination. Anaerobic treatment technologies that utilize Dehalococcoides spp. can be an effective tool to remediate these contaminated sites. Therefore, the aim of this study was to develop, optimize and validate peptide nucleic acid (PNA) probes for the detection of Dehalococcoides spp. in both pure and mixed cultures. PNA probes were designed by adapting previously published DNA probes targeting the region of the point mutations described for discriminating between the Dehalococcoides spp. strain CBDB1 and strain 195 lineages. Different fixation, hybridization and washing procedures were tested. The results indicated that the PNA probes hybridized specifically and with a high sensitivity to their corresponding lineages, and that the PNA probes developed during this work can be used in a duplex assay to distinguish between strain CBDB1 and strain 195 lineages, even in complex mixed cultures. This work demonstrates the effectiveness of using PNA fluorescence in situ hybridization to distinguish between two metabolically and genetically distinct Dehalococcoides strains, and they can have strong implications in the monitoring and differentiation of Dehalococcoides populations in laboratory cultures and at contaminated sites.氯化溶剂包括四氯乙烯（又称全氯乙烯，perchloroethene）与三氯乙烯（trichloroethene），是一类广泛应用的工业溶剂。此类化学品的不当使用与处置已造成大范围环境污染。以脱卤球菌属（Dehalococcoides spp.）为核心的厌氧处理技术，可作为修复此类污染场地的有效手段。因此，本研究旨在开发、优化并验证肽核酸（PNA，peptide nucleic acid）探针，以实现纯培养与混合培养体系中脱卤球菌属的检测。本研究基于已发表的DNA探针，针对用于区分脱卤球菌属CBDB1菌株与195菌株谱系的点突变区域进行改造，以此设计PNA探针，并对多种固定、杂交及洗涤流程进行了测试。实验结果表明，本研究开发的PNA探针可与对应谱系特异性结合且具有较高灵敏度；即便在复杂混合培养体系中，该探针也可通过双重杂交实验区分CBDB1菌株与195菌株谱系。本研究证实了肽核酸荧光原位杂交（PNA fluorescence in situ hybridization）技术可有效区分两种代谢与遗传特征均存在差异的脱卤球菌菌株，该技术有望在实验室培养体系及污染场地中脱卤球菌种群的监测与分型工作中发挥重要应用价值。