Supplementary Material for: Detection of <b><i>Dehalococcoides</i></b> 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 <i>Dehalococcoides</i> 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 <i>Dehalococcoides</i> 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 <i>Dehalococcoides</i> 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 <i>Dehalococcoides</i> strains, and they can have strong implications in the monitoring and differentiation of <i>Dehalococcoides</i> populations in laboratory cultures and at contaminated sites.

氯化溶剂涵盖四氯乙烯（全氯乙烯、三氯乙烯），是一类应用广泛的工业溶剂。此类化学品使用与处置不当已造成大范围环境污染。利用脱卤球菌属（*Dehalococcoides*）多个物种的厌氧处理技术，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（peptide nucleic acid, PNA）探针，以实现纯培养与混合培养体系中脱卤球菌属（*Dehalococcoides*）的检测。本研究通过改造已发表的DNA探针设计PNA探针，这些探针靶向用于区分脱卤球菌属菌株CBDB1与195谱系的点突变区域。研究对多种固定、杂交及洗涤流程进行了测试。实验结果表明，本研究所开发的PNA探针可与对应谱系特异性结合，且具备优异的灵敏度；即便在复杂混合培养体系中，该探针也可通过双重杂交检测体系区分菌株CBDB1与195谱系。本研究证实了利用PNA荧光原位杂交（fluorescence in situ hybridization, FISH）技术区分两种代谢与遗传特征均存在差异的脱卤球菌菌株的可行性，该成果可为实验室培养体系及污染场地中脱卤球菌种群的监测与区分提供重要参考依据。