Danko Anthony S, Fontenete Silvia J, de Aquino Leite Daniel, Leitão Patrícia O, Almeida Carina, Schaefer Charles E, Vainberg Simon, Steffan Robert J, Azevedo Nuno F
Centro de Investigação em Geo-Ambiente e Recursos (CIGAR), Departamento de Engenharia de Minas, Faculdade de Engenharia, Porto, Portugal.
J Mol Microbiol Biotechnol. 2014;24(3):142-9. doi: 10.1159/000362790. Epub 2014 Jun 26.
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.
包括四氯乙烯（全氯乙烯和三氯乙烯）在内的氯化溶剂是广泛使用的工业溶剂。这些化学品的不当使用和处置导致了广泛的污染。利用脱卤球菌属的厌氧处理技术可能是修复这些受污染场地的有效工具。因此，本研究的目的是开发、优化和验证用于检测纯培养物和混合培养物中脱卤球菌属的肽核酸（PNA）探针。通过改编先前发表的针对所述点突变区域的DNA探针来设计PNA探针，以区分脱卤球菌属菌株CBDB1和菌株195谱系。测试了不同的固定、杂交和洗涤程序。结果表明，PNA探针与其相应谱系特异性杂交且灵敏度高，并且本研究中开发的PNA探针可用于双重分析，以区分菌株CBDB1和菌株195谱系，即使在复杂的混合培养物中也是如此。这项工作证明了使用PNA荧光原位杂交区分两种代谢和遗传上不同的脱卤球菌菌株的有效性，它们对实验室培养物和受污染场地中脱卤球菌种群的监测和区分可能具有重要意义。
