Biomonitoring of Dehalococcoides mccartyi: Comparative Study Between CARD-FISH and Real-Time PCR

摘要

Background/Objectives. Dehalococcoides (Dhc) is the biomarker of chlorinated solvents contamination and its detectability during natural attenuation and/or engineered bioremediation processes by means of standardized molecular biological tools (MBT), is increasingly required. PCR-based methods (qPCR, RT-qPCR) are the method of choice for Dhc quantification and activity levels estimation. Although specificity and sensitivity have been improved, qPCR limitations still remain and additional quantitative MBTs may greatly assist the accurate quantification of Dhc by providing consensus enumeration and establishing uncertainty values. In situ hybridization techniques may represent a valid improvement of qPCR quantification of Dhc: they are applied directly to the aquifer samples overcoming the limitations of the nucleic acid extraction and allowing for the rapid and exact estimation of cell abundance. In this study, CAtalysed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) and qPCR were directly compared, in order to evaluate the advantages and drawbacks related to Dhc quantification in environmental samples over a wide concentration range. We have also exploited the application potential of FISH analysis to quantify rapidly and efficiently metabolically active Dhc cells by comparing data generated from FISH analysis to those generated from PCR-based techniques directed at assessing cell activities (Reverse Transcription qPCR (RT-qPCR).