The abundance of total 16S rRNA genes, putative dechlorinating Chloroflexi 16S rRNA genes, Dehalococcoides-like 16S rRNA genes, reductive dehalogenase (rdhA) gene RD14 from D. mccartyi strain CG5 and biphenyl dioxygenase gene bphA were estimated with an ABI QuantStudio Flex 7 Real-Time PCR System (Applied Biosystems, Grand Island, NY) in 384 well plate format. The primer sets used for qPCR were 16SUf/16SUr (total 16S rRNA genes), chl3487f/dehal884r (putative dechlorinating Chloroflexi 16S rRNA genes), dhc793f/dhc946r (Dehalococcoides 16S rRNA genes), CG5-14F/CG5-14R (D. mccartyi strain CG5 rdhA gene), and bph463f/674r (bphA genes). Each 20 μL qPCR contained 10 μL Power SYBR Green PCR Master Mix (Invitrogen, Carlsbad, CA), 0.5 μg bovine serum albumin (to alleviate the effects of PCR inhibitors), qPCR primers (concentration range 0.1 – 1 µM) and either standard curve templates, sample DNA templates (mass range 1 – 10 ng), or no template in the case of NTCs.
Plates for qPCR (384 wells) were loaded with 20 μL reaction mixtures and placed into the qPCR system for thermocycling. The qPCR thermocycling conditions were as follows: 10 min at 95 °C, followed by 40 cycles of 95 °C (15 s), and 60 °C (1 min), concluding with a PCR product melt-curve procedure, where the temperature was raised from 65 °C to 95 °C in 0.25 °C increments. ABI QuantStudio Real-Time PCR Software (Applied Biosystems, Grand Island, NY) was used to obtain amplification data for standards, samples and NTCs, analyse qPCR standard calibration curve parameters, determine gene copy numbers in unknown samples by analysis of amplification data, and perform qPCR quality assurance and quality control procedures (QA/QC) by analysis of NTC amplification data and melt curve data.