Quantification of sulfate-reducing bacteria in industrial wastewater, by real-time polymerase chain reaction (PCR) using dsrA and apsA genes

Abstract

Real-time polymerase chain reaction (PCR) is considered a highly sensitive method for the quantification of microbial organisms in environmental samples. This study was conducted to evaluate real-time PCR with SybrGreen detection as a quantification method for sulfate-reducing bacteria (SRB) in industrial wastewater produced by several chemical industries. We designed four sets of primers and developed standard curves based on genomic DNA of Desulfovibrio vulgaris from pure culture and on plasmids containing dissimilatory sulfate reductase (dsrA) or adenosine-5'-phosphosulfate reductase (apsA) genes of SRB. All the standard curves, two for dsrA and two for apsA genes, had a linear range between 0.95 x 10(2) and 9.5 x 10(6) copies/microL and between 1.2 x 10(3) and 1.2 x 10(7) copies/microL, respectively. The theoretical copy numbers of the tenfold dilutions of D. vulgaris genomic DNA were best estimated (between 2.7 to 10.5 times higher than theoretical numbers) by the standard curve with DSR1F and RH3-dsr-R primers. To mimic the effect of foreign DNA in environmental samples, serial dilutions of D. vulgaris genomic DNA were mixed with Escherichia coli chromosomal DNA (40 ng per assay). This influenced neither PCR amplification nor the quantification of target DNA. Industrial wastewater was sampled during a 15-month period and analyzed for the presence of SRB, based on dsrA gene amplification. SRB displayed a higher abundance during the summer (about 10(7)-10(8) targets mL(-1)) and lower during the winter (about 10(4)-10(5) targets mL(-1)). The results indicate that our real-time PCR approach can be used for detection of uncultured SRB and will provide valuable information related to the abundance of SRB in durable environmental samples, such as complex and saline industrial wastewaters.