Application of quantitative real-time PCR for quantification of Rhodococcus sp. EH831 in a polyurethane biofilter
Lee EH, Cho KS, Ryu HW
Department of Environmental Science and Engineering, Ewha Womans University, Seoul, Republic of Korea.
Rhodococcus sp. EH831 is a microbial species that can degrade volatile organic compounds. We optimized a method for monitoring quantitative real-time PCR (qRT-PCR) of EH831 that was incorporated into a polyurethane (PU) biofilter. When the genomic DNA of EH831 was directly extracted from a PU sample with immobilized EH831, the recovery efficiency was very low due to DNA absorption into the PU. DNA amplification during PCR was also inhibited by PU impurities. Therefore, a pre-treatment step was necessary. We successfully recovered cells from the PU by squeezing the matrix, adding sterilized water, and vortexing. The recovery efficiency ranged from 105 to 144%, and there was no statistically significant difference. We designed a novel TaqMan probe for EH831 and demonstrated its high specificity for EH831. The detection range for EH831 was 10(5)-10(11) CFU ml(-1). The method described in this study can be used to investigate the relationship between quantitative analysis of Rhodococcus sp. EH831 and PU biofilter performance.
Keywords:Rhodococcus sp. EH831 is a microbial species,monitoring quantitative real-time PCR,squeezing the matrix, adding sterilized water, and vortexing.