Assessment and modification of degenerate qPCR primers that amplify functional genes from etheneotrophs and vinyl chloride-assimilators


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Abstract

Aims:Degenerate qPCR primer sets that target the functional genes etnC and etnE in etheneotrophs and vinyl chloride-assimilating bacteria were assessed and modified in an effort to improve performance.Methods and Results:Functional gene abundance in four pure cultures was estimated by qPCR using novel (MRTC and MRTE) and existing (RTC and RTE) degenerate primer sets and compared to abundances estimated with nondegenerate gene-specific primers (GSPs). Functional gene abundance in groundwater DNA extracted from several contaminated sites was also estimated with MRTC and MRTE primers.Conclusions:MRTC primers displayed significantly improved etnC quantification in both pure cultures and environmental samples.Significance and Impact of the Study:Application of MRTC and MRTE primer sets will enhance microbial ecology studies involving etheneotrophs and qPCR analyses that support vinyl chloride bioremediation strategies.

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