Endpoint Recombinase Polymerase Amplification (RPA) Assay for Enumeration of Thiocyanate-degrading Bacteria

Abstract

An endpoint recombination amplification reaction (RPA) assay for assessing the abundance of the gene encoding thiocyanate dehydrogenase (TcDH) in Thiohalobacter has been developed. The RPA reaction was performed at 37°C for 30‍ ‍min, terminated by the addition of sodium dodecyl sulfate (SDS) solution, and the DNA concentration of the RPA product was fluorometrically measured. The abundance of TcDH in 22 activated sludge samples and 7 thiocyanate-degrading enrichment cultures ranged between 2.5×10³ and 1.5×10⁶ copies μL^-1, showing a linear relationship (R²=0.83) with those measured using a conventional quantitative PCR assay.

Keywords: endpoint assay; gene quantification; recombination amplification reaction (RPA); thiocyanate dehydrogenase.

Fig. 1.

Influence of the incubation period on the quantitative detection of the thiocyanate dehydrogenase (TcDH) gene by the endpoint recombinase polymerase amplification (RPA) assay. The initial copy number of TcDH was in the range of 2.6×10¹ to 10⁷ copies μL^–1, and the RPA reaction was performed at 37°C. After an incubation for 10, 20, and 30‍ ‍min, the RPA product was subjected to the fluorometric assessment of DNA concentrations. DNA concentrations are shown as relative fluorescence units (RFU).

Fig. 2.

Correlation between the copy number of the thiocyanate dehydrogenase (TcDH) gene in activated sludge and enrichment cultures assessed by endpoint recombinase polymerase amplification (RPA) and quantitative PCR (qPCR) assays. Genomic DNA from 22 activated sludge samples and 7 SCN^–-degrading enrichment cultures was subjected to assays, and a linear correlation (R²=0.832) was observed between the copy numbers of TcDH assessed by endpoint RPA and qPCR assays.