Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix

• Published in: Journal of microbiological methods Vol. 97; pp. 6 - 14
• Main Authors: Vanysacker, Louise, Denis, Carla, Roels, Joris, Verhaeghe, Kirke, Vankelecom, Ivo F.J.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2014
• Subjects: Actinobacteria - genetics; Actinobacteria - physiology; Bacterial Load - methods; Microthrix calida; Microthrix parvicella; Real-time PCR; Real-Time Polymerase Chain Reaction - standards; Sensitivity and Specificity; Sewage - microbiology; Sludge bulking; TaqMan; Water Purification - methods; Microthrix parvicella; Sludge bulking; Microthrix calida; Real-time PCR; TaqMan
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2013.11.016

Candidatus Microtrhix parvicella is one of the most common filamentous bacteria reported to be involved in bulking and foaming problems in activated sludge plants worldwide. In order to detect and quantify both M. parvicella and Microthrix calida by quantitative PCR (qPCR), primers targeting 16S rDNA genes were designed. The qPCR reaction was optimized by using the TaqMan technology and an internal positive control was included to ensure the absence of PCR inhibitors. A total of 29 samples originating from different wastewater treatment plants were analyzed and the results were compared by using conventional microscopy, fluorescent in situ hybridization and an existing SYBR Green-based assay. Our assay showed a 100% specificity for both M. parvicella and M. calida, a sensitivity of 2.93×109 to 29 copy numbers/reaction, an amplification efficiency of 93% and no PCR inhibition. By performing a spiking experiment including different Microthrix concentrations, recovery rates ranging from 65 to 98% were obtained. A positive correlation with the SYBR Green assay (R2=0.85) was found and most of the samples were in accordance with the microscopical observation. In comparison with SYBR Green assay, the probe-based TaqMan assay had a much lower detection limit. Compared with microscopy, some samples had a lower or higher enumeration when using qPCR. In conclusion, a qPCR method is forwarded here that could be useful as an early warning tool for fast and reliable detection of Microthrix in for instance sludge bulking events. •A qPCR was designed in order to detect and quantify both Microthrix parvicella and M. calida, two species frequently involved in sludge bulking.•The qPCR is based on the TaqMan chemistry and contained an internal positive control.•The TaqMan assay had a low detection limit.•The quantified samples were in accordance with microscopical enumeration.•The method can be useful as an early warning tool for in sludge bulking events.