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

Abstract

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 × 10 9 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 (R 2 = 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.

Development and Validation of a Harmonized TaqMan-Based Triplex Real-Time RT-PCR Protocol for the Quantitative Detection of Normalized Gene Expression Profiles of Seven Porcine Cytokines

“…Consequently, PCR products are not proportional to the amount of initial target when visualised on a gel [37] . The most widely used SYBR-Green I assays [38] – [42] have the disadvantages of potentially generating primer-dimers, the indiscriminately binding to all double-stranded DNA which might lead to a formation of secondary structures, and to a possibly limiting primer-concentration as well as to an overestimation of target-DNA [43] . To overcome these problems and to add specificity, a fluorogenic probe based approach was chosen in the presented study.…”

A novel fluorescent long-chain fatty acid-substituted dye: labeling and biodegrading ofMicrothrix parvicella

“…The literature shows that excessive proliferation of Microthrix parvicella (M. parvicella) is responsible for 90% of sludge-bulking. [1][2][3][4][5] Therefore, if M. parvicella can be targeted, then methods for early identication and treatment could prevent or solve the issue of sludgebulking. 6 Traditional sludge volume index (SVI) and morphological recognition 7,8 have been used with uorescence in situ hybridization (FISH) to identify M. parvicella, 9,10 but this has many disadvantages, including complex pretreatment, low cell permeability, destructiveness, weak uorescence signal, long turnaround times, complicated processing, narrow application scope, and high expense.…”

On-filter direct amplification of Legionella pneumophila for rapid assessment of its abundance and viability

“…Though not yet in effect, Michigan is also considering implementing new L. pneumophila and Legionella spp testing regulations near hospitals (Associated Press, 2016). At present, culturing on buffered charcoal yeast extract agar (Feeley et al, 1979) and qPCR (Krøjgaard et al, 2011; Mentasti et al, 2015; Vanysacker et al, 2014) are the two main techniques adapted for quantification. Culturing will assess viability but it is slow (4 to 10 days).…”