Effect of Quantitative Polymerase Chain Reaction Data Analysis Using Sample Amplification Efficiency on Microbial Source Tracking Assay Performance and Source Attribution

Abstract

The widely used microbial source tracking (MST) technique, quantitative polymerase chain reaction (qPCR), quantifies host-specific gene abundance in polluted water to identify and prioritize contamination sources. This study characterized the effects of a qPCR data analysis using the sample PCR efficiencies (the LinRegPCR model) on gene abundance and compared them with the standard curve-based method (the mixed model). Five qPCR assays were evaluated: the universal GenBac3, human-specific HF183/BFDrev and CPQ_056, swine-specific Pig-2-Bac, and cattle-specific Bac3qPCR assays. The LinRegPCR model increased the low-copy amplification, especially in the HF183/BFDrev assay, thus lowering the specificity to 0.34. Up to 1.41 log 10 copies/g and 0.41 log 10 copies/100 mL differences were observed for composite fecal and sewage samples (n = 147) by the LinRegPCR approach, corresponding to an 18.2% increase and 6.4% decrease, respectively. Freshwater samples (n = 48) demonstrated a maximum of 1.95 log 10 copies/100 mL difference between the two models. Identical attributing sources by both models were shown in 54.55% of environmental samples; meanwhile, the LinRegPCR approach improved the inability to identify sources by the mixed model in 29.55% of the samples. This study emphasizes the need for a standardized data analysis protocol for qPCR MST assays for interlaboratory consistency and comparability.

Human Fecal Pollution Monitoring and Microbial Risk Assessment for Water Reuse Potential in a Coastal Industrial–Residential Mixed-Use Watershed

“…The MST marker abundance was investigated in surface water near the mixed land use of industrial and residential areas. Fecal contamination was indicated by GenBac3 level in this study (four to eight orders of magnitude in log 10 copies/100 ml), which were slightly lower than those found in untreated sewage in Thailand (five to nine orders in log 10 copies/100 ml) (Kongprajug et al, 2020), but slightly higher than the Tha Chin River in Central Thailand (three to seven orders in log 10 copies/100 ml) (Kongprajug et al, 2019a). These GenBac3 levels were aligned with levels found in other geographical regions in urban rivers (five to seven orders), urban recreational beaches (two to five orders), and karst springs (four to seven orders) (Molina et al, 2014;Ohad et al, 2015;Devane et al, 2019).…”

“…A general fecal marker, GenBac3 (Siefring et al, 2008), two human-specific fecal markers, human polyomavirus BK and JC (HPyVs) (McQuaig et al, 2009) and crAssphage (Stachler et al, 2017), and adenovirus types 40/41 (HAdV40/41) (Ko et al, 2005) were used in this study (Supplementary Table 2). The abovementioned MST markers were selected because they have been investigated and validated in Thailand (Kongprajug et al, 2019a(Kongprajug et al, , 2020Sangkaew et al, 2021), and HAdV40/41 was chosen because it has been identified as the second most prevalent following the subgroup C from child patients with acute gastroenteritis in Thailand (Kumthip et al, 2019). A 20-µl qPCR reaction contained 0.8 µl of each 10 µM forward and reverse primers, 0.4 µl of 10 µM probe, 2 µl of extracted DNA, 6 µl of 1 µg/µl BSA, and 10 µl of the 2X iTaq Universal Probes Supermix (Bio-Rad, United States).…”

“…The qPCR protocol was conducted according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al, 2009). For each instrumental run containing environmental samples, the DNA standard, at a concentration of 5 × 10 4 -5 × 10 5 copies/reaction, was run in triplicate as a calibration control according to a mixed model (Sivaganesan et al, 2010;Kongprajug et al, 2020). No-template controls (NTCs) in triplicate were also included in every qPCR instrumental run.…”

“…The assay limit of detection (ALOD) was the lowest concentration in copies/reactions that showed positive detection in all 10 standard replicates. The assay limit of quantification (ALOQ) was considered to be the lowest concentration in copies/reactions of the target gene that could be correctly quantified-in this case, the lowest concentration in the standard curve with a standard deviation of C q of less than 0.5 (Haugland et al, 2010;Kongprajug et al, 2020). The method limit of quantification (MLOQ) was calculated for each sample as copies/100 ml by incorporating the sample's filtration volume and DNA extracted volume.…”

Effects of face masks and ventilation on the risk of SARS-CoV-2 respiratory transmission in public toilets: a quantitative microbial risk assessment

“…However, heterogeneity in published standard curves for SARS-CoV-2 has been observed (Bivins, Kaya, et al, 2021). Moreover, variations in technical and laboratory analyses (e.g., data analysis methods and control materials) could intensify biases, leading to variability in the calculated virus concentrations (Bivins, Kaya, et al, 2021; Kongprajug, Chyerochana, et al, 2020). Adhering to standards and quality control measures is therefore underlined in order to support data sharing and referencing for future research, especially for emerging infectious diseases.…”