TY - JOUR
T1 - Effect of Quantitative Polymerase Chain Reaction Data Analysis Using Sample Amplification Efficiency on Microbial Source Tracking Assay Performance and Source Attribution
AU - Kongprajug, Akechai
AU - Chyerochana, Natcha
AU - Mongkolsuk, Skorn
AU - Sirikanchana, Kwanrawee
N1 - Funding Information:
This research was financially supported by the Thailand Research Fund under contract no. SRI5930305 and the Chulabhorn Research Institute. Miss Chuthamas Phongphila is thankfully acknowledged for her assistance in laboratory preparation.
Publisher Copyright:
Copyright © 2020 American Chemical Society.
PY - 2020/7/7
Y1 - 2020/7/7
N2 - 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 log10 copies/g and 0.41 log10 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 log10 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.
AB - 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 log10 copies/g and 0.41 log10 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 log10 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.
UR - http://www.scopus.com/inward/record.url?scp=85088206631&partnerID=8YFLogxK
U2 - 10.1021/acs.est.0c01559
DO - 10.1021/acs.est.0c01559
M3 - Article
C2 - 32484662
AN - SCOPUS:85088206631
SN - 0013-936X
VL - 54
SP - 8232
EP - 8244
JO - Environmental Science and Technology
JF - Environmental Science and Technology
IS - 13
ER -