Quantitative real-time PCR analysis of total and propidium monoazide-resistant fecal indicator bacteria in wastewater

ABSTRACT Real-time PCR was applied to quantify the abundance of human adenoviruses in two southern California urban rivers, the San Gabriel and Los Angeles. A total of 114 river samples from five different locations were collected over a 1-year period and analyzed for human adenoviruses, along with fecal indicator bacteria and coliphages. Adenoviruses were detected by real-time PCR in ∼16% of the samples, with concentrations ranging from 102 to 104 genomes per liter. However, a plaque assay using two human tissue culture cell lines, HEK-293A and A549, yielded negative results, suggesting that adenoviruses detected by real-time PCR are likely noninfectious. Enterovirus genome was detected in ∼7% of the samples by reverse transcription-PCR. Analysis by Spearman's rho rank order correlation showed significant correlations between fecal indicator bacteria and indicator virus (total coliform, fecal coliform, enterococcus, and coliphage values). However, no significant correlations were found between human adenoviruses quantified by real-time PCR and culturable coliphages or fecal indicator bacteria. Kruskal-Wallis chi-square analysis showed significant seasonal variability of all fecal indicator bacteria and coliphages, while no significant variability was observed for adenoviruses or enteroviruses. This study presents the first quantitative measurement of human adenovirus genomes in urban rivers and their statistical relationship to fecal indicator bacteria and coliphages. The uncoupling between high-number genome copies of adenoviruses detected by real-time PCR and the absence of infectivity detected by tissue culture suggests that genome-based detection methods are inadequate for direct assessment of human health risk.

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rpoB and efp are stable candidate reference genes for quantitative real-time PCR analysis in Saccharopolyspora spinosa

Biotechnology & Biotechnological Equipment ◽

10.1080/13102818.2021.1899852 ◽

2021 ◽

Vol 35 (1) ◽

pp. 619-632

Author(s):

Xiaomeng Liu ◽

Yunpeng Zhang ◽

Kexue Huang ◽

Tie Yin ◽

Qi Li ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Saccharopolyspora Spinosa

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Quantification of plasmid DNA standards for U.S. EPA fecal indicator bacteria qPCR methods by droplet digital PCR analysis

Journal of Microbiological Methods ◽

10.1016/j.mimet.2018.07.005 ◽

2018 ◽

Vol 152 ◽

pp. 135-142 ◽

Cited By ~ 5

Author(s):

Mano Sivaganesan ◽

Manju Varma ◽

Shawn Siefring ◽

Richard Haugland

Keyword(s):

Plasmid Dna ◽

Fecal Indicator Bacteria ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Indicator Bacteria ◽

Pcr Analysis ◽

Fecal Indicator

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Application of quantitative real-time PCR compared to filtration methods for the enumeration of Escherichia coli in surface waters within Vietnam

Journal of Water and Health ◽

10.2166/wh.2016.173 ◽

2016 ◽

Vol 15 (1) ◽

pp. 155-162 ◽

Cited By ~ 5

Author(s):

Pierangeli G. Vital ◽

Nguyen Thi Van Ha ◽

Le Thi Hong Tuyet ◽

Kenneth W. Widmer

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Surface Waters ◽

Real Time Pcr ◽

Membrane Filtration ◽

Quantitative Real Time Pcr ◽

Wet Season ◽

Culture Methods ◽

Fecal Indicator ◽

Filtration Method

Surface water samples in Vietnam were collected from the Saigon River, rural and suburban canals, and urban runoff canals in Ho Chi Minh City, Vietnam, and were processed to enumerate Escherichia coli. Quantification was done through membrane filtration and quantitative real-time polymerase chain reaction (PCR). Mean log colony-forming unit (CFU)/100 ml E. coli counts in the dry season for river/suburban canals and urban canals were log 2.8 and 3.7, respectively, using a membrane filtration method, while using Taqman quantitative real-time PCR they were log 2.4 and 2.8 for river/suburban canals and urban canals, respectively. For the wet season, data determined by the membrane filtration method in river/suburban canals and urban canals samples had mean counts of log 3.7 and 4.1, respectively. While mean log CFU/100 ml counts in the wet season using quantitative PCR were log 3 and 2, respectively. Additionally, the urban canal samples were significantly lower than those determined by conventional culture methods for the wet season. These results show that while quantitative real-time PCR can be used to determine levels of fecal indicator bacteria in surface waters, there are some limitations to its application and it may be impacted by sources of runoff based on surveyed samples.

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