Abstract
Abstract Waterborne pathogens are the primary concern for the safe reuse of wastewater. Although digital PCR (dPCR) is considered promising for absolutely quantitating genes, the detection efficiency of dPCR is affected by many factors. This study tested eight virulence genes of pathogenic bacteria on a control plasmid and reclaimed water samples with reported primer–probe sets and designed ones on quantitative PCR (qPCR) and dPCR. Probe efficiency, data analysis, and PCR inhibition were found to affect the detection efficiency of dPCR. Firstly, poor probe quality, which is determined by probe quenching and activation efficiencies, was the main cause of PCR failure. Secondly, even if the PCR was successful, the probe quality and signal intensity could still affect the quantitative process. Manual analysis of dPCR data on the weak signal intensity would significantly reduce errors. And lastly, the sensitivity of PCR inhibition was lower in dPCR than qPCR, but inhibition still existed. The dPCR produced various detection efficiencies for different targets in one sample indicating inconstant inhibitory effects. Dilution was still the proper approach to overcome inhibition, but decreased the detection limit. More studies are required to ensure accurate waterborne pathogen quantitation by dPCR.
Highlights
The intense pressure on water resources globally has led to efforts in applying reclaimed water for many purposes
This study investigated the influence of PCR reaction conditions, analytical methods, and PCR inhibitions on the detection efficiency of absolute quantification of waterborne pathogenic bacteria by detecting the eight virulence genes in digital PCR (dPCR), and provided corresponding solutions
All minor groove binder (MGB) sets and a commercial GAPDH control were tested on a dPCR instrument, since MGB sets had stronger signal and smaller Ct values
Summary
The intense pressure on water resources globally has led to efforts in applying reclaimed water for many purposes. Reclaimed water is perceived as risky due to the occurrence of waterborne pathogens. Waterborne pathogens can be classified as bacteria, molds, viruses, and parasites. They can cause cellular damage and lead to clinical outcomes of morbidity or mortality. According to the statistics of the United States Centers for Disease Control and Prevention, public health officials of 46 states reported approximately 493 outbreaks associated with recreational water, and among 363 outbreaks, 57 (16%) caused by Legionella spp., and 47 (13%) by Pseudomonas spp. during the years 2000–2014. Six out of eight reported deaths were caused by Legionella spp. It seems that among the pathogens, bacteria are the common etiological agents for illness
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