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Advancements in mitigating interference in quantitative polymerase chain reaction (qPCR) for microbial water quality monitoring.

ABSTRACT: The United States Environmental Protection Agency's (EPA)1 2012 Recreational Water Quality Criteria included an Enterococcus spp. quantitative polymerase chain reaction (qPCR) method as a supplemental indicator-method. In 2012, performance of qPCR for beach monitoring remained limited, specifically with addressing interference. A systematic literature search of peer-reviewed publications was conducted to identify where Enterococcus spp. and E. coli qPCR methods have been applied in ambient waters. In the present study, we evaluated interference rates, contributing factors resulting in increased interference in these methods, and method improvements that reduced interference. Information on qPCR methods of interest and interference controls were reported in 16 papers for Enterococcus spp. and 13 papers for E. coli. Of the Enterococcus spp. qPCR methods assessed in this effort, the lowest frequencies of interference were reported in samples using Method 1609. Low frequencies of sample interference were also reported EPA's modified E. coli qPCR method, which incorporates the same reagents and interference controls as Method 1609. The literature indicates that more work is needed to demonstrate the utility of E. coli qPCR for widespread beach monitoring purposes, whereas more broad use of Method 1609 for Enterococcus spp. is appropriate when the required and suggested controls are employed.

SUBMITTER: Nappier SP

PROVIDER: S-EPMC6555561 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Project description:Background:Culture-independent methods such as quantitative polymerase chain reaction (qPCR) are more sensitive for detecting pathogens than conventional culture. This study aimed to test the clinical potential of a multiple target qPCR array in identifying sputum pathogens, compared to traditional culture. Methods:Forty chronic obstructive pulmonary disease (COPD) patients provided spontaneous sputum and blood samples during an exacerbation event (n=25 patients) and in stable state (n=15 patients). Sputum was processed and analysed by microscopy, culture and sensitivity testing (MCS) to identify living microbial isolates, and multiple target qPCR (44 targets for bacterial and fungal pathogens and antibiotic resistance genes), and 16S rRNA gene sequencing. Results:Six microbial isolates (5 bacterial, 1 fungal) were cultured from 20 exacerbation and 10 stable patient sputum samples. Four of these microbial isolates had their presence in patient sputum confirmed by qPCR. All bacterial targets detected by qPCR were further confirmed by 16S rRNA gene sequencing at a genus level. qPCR identified significantly more bacterial pathogens than culture (P<0.001). The most prevalent bacterial species identified by qPCR were Streptococcus pneumoniae (72% of patients), Pseudomonas aeruginosa (40%), Prevotella oris (32%) and Haemophilus influenzae (17%). Microbial species diversity and richness were not significantly different between samples obtained from exacerbating and clinically stable cases. 16S rRNA gene sequencing identified Pseudomonas 4408227 (P=0.022, FDR =0.043 AUC =0.72) as a significantly different bacterial OTU (operational taxonomic units) in exacerbation sputum samples compared to stable state samples. Conclusions:Multiple target qPCR was more sensitive for detection of sputum pathogens in COPD patients than conventional culture. 16S rRNA gene sequencing confirmed the identity at a genus level of all bacterial targets detected by qPCR, as well as identifying bacterial OTUs that could potentially be used to distinguish between exacerbation and stable COPD disease states. Multiple target qPCR pathogen detection in the sputum of COPD patients warrants further investigation to determine how it may influence COPD clinical management.

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Advancements in mitigating interference in quantitative polymerase chain reaction (qPCR) for microbial water quality monitoring.

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