Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods

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h Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters. N umerous host-associated indicators are used to identify human fecal pollution in ambient surface waters, with many relying on molecular methods such as quantitative real-time PCR (qPCR). The most widely used methods target the HF183 16S rRNA gene cluster of members of the genus Bacteroides initially identified in human fecal samples collected from the United States Pacific Northwest (1) and targeted by PCR to distinguish humanassociated contamination (2). Over the past decade, researchers have developed (2-9) and implemented (10-15) many PCR-based methods targeting the HF183 cluster worldwide.In most HF183 PCR applications, the forward primer targets the Bacteroides HF183 cluster, reported to include Bacteroides dorei (3). The reverse primer typically hybridizes to a wider diversity of Bacteroidales so as not to further restrict the range of targeted bacteria. Researchers have employed this strategy for endpoint PCR applications (2), as well as a variety of quantitative real-time PCR chemistries, such as the SYBR green (8, 16) and TaqMan (4, 5, 9, 17) chemistries.In recent performance evaluation studies, PCR-based methods targeting the HF183 cluster, in particular, the TaqMan HF183/ BFDrev assay (3), consistently outperformed other tested approaches in terms of specificity and sensitivity (18-21). TaqMan chemistry utilizes an internal oligonucleotide probe, greatly reducing the chance of false-positive results due to the accumulation of unintended amplification by-products (e.g., primer dimerization [PD] molecules). The TaqMan HF183/BFDrev qPCR assay was recently tested in a five-laboratory repeatability study and shown to be highly reproducible across laboratories when key components of the protocol were standardized (22). However, the ...

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

Green

Haugland

Varma

et al. 2014

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215

“…A useful human-associated qPCR method should exhibit comparable performance when tested from one laboratory to the next. It is well documented that uniformity in qPCR performance is accomplished through the standardization of protocols, good laboratory practice, and the implementation of data acceptance criteria (36)(37)(38)(39). Data acceptance parameters such as E, calculated from the calibration curve slope, calibration curve R 2 , LLOQ, and extraneous DNA controls, as well as evidence for the absence of amplification inhibition, among others, were measured across 14 laboratories employing a standardized procedure.…”

Section: Discussionmentioning

confidence: 99%

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

Shanks

Kelty

Oshiro

et al. 2016

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There is growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data quality across laboratories. Data quality is typically determined through a series of specifications that ensure good experimental practice and the absence of bias in the results due to DNA isolation and amplification interferences. However, there is currently a lack of consensus on how best to evaluate and interpret human fecal source identification qPCR experiments. This is, in part, due to the lack of standardized protocols and information on interlaboratory variability under conditions for data acceptance. The aim of this study is to provide users and reviewers with a complete series of conditions for data acceptance derived from a multiple laboratory data set using standardized procedures. To establish these benchmarks, data from HF183/BacR287 and HumM2 human-associated qPCR methods were generated across 14 laboratories. Each laboratory followed a standardized protocol utilizing the same lot of reference DNA materials, DNA isolation kits, amplification reagents, and test samples to generate comparable data. After removal of outliers, a nested analysis of variance (ANOVA) was used to establish proficiency metrics that include lab-to-lab, replicate testing within a lab, and random error for amplification inhibition and sample processing controls. Other data acceptance measurements included extraneous DNA contamination assessments (notemplate and extraction blank controls) and calibration model performance (correlation coefficient, amplification efficiency, and lower limit of quantification). To demonstrate the implementation of the proposed standardized protocols and data acceptance criteria, comparable data from two additional laboratories were reviewed. The data acceptance criteria proposed in this study should help scientists, managers, reviewers, and the public evaluate the technical quality of future findings against an established benchmark. Fecal pollution remains a significant challenge for ambient water quality managers worldwide. In the United States alone, it is estimated that 39.2% of all rivers, lakes, and streams are unsafe for recreational use, with fecal pathogens as the number one cause of impairment (1). General fecal indicator bacteria (FIB), such as Escherichia coli and enterococci, shed by nearly all warm-blooded animals, are routinely used to assess water quality. However, general FIB cannot discriminate between different animal groups, making it challenging to pinpoint the origin of fecal pollution. Researchers and managers alike recognize the advantages of animal source information to help solve long-standing ambient water quality problems. As a result, a number of fecal source identification technologies have been developed (2-9). These methods have been employed to address challenges such as the identification ...

“…Many of these MST methods are based on quantitative PCR (qPCR) assays targeting the bacterial rRNA genes present within water DNA extracts (12,13). However, the value of DNA-based monitoring in microbial ecology studies is limited by the possibility of DNA being associated with dead cells or the extent to which "naked DNA" may survive in the environment once bacteria are lysed (14)(15)(16).…”

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confidence: 99%

Distribution of Human-Specific Bacteroidales and Fecal Indicator Bacteria in an Urban Watershed Impacted by Sewage Pollution, Determined Using RNA- and DNA-Based Quantitative PCR Assays

Kapoor

Pitkänen

Ryu

et al. 2015

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The identification of fecal pollution sources is commonly carried out using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as "naked DNA" in the environment. Furthermore, it has been shown that rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) assays can be more sensitive than rRNA gene-based qPCR assays since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells. To this end, we compared the detection frequency of host-specific markers and fecal bacteria using RNA-based RT-qPCR and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. As a group, fecal bacteria were more frequently detected in most sites using RNA-based methods. Specifically, 8, 87, and 85% of the samples positive for general enterococci, Enterococcus faecalis, and Enterococcus faecium markers, respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. On average, two human-specific Bacteroidales markers were not detected when using DNA in 12% of the samples, while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays than in qPCR assays. The human-specific Bacteroidales markers exhibited moderate correlation with conventional fecal indicators using RT-qPCR results, suggesting the persistence of nonhuman sources of fecal pollution or the presence of false-positive signals. In general, the results from this study suggest that RNA-based assays can increase the detection sensitivity of fecal bacteria in urban watersheds impacted with human fecal sources. Sewage overflows and stormwater runoff introduce high levels of fecal bacteria into surface waters and are considered the primary cause of water quality impairments in urban watersheds, particularly those affected by combined sewer overflows (CSOs) (1, 2). Sewage contamination of surface waters poses a serious risk to human and environmental health via waterborne disease outbreaks (3-5), deterioration of recreational and drinking water quality (6, 7), and degradation of aquatic ecology (8, 9). Hence, identifying the primary source(s) of fecal contamination is imperative to enable best management practices for mitigating pollution and public health risks.Microbial source tracking (MST) methods targeting fecal bacteria have recently been used to identify the sources of fecal contamination impacting water systems (10, 11). Many of these MST methods are based on quantitative PCR (qPCR) assays targeting the bacterial rRNA genes present within water DNA extracts (12, 13). However, the value of DNA-based monitoring in microbial ecology studies is limited by the possibility of DNA being associated with dead cells or the extent to which "naked DNA" may survive in the environment once bacteria are lysed (14-16). These facts pose a significant challenge to the environmental fate and transport of fecal bacteria which are often assess...