Effectiveness of qPCR permutations, internal controls and dilution as means for minimizing the impact of inhibition while measuring Enterococcus in environmental waters

Cao, Yiping; Griffith, John F.; Dorevitch, Samuel; Weisberg, Stephen B.

doi:10.1111/j.1365-2672.2012.05305.x

Cited by 120 publications

(123 citation statements)

References 21 publications

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“…Salmon sperm DNA has been used as a dual control for recovery through sample processing and inhibition (54). Cao et al (9) compared the efficacy of four internal controls to that of sample dilution and concluded the following: (i) sample dilution provided the most reliable and readily interpretable evidence for inhibition, (ii) PCR chemistry, including the specific polymerase master mix used, had a major influence on the frequency of inhibited samples, and (iii) the choice of method to assess inhibition is not yet agreed upon and may vary by site or study objective.…”

Section: Discussionmentioning

confidence: 99%

Performance of Two Quantitative PCR Methods for Microbial Source Tracking of Human Sewage and Implications for Microbial Risk Assessment in Recreational Waters

Staley

Gordon

Schoen

et al. 2012

Appl Environ Microbiol

141

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Before new, rapid quantitative PCR (qPCR) methods for assessment of recreational water quality and microbial source tracking (MST) can be useful in a regulatory context, an understanding of the ability of the method to detect a DNA target (marker) when the contaminant source has been diluted in environmental waters is needed. This study determined the limits of detection and quantification of the human-associated Bacteroides sp. (HF183) and human polyomavirus (HPyV) qPCR methods for sewage diluted in buffer and in five ambient, Florida water types (estuarine, marine, tannic, lake, and river). HF183 was quantifiable in sewage diluted up to 10 ؊6 in 500-ml ambient-water samples, but HPyVs were not quantifiable in dilutions of >10 ؊4 . Specificity, which was assessed using fecal composites from dogs, birds, and cattle, was 100% for HPyVs and 81% for HF183. Quantitative microbial risk assessment (QMRA) estimated the possible norovirus levels in sewage and the human health risk at various sewage dilutions. When juxtaposed with the MST marker detection limits, the QMRA analysis revealed that HF183 was detectable when the modeled risk of gastrointestinal (GI) illness was at or below the benchmark of 10 illnesses per 1,000 exposures, but the HPyV method was generally not sensitive enough to detect potential health risks at the 0.01 threshold for frequency of illness. The tradeoff between sensitivity and specificity in the MST methods indicates that HF183 data should be interpreted judiciously, preferably in conjunction with a more host-specific marker, and that better methods of concentrating HPyVs from environmental waters are needed if this method is to be useful in a watershed management or monitoring context. Fecal indicator bacteria (FIB), including fecal coliforms, Escherichia coli, and enterococci, have been approved indicators of sewage contamination in recreational waters for decades (49, 51). Swimmers exposed to waters contaminated with sewage are at a greater risk of infection by human pathogens, and subsequent gastrointestinal or other illnesses, than those exposed to unimpacted waters (57). The use of FIB to detect sewage contamination, however, relies on the assumptions that elevated FIB concentrations are representative of pathogen presence and that the fate of these bacteria mimics that of pathogens. Unfortunately, previous studies have shown that concentrations of FIB do not necessarily correlate well with bacterial, protozoan, and viral pathogens (2,20).To address the limitations of FIB, microbial source tracking (MST) methods have been developed to identify human fecal contamination (e.g., sewage) in recreational waters (7, 32). These methods are culture independent and are therefore more rapid than FIB methodologies that require a culture step. The potential for same-day results provides the possibility of advisories that reflect current conditions, rather than day-old ones, which allows more-immediate action to protect against public health risks posed by recreational water use (12, 56). The selecti...

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Section: Discussionmentioning

confidence: 99%

Performance of Two Quantitative PCR Methods for Microbial Source Tracking of Human Sewage and Implications for Microbial Risk Assessment in Recreational Waters

Staley

Gordon

Schoen

et al. 2012

Appl Environ Microbiol

141

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“…Samples were considered inhibited if the ΔC q between 5-fold dilutions was less than 1.3, which is one cycle less than expected amplification assuming 100% efficiency, as recommended by Cao and others. 19 Statistical analysis. Stata version 13.1 (StataCorp LP, College Station, TX) was used for all data analysis with the primary goal of determining if child soil ingestion was associated child diarrhea in the past 7 days.…”

Section: Methodsmentioning

confidence: 99%

“…19 For samples classified as quantifiable (C q < 32), 5-fold dilutions were completed using each sample's DNA extract. For samples that were classified as DNQ or ND, a subset of these samples was evaluated for inhibition by spiking 3 × 10 3 copies/μL into the DNA extract before completing 5-fold dilutions to measure the linearity of response.…”

Section: Methodsmentioning

confidence: 99%

Soil Ingestion is Associated with Child Diarrhea in an Urban Slum of Nairobi, Kenya

Bauza

Ocharo

Nguyen

et al. 2017

The American Society of Tropical Medicine and Hygiene

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Abstract. Diarrhea is a leading cause of mortality in children under 5 years of age. We conducted a crosssectional study of 54 children aged 3 months to 5 years old in Kibera, an urban slum in Nairobi, Kenya, to assess the relationship between caregiver-reported soil ingestion and child diarrhea. Diarrhea was significantly associated with soil ingestion (adjusted odds ratio = 9.9, 95% confidence interval = 2.1-47.5). Soil samples from locations near each household were also collected and analyzed for Escherichia coli and a human-associated Bacteroides fecal marker (HF183). Escherichia coli was detected in 100% of soil samples (mean 5.5 log colony forming units E. coli per gram of dry soil) and the Bacteroides fecal marker HF183 was detected in 93% of soil samples. These findings suggest that soil ingestion may be an important transmission pathway for diarrheal disease in urban slum settings.

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“…Previous studies identified that neat DNA extracts and 10-fold dilutions of sample must be run in parallel when performing the T. orientalis cPCR assays to account for PCR-inhibiting contaminants present within nucleic acid extracts prepared from blood (6,19,27). To overcome this, we used a DNA polymerase master mix that has been demonstrated to be effective at reducing contaminant-based inhibition (TaqMan environmental master mix) (42). We assessed the efficacy of this master mix in reducing or eliminating the effects of PCR inhibition during development of the UIC assay.…”

Section: Fig 1 the U Component Of The Uic Assay Is Quantitative (A) mentioning

confidence: 99%

Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant Subtypes

et al. 2015

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T heileria orientalis is a vector-borne hemoprotozoan that infects cattle and buffalo and is generally spread by ticks of the Haemaphysalis genus (1-3). Historically, this organism has been referred to as Theileria sergenti, Theileria buffeli, or the T. orientalis/T. sergenti/T. buffeli complex; however, the name T. sergenti is now considered invalid (4), and T. orientalis is commonly used to refer to all (5). From a clinical perspective, T. orientalis can cause anemia, lethargy, jaundice, fever, abortion, and mortality in cattle (6). Clinical infection can also result in decreased milk production in dairy cattle (7). T. orientalis is a conditional pathogen, and while pathogenic forms are largely limited to Eastern Asia and Australasia (5, 6, 8-10), it is frequently detected in asymptomatic animals; these benign forms are globally spread (5,(11)(12)(13)(14). Analysis of the most common genotyping locus (p32), encoding the variable major piroplasm surface protein (MPSP), currently identifies 11 distinct T. orientalis genotypes (13,15,16). Of these genotypes, type 2 (Ikeda) and to a lesser extent type 1 (Chitose) are typically found in association with clinical disease (6,9,10,(17)(18)(19)(20)(21)(22). The presence of pathogenic and benign forms of T. orientalis greatly complicates its clinical diagnosis, with standard blood film analysis unable to identify the pathogenic genotypes.Multiple conventional PCR (cPCR) assays have been published for the identification of T. orientalis in blood samples (9,21,23,24). The most commonly cited assays detect and genotype T. orientalis by amplifying unique regions of the MPSP gene (21). These assays use two universal primers to detect T. orientalis infection and specific forward primers to identify the Ikeda, Chitose, and Buffeli genotypes. While this method is highly sensitive and has been validated in prior studies (19,21), it is not multiplexed and is highly susceptible to PCR inhibitors (6), which are often found in nucleotide extractions obtained from blood (25,26). To overcome inhibition, undiluted and diluted nucleotide extracts can be examined in parallel to prevent false-negative results (6,19,27). However, if multiple reactions per sample are required to determine the presence of infection, the procedure becomes both expensive and time-consuming. Furthermore, cPCR does not give an accurate representation of parasite load and therefore cannot provide an indication of the severity of T. orientalis infection. A recently developed multiplexed tandem PCR (28) is able to discriminate between four genotypes of T. orientalis, but it is only semiquantitative and therefore cannot be used to define a clinical threshold.Hydrolysis probe quantitative PCR (qPCR) assays employ sequence-specific, fluorescently labeled probes attached to duplex-

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Effectiveness of qPCR permutations, internal controls and dilution as means for minimizing the impact of inhibition while measuring Enterococcus in environmental waters

Cited by 120 publications

References 21 publications

Performance of Two Quantitative PCR Methods for Microbial Source Tracking of Human Sewage and Implications for Microbial Risk Assessment in Recreational Waters

Performance of Two Quantitative PCR Methods for Microbial Source Tracking of Human Sewage and Implications for Microbial Risk Assessment in Recreational Waters

Soil Ingestion is Associated with Child Diarrhea in an Urban Slum of Nairobi, Kenya

Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant Subtypes

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