Evaluation of uncertainty in quantitative real-time PCR

Love, John L.; Scholes, Paula; Gilpin, Brent; Savill, M.; Lin, Shen; Samuel, Laly

doi:10.1016/j.mimet.2006.04.005

Cited by 24 publications

(22 citation statements)

References 7 publications

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“…S2). We retested microcosm DNAs that tested positive for Camp with Campylobacter jejuni ‐specific assays mapA (Price et al ., 2006) and groEL (Love et al ., 2006), and both were below assay limits of detection. A search for database sequences matching Camp primers and probe found no exact matches outside the genus Campylobacter (NCBI nr/nt).…”

Section: Resultsmentioning

confidence: 99%

Differential decay of human faecal Bacteroides in marine and freshwater

Green

Shanks

Sivaganesan

et al. 2011

Environmental Microbiology

104

116

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Genetic markers from Bacteroides and other faecal bacteria are being tested for inclusion in regulations to quantify aquatic faecal contamination and estimate public health risk. For the method to be used quantitatively across environments, persistence and decay of markers must be understood. We measured concentrations of contaminant molecular markers targeting Enterococcus and Bacteroides spp. in marine and freshwater microcosms spiked with human sewage and exposed to either sunlight or dark treatments. We used Bayesian statistics with a delayed Chick-Watson model to estimate kinetic parameters for target decay. DNA- and RNA-based targets decayed at approximately the same rate. Molecular markers persisted (could be detected) longer in marine water. Sunlight increased the decay rates of cultured indicators more than those of molecular markers; sunlight also limited persistence of molecular markers. Within each treatment, Bacteroides markers had similar decay profiles, but some Bacteroides markers significantly differed in decay rates. The role of extracellular DNA in persistence appeared unimportant in the microcosms. Because conditions were controlled, microcosms allowed the effects of specific environmental variables on marker persistence and decay to be measured. While marker decay profiles in more complex environments would be expected to vary from those observed here, the differences we measured suggest that water matrix is an important factor affecting quantitative source tracking and microbial risk assessment applications.

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Section: Resultsmentioning

confidence: 99%

Differential decay of human faecal Bacteroides in marine and freshwater

Green

Shanks

Sivaganesan

et al. 2011

Environmental Microbiology

104

116

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“…The assay sensitivity was evaluated by 10-fold dilution of a synthetic capped mRNA transcript to take into account both the RT and the PCR step. The obtained sensitivity of 10 copies per reaction under optimal conditions represents approximately 2500 copies of target per g of feces, although this estimation has important limitations (Love et al, 2006). The obtained Ct-values for the 208 BoNoV positive samples ranged from 6.9 to 39.7 (Fig.…”

Section: Discussionmentioning

confidence: 97%

SYBR Green based real-time RT-PCR assay for detection and genotype prediction of bovine noroviruses and assessment of clinical significance in Norway

Jor

Myrmel

Jonassen

2010

Journal of Virological Methods

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A novel SYBR Green based real-time RT-PCR assay for detection of genogroup III bovine noroviruses (BoNoV) was developed and the assay applied to 419 faecal samples from calves with and without diarrhoea. The samples were obtained from 190 Norwegian dairy and beef herds. BoNoV was detected in 49.6% of the samples from 61.1% of the herds indicating that BoNoV is ubiquitous in Norway. The overall prevalence was not significantly different in diarrhoea and non-diarrhoea samples. Analyses of polymerase gene sequences revealed both genotype III/1 and III/2 with genotype III/2 (Newbury2-like) being the most prevalent. Detected capsid sequences were restricted to Newbury2-like and the chimeric Bo/Thirsk10/00/UK strain. The RNA polymerase genotypes of the circulating BoNoVs in Norway were predicted by melting temperature analysis. Additional data from a challenge experiment suggest that a high proportion of young calves are shedding low levels of BoNoV for a prolonged time after recovering from the associated diarrhoea. The findings may explain some of the discrepancies in detection rates from previous studies and explain why some studies have failed to detect significant prevalence differences between calves with and without diarrhoea. It may also shed new light on some epidemiological aspects of norovirus infections.

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“…Hence, coverage of the target bacterial genera in the present study may have been restricted by the availability of sequence data for the species within each genus. Also, what is ultimately achieved from the quantification of bacteria in a qPCR-based study is relative to the processed standard curve, and any unanticipated error through standard curve construction can adversely affect the interpretation of results [57, 58]. Also, it would impose a considerable cost and time to individually assess all samples for more than a few bacteria by real-time PCR.…”

Section: Discussionmentioning

confidence: 99%

pH gradient and distribution of streptococci, lactobacilli, prevotellae, and fusobacteria in carious dentine

et al. 2013

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Objectives Caries process comprises acidogenic and aciduric bacteria that are responsible for lowering the pH and subsequent destruction of hydroxyapatite matrix in enamel and dentine. The aim of this study was to identify the correlation between the pH gradient of a carious lesion and proportion and distribution of four bacterial genera; lactobacilli, streptococci, prevotellae, and fusobacteria with regard to total load of bacteria. Materials and methods A total of 25 teeth with extensive dentinal caries were sampled in sequential layers. Using quantitative real-time PCR of 16S rRNA gene, we quantified the total load of bacteria as well as the proportion of the abovementioned genera following pH measurement of each sample with a fine microelectrode. Results We demonstrated the presence of a pH gradient across the lesion with a strong association between the quantity of lactobacilli and the lowest pH range (pH 4.5–5.0; p = 0.003). Streptococci had a tendency to occupy the most superficial aspect of the carious lesion but showed no correlation to any pH value. Prevotellae showed clear preference for the pH range 5.5–6.0 (p = 0.042). The total representation of these four genera did not reach more than one quarter of the total bacterial load in most carious samples. Conclusion We revealed differential colonization behavior of bacteria with respect to pH gradient and a lower than expected abundance of lactobacilli and streptococci in established carious lesions. The data indicate the numerical importance of relatively unexplored taxa within the lesion of dentinal caries. Clinical relevance The gradient nature of pH in the lesion as well as colonization difference of examined bacterial taxa with reference to pH provides a new insight in regard to conservative caries management.

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Evaluation of uncertainty in quantitative real-time PCR

Cited by 24 publications

References 7 publications

Differential decay of human faecal Bacteroides in marine and freshwater

Differential decay of human faecal Bacteroides in marine and freshwater

SYBR Green based real-time RT-PCR assay for detection and genotype prediction of bovine noroviruses and assessment of clinical significance in Norway

pH gradient and distribution of streptococci, lactobacilli, prevotellae, and fusobacteria in carious dentine

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