RESUMO
AIMS: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. METHODS AND RESULTS: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. CONCLUSIONS: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms.
Assuntos
Bacillus anthracis/genética , DNA Bacteriano/isolamento & purificação , Francisella tularensis/genética , Reação em Cadeia da Polimerase/métodos , Kit de Reagentes para Diagnóstico/estatística & dados numéricos , Vibrio cholerae/genética , DNA Bacteriano/análise , DNA Bacteriano/genética , Indicadores e Reagentes/normas , Reação em Cadeia da Polimerase/normas , Kit de Reagentes para Diagnóstico/normas , Análise de SobrevidaRESUMO
AIMS: To compare the performance of traditional methods to quantitative polymerase chain reaction (qPCR) for detecting five biological agents in large-volume drinking-water samples concentrated by ultrafiltration (UF). METHODS AND RESULTS: Drinking-water samples (100 l) were seeded with Bacillus anthracis, Cryptospordium parvum, Francisella tularensis, Salmonella Typhi, and Vibrio cholerae and concentrated by UF. Recoveries by traditional methods were variable between samples and between some replicates; recoveries were not determined by qPCR. Francisella tularensis and V. cholerae were detected in all 14 samples after UF, B. anthracis was detected in 13, and C. parvum was detected in 9 out of 14 samples. Numbers found by qPCR after UF were significantly or nearly related to those found by traditional methods for all organisms except for C. parvum. A qPCR assay for S. Typhi was not available. CONCLUSIONS: qPCR can be used to rapidly detect biological agents after UF as well as traditional methods, but additional work is needed to improve qPCR assays for several biological agents, determine recoveries by qPCR, and expand the study to other areas. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first study to compare the use of traditional and qPCR methods to detect biological agents in large-volume drinking-water samples.
Assuntos
Bactérias/isolamento & purificação , Contagem de Colônia Microbiana/métodos , Reação em Cadeia da Polimerase/métodos , Ultrafiltração , Microbiologia da Água , Água/parasitologia , Bacillus anthracis , Bactérias/genética , Técnicas Bacteriológicas , Cryptosporidium parvum/genética , Cryptosporidium parvum/isolamento & purificação , DNA Bacteriano/análise , Francisella tularensis , Microscopia de Fluorescência , Biologia Molecular/métodos , Salmonella typhi , Vibrio choleraeRESUMO
AIMS: The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water. METHODS AND RESULTS: Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis (r = 0.62 and 0.77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci. CONCLUSIONS: The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications.
