Method B: Bacteroidales in Water by TaqMan(Trade Name) Quantitative Polymerase Chain Reaction (qPCR) Assay

摘要

Bacteria of the Bacteroidales order are commonly found in the feces of humans and other warm-blooded animals. Although these organisms can be persistent in the environment, the presence of Bacteroidales in water is an indication of fecal pollution and the possible presence of enteric pathogens. Method B describes a quantitative polymerase chain reaction (qPCR) procedure for the detection of DNA from Bacteroidales bacteria in ambient water matrices based on the amplification and detection of a specific region of the 16S ribosomal RNA gene from these organisms. Results can be obtained by this method in 3-4 hours, allowing same-day notification of recreational water quality. Recent epidemiological studies at fresh water recreational beaches (Reference 17.7) have demonstrated similar or improved positive correlations between Bacteroidales DNA measurements by this method and swimming associated gastrointestinal (GI) illness rates. In Method B, water samples are filtered to collect Bacteroidales on polycarbonate membrane filters. Following filtration, total DNA is solubilized from the filter retentate using a bead beater. Bacteroidales target DNA sequences present in the clarified homogenate are detected by the real time polymerase chain reaction technique using TaqMan(Trade Name) Universal Master Mix PCR reagent and the TaqMan(Trade Name) probe system. The TaqMan(Trade Name) system signals the formation of PCR products by a process involving enzymatic hydrolysis of a fluorogenically-labeled oligonucleotide probe when it hybridizes to the target sequence. Method B uses an arithmetic formula, the comparative cycle threshold (CT) method, to calculate the ratio of Bacteroidales 16S rRNA gene target sequences (target sequences) recovered in total DNA extracts from water samples relative to those in similarly-prepared extracts of calibrator samples containing a known quantity of Bacteroidales cells. Mean estimates of the absolute quantities of target sequences in the calibrator sample extracts are then used to determine the absolute quantities of target sequences in the water samples. CT values for sample processing control (SPC) sequences added in equal quantities to both the water filtrate and calibrator samples before DNA extraction are used to normalize results for potential differences in DNA recovery or to signal inhibition or fluorescence quenching of the PCR analysis caused by a sample matrix component or possible technical error.