Molecular beacons: a real-time polymerase chain reaction assay for detecting Escherichia coli from fresh produce and water.
Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. The development of a real-time polymerase chain reaction (PCR) assay to detect the presence of Escherichia coli using these fluorogenic reporter molecules is reported. MBs were designed to recognize a 19-bp region of the uid A gene, coding for an enzyme beta-glucuronidase. The specificity of the MB-based PCR assay was evaluated for various E. coli strains as well as bacteria species that are present in nature. The capability of the assay to detect E. coli in drinking water and produce was demonstrated. Positive detection of E. coli was demonstrated when >10(1) CFU mL(-1) (colony forming unit) was present in the water samples and fresh produce after 18 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli in food and environmental samples.
Sandhya
S
S
Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521, United States. sswami_in@yahoo.com
Chen
Wilfred
W
Mulchandani
Ashok
A
D015374:Biosensing Techniques / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D005456:Fluorescent Dyes / Q000737:chemistry*; D005516:Food Microbiology; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D015345:Oligonucleotide Probes / Q000737:chemistry; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D018724:Spinacia oleracea / Q000737:chemistry / Q000382:microbiology*; D013696:Temperature; D014871:Water Microbiology
D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes