Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 30, 2003
Publication Date: August 11, 2003
Citation: FRATAMICO, P.M. REAL-TIME PCR. MEETING ABSTRACT. 2003. Technical Abstract: Assays based on the polymerase chain reaction (PCR) are now accepted methods for rapidly confirming the presence or absence of specific pathogens in foods. The amplified DNA fragments are visualized by agarose gel electrophoresis, which also allows determination of the PCR product size, or by hybridizing the amplification product with a labeled nucleic acid probe. Combining the PCR with a hybridization step enhances assay sensitivity and specificity. Since the PCR is very sensitive, small amounts of DNA carried from one run to the next may lead to false positive results. In recent years, real-time PCR-based methods have been developed, which have the advantages of having the ability to quantify the amount of initial template and eliminate the need for sample post-processing steps. Real-time PCR systems rely upon detection and quantitation of signal generated from a fluorescent reporter that increases in direct proportion to the amount of PCR product produced. These systems can employ the SYBR Green dye that binds double-stranded DNA, or dual-labeled (TaqMan) probes or molecular beacons, which are oligonucleotides that contain fluorescent and quenching dyes at the 5' and 3' ends, respectively. TaqMan probes bind to an internal region of the PCR product, and during replication of the template, the polymerase cleaves the probe separating the reporter and quenching dyes. Molecular beacons are oligonucleotides with a hairpin structure that hybridize to a complementary sequence within the PCR product distancing the fluorophore and the quencher yielding fluorescence. It is possible to perform multiplex real-time PCR assays, in which multiple sequences are amplified simultaneously in a single reaction, using probes labeled with dyes that have unique emission spectra. A multiplex real-time PCR assay employing TaqMan probes was developed to detect E. coli O157:H7 in foods. Target sequences (four) of the E. coli O157:H7 fliCh7, rfbEO157:H7, stx1 and stx2 genes were amplified simultaneously; the sizes of the PCR products were 171, 114, 199, and 157 bp, respectively. The probe for the fliCh7 PCR product was labeled with 6-carboxy-4,7,2',7'-tetrachlorofluorescein (TET) and the Black Hole Quencher 1, the probe for rfbEO157:H7 with 6-carboxyfluorescein (FAM) and Black Hole Quencher 1, and the probes for stx1 and stx2 both with Texas Red and Black Hole Quencher 2. Ground beef samples (25 g) were inoculated with ca.1 to 5 CFU of a cocktail of three strains of E. coli O157:H7, stored at 4ºC for 72 h or at -20ºC for 2 weeks, then subjected to enrichment in Rapid-Chek E. coli O157:H7 enrichment medium (225 ml) for 8 and 20 h at 42ºC at 150 rpm. E. coli O157:H7 was detected in enrichments incubated for 8 h using the real-time multiplex PCR assay. Thus, the assay can be employed for rapid detection of E. coli O157:H7 in ground beef, and potentially other types of samples as well.