The $' nuclease PCR assay detects the accumulation of specific PCR product by hybridization and cleavage of a double-labeled fiuorogenic probe during the amplification reaction. The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. An increase In reporter fluorescence intensity indicates that the probe has hybridized to the target PCR product and has been cleaved by the 5'-~3' nucleolytic activity of Toq DNA polymerase. In this study, probes with the quencher dye attached to an internal nucleotide were compared with probes with the quencher dye attached to the 3'-end nucleotlde. In all cases, the reporter dye was attached to the S' end. All intact probes showed quenching of the reporter fluorescence. In general, probes with the quencher dye attached to the 3'-end nucleotide exhibited a larger signal in the 5' nuclease PCR assay than the internally labeled probes, it is proposed that the larger signal is caused by increased likelihood of cleavage by Taq DNA polymerase when the probe is hybridized to a template strand during PCR. Probes with the quencher dye attached to the 3'-end nucleotide also exhibited an increase in reporter fluorescence intensity when hybridized to a complementary strand. Thus, oligonucleotides with reporter and quencher dyes attached at opposite ends can be used as homogeneous hybridization probes.
Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC)eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5′ nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5′ nuclease assay system was sufficient to correctly identify all E. coliO157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed,eaeA-targeted 5′ nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coliO157:H7 when ≥103 CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when ≥104 CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to ≥102 CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when ≥104 CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed,eaeA-targeted 5′ nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.
A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.
We evaluated the TaqMan Salmonella amplification/detection kit from PE Applied Biosystems, which uses a polymerase chain reaction (PCR) assay for rapid detection of Salmonella in food samples. This system uses the 5' nuclease activity of Taq DNA polymerase, which digests an internal fluorogenic probe to monitor the amplification of the target gene. The system's sensitivity and specificity were evaluated using 42 serotypes of 68 Salmonella strains isolated from fecal samples from patients in Tokyo, Japan, and 39 non-Salmonella strains in 22 genera. There were no false-negative or false-positive results. This PCR assay can detect 3 CFU per PCR tube of Salmonella in pure culture (120 CFU/ml of TSB culture). PCR signals were attenuated with artificially contaminated shrimp, but a similar detection limit was obtained. TaqMan's performance was tested with 100 meat and chicken samples purchased from stores in Tokyo. Overall, two of the DNA extraction protocols (the Chelex and EnviroAmp methods) worked equally well, with some exceptions. Of the 100 samples analyzed, 10 were positive for Salmonella with both conventional culture methods and the kit and 89 were negative with both. One sample was negative by the culture method but positive by the kit assay. These results indicate that TaqMan is a reliable and rapid method for Salmonella analysis in the food industry. With this system, food samples can be analyzed for Salmonella in less than 20 h.
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