ABSTRACT
A quantitative PCR (qPCR) assay with two primers and a TaqMan probe targeting conserved regions of the specific ipaH gene of Shigella species and enteroinvasive Escherichia coli (EIEC) were developed. This qPCR assay was used to identify 206 Shigella strains (including four Shigella species with all serotypes and two provisional Shigella species), 3 EIEC strains, and 113 non-Shigella strains with 100% accuracy. Pure cultures of six Shigella reference strains were used to derive standard curves to determine the detection limit and efficiency of the qPCR method. The ipaH qPCR assay had an equally low detection limit (0.12 to 0.74 CFU per PCR) for the four Shigella species tested. The average qPCR efficiency was 99.29% (95.36 to 103.92%). The detection limit of the qPCR assay tested on 15 varieties of inoculated fresh produce ranged from 0.4 to 16 CFU/100 ml of buffer rinse. This qPCR assay took the variation of wild-type nucleotides into consideration and was used successfully to screen fresh produce. This highly sensitive qPCR assay can be completed within 24 h and has potential use as a screening tool for all four Shigella species and EIEC in food samples.
Subject(s)
DNA, Bacterial/analysis , Food Contamination/analysis , Polymerase Chain Reaction/methods , Shigella/isolation & purification , Vegetables/microbiology , Base Sequence , Colony Count, Microbial , Consumer Product Safety , Escherichia coli/classification , Escherichia coli/genetics , Escherichia coli/isolation & purification , Food Microbiology , Humans , Molecular Sequence Data , Sensitivity and Specificity , Sequence Alignment , Serotyping , Shigella/classification , Shigella/genetics , Species SpecificityABSTRACT
The efficacy of a 24-h Salmonella real-time, or quantitative, PCR (qPCR) detection method was assessed through a collaborative effort involving eight Federal and state laboratories. Eleven foods including mashed potatoes, soft cheese, chili powder, chocolate, eggs, sprouts, apple juice, fish, shrimp, ground beef, and ground chicken were tested. For each food, seven blind samples were distributed to each participant for testing. These included six samples equivalently inoculated with 1 to 5 CFU/25 g of various serotypes of Salmonella (Gaminara, Weltevreden, Heidelberg, Senftenberg, Enteritidis, Newport, Typhimurium, and Kentucky for each food) and 10 to 50 CFU/25 g of the competitor Enterobacter cloacae. The seventh sample was inoculated with 10 to 50 CFU/25 g of the competitor, E. cloacae, only. These samples were tested for Salmonella by using four methods in parallel: (i) 24-h qPCR method detecting Salmonella from modified buffered peptone water enrichment medium; (ii) 48-h qPCR method detecting Salmonella from a secondary selective enrichment broth; (iii) modified Bacteriological Analytical Manual method; and (iv) VIDAS, an immunoassay system. The results of the statistical analysis showed there was no significant (P > or = 0.05) difference between either of the qPCR methods and the modified Bacteriological Analytical Manual method for 10 of 11 foods. For the one exception, sprouts, detection by qPCR required 48 h. Both qPCR methods showed a detection limit of 0.08 to 0.2 CFU/g. These results provide a solid basis for using this 24-h qPCR rapid screening method to detect Salmonella in foods.
Subject(s)
DNA, Bacterial/analysis , Food Contamination/analysis , Laboratories/standards , Polymerase Chain Reaction/standards , Salmonella/isolation & purification , Colony Count, Microbial/methods , Colony Count, Microbial/standards , Food Microbiology , Humans , Phylogeny , Polymerase Chain Reaction/methods , Reproducibility of Results , Salmonella/classification , Sensitivity and Specificity , Species Specificity , Time FactorsABSTRACT
The current AOAC Method 966.24 for enumeration of Escherichia coli in foods uses a most probable number (MPN) procedure with extensive confirmation steps. Two new methods based on membrane filtration (MF) were compared to the MPN reference method for detection of high levels of E. coli in 5 food types, some of which represent categories for which the U.S. Food and Drug Administration (FDA) mandates additional testing if an action level of 10(4)/g E. coli is exceeded. Ground beef, which is not FDA regulated, was also tested. The 5 food types were all inoculated at 3 levels: 10(2)/g, > or = 10(4)/g, and > or = 10(5)/g E. coli. An MF protocol using either m-ColiBlue24 (CB) or lauryl sulfate tryptose plus BCIG (LST/BCIG) was an effective potential alternative to the reference method. Sensitivity and specificity for both CB and LST/BCIG were 98 and 100%, respectively. Agreement between MPN and both CB and LST/BCIG was 98%. The 2 proposed methods allow completion of both presumptive and confirmatory steps in 1-3 days, whereas the reference method requires as many as 11 days. Exclusivity testing with 50 non-E. coli strains indicated 100% were correctly ruled out by the proposed protocols. Inclusivity testing was used to determine whether typical results were obtained after incubation of E. coli cultures on CB or LST/BCIG for 24 h. Of 50 E. coli strains tested, 100% yielded typical results after incubation on CB, and 98% yielded typical results after incubation on LST/BCIG.
