Rapid and Accurate Diagnosis Based on Real-Time PCR Cycle Threshold Value for the Identification of Campylobacter jejuni, astA Gene-Positive Escherichia coli, and eae Gene-Positive E. coli.

We previously developed a multiplex real-time PCR assay (Rapid Foodborne Bacterial Screening 24 ver.5, [RFBS24 ver.5]) for simultaneous detection of 24 foodborne bacterial targets. Here, to overcome the discrepancy of the results from RFBS24 ver.5 and bacterial culture methods (BC), we analyzed 246 human clinical samples from 49 gastroenteritis outbreaks using RFBS24 ver.5 and evaluated the correlation between the cycle threshold (CT) value of RFBS24 ver.5 and the BC results. The results showed that the RFBS24 ver.5 was more sensitive than BC for Campylobacter jejuni and Escherichia coli harboring astA or eae, with positive predictive values (PPV) of 45.5-87.0% and a kappa coefficient (KC) of 0.60-0.92, respectively. The CTs were significantly different between BC-positive and -negative samples (p ＜ 0.01). All RFBS24 ver.5-positive samples were BC-positive under the lower confidence interval (CI) limit of 95% or 99% for the CT of the BC-negative samples. We set the 95% or 99% CI lower limit to the determination CT (d-CT) to discriminate for assured BC-positive results (d-CTs: 27.42-30.86), and subsequently the PPVs (94.7%-100.0%) and KCs (0.89-0.95) of the 3 targets were increased. Together, we concluded that the implication of a d-CT-based approach would be a valuable tool for rapid and accurate diagnoses using the RFBS24 ver.5 system.

Comparison of two methods of bacterial DNA extraction from human fecal samples contaminated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni.

In this study, 2 methods of DNA extraction were evaluated for use in conjunction with the screening system Rapid Foodborne Bacterial Screening 24 (RFBS24), which employs multiplex real-time SYBR Green polymerase chain reaction (SG-PCR) and can simultaneously detect 24 target genes of foodborne pathogens in fecal DNA samples. The QIAamp DNA Stool mini kit (Qkit) and Ultra Clean Fecal DNA Isolation Kit (Ukit) were used for bacterial DNA extraction from fecal samples artificially inoculated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni. SG-PCR and simplex real-time quantitative PCR (S-qPCR) analyses revealed higher copy numbers (8-234 times) of DNA in samples obtained using Ukit compared with those obtained using Qkit, resulting in lower cycle threshold values for the Ukit samples of the 4 bacteria on SG-PCR analysis. Fecal DNA samples from patients infected during foodborne outbreaks of Salmonella and Campylobacter were also prepared by Qkit and Ukit methods and subjected to RFBS24 analyses. Higher numbers of RFBS24 bacterial target genes were detected in DNA samples obtained using Ukit compared with those obtained using Qkit. Thus, the higher DNA extraction efficiency of the Ukit method compared with Qkit renders the former more useful in achieving improved detection rates of these 4 bacteria in fecal samples using SG-PCR.

Comprehensive and rapid real-time PCR analysis of 21 foodborne outbreaks.

A set of four duplex SYBR Green I PCR (SG-PCR) assay combined with DNA extraction using QIAamp DNA Stool Mini kit was evaluated for the detection of foodborne bacteria from 21 foodborne outbreaks. The causative pathogens were detected in almost all cases in 2 hours or less. The first run was for the detection of 8 main foodborne pathogens in 5 stool specimens within 2 hours and the second run was for the detection of other unusual suspect pathogens within a further 45 minutes. After 2 to 4 days, the causative agents were isolated and identified. The results proved that for comprehensive and rapid molecular diagnosis in foodborne outbreaks, Duplex SG-PCR assay is not only very useful, but is also economically viable for one-step differentiation of causative pathogens in fecal specimens obtained from symptomatic patients. This then allows for effective diagnosis and management of foodborne outbreaks.

[Study of real-time PCR assays for rapid detection of food-borne pathogens].

A duplex real-time SYBR Green LightCycler PCR (LC-PCR) assay with DNA extraction using QIAamp DNA Stool Minikit was evaluated for detection of 8 of 19 species of food-borne pathogens, including Plesiomonas shigelloides, Providencia alcalifaciens, in five stool specimens. The time frame was within 2h or less. The protocol used the same LC-PCR with 22 pairs of specific primers. The rapid amplification and reliable detection of specific genes were determined by this LC-PCR assay from 10 cases of food-borne outbreaks in Shimane Prefecture from 2002 to 2005. These cases included Campylobacter jejuni (4), Clostridium perfringens (2), enteropathogenic Escherichia coli and astA positive E. coli (1), and astA positive E. coli, enterohemorrhagic E. coli 026, and Bacillus cereus (1 each). Rapid amplification and reliable detection of specific genes of food-or water-borne pathogenic bacteria in fecal samples should facilitate the diagnosis and management of food-borne outbreaks.

Duplex real-time SYBR green PCR assays for detection of 17 species of food- or waterborne pathogens in stools.

A duplex real-time SYBR Green LightCycler PCR (LC-PCR) assay with DNA extraction using the QIAamp DNA Stool Mini kit was evaluated with regard to detection of 8 of 17 species of food- or waterborne pathogens in five stool specimens in 2 h or less. The protocol used the same LC-PCR with 20 pairs of specific primers. The products formed were identified based on a melting point temperature (T(m)) curve analysis. The 17 species of food- or waterborne pathogens examined were enteroinvasive Escherichia coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, Salmonella spp., Shigella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, Campylobacter jejuni, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Aeromonas spp., Staphylococcus aureus, Clostridium perfringens, and Bacillus cereus. No interference with the LC-PCR assay was observed when stool specimens were artificially inoculated with each bacterial species. The detection levels were approximately 10(5) food- or waterborne pathogenic bacteria per g of stool. The protocol for processing stool specimens for less than 10(4) food- or waterborne pathogenic bacteria per g of stool requires an overnight enrichment step to achieve adequate sensitivity. However, the rapid amplification and reliable detection of specific genes of greater than 10(5) food- or waterborne pathogenic bacteria per g in samples should facilitate the diagnosis and management of food- or waterborne outbreaks.