Real-Time PCR Detection of
            <i>Salmonella</i>
            in Suspect Foods from a Gastroenteritis Outbreak in Kerr County, Texas

Daum, Luke T.; Barnes, William J.; McAvin, James C; Neidert, Margaret S.; Cooper, Lynn A.; Huff, William B.; Gaul, Linda; Riggins, W. S.; Morris, Sandra A.; Salmen, A; Lohman, Kenton L.

doi:10.1128/jcm.40.8.3050-3052.2002

Cited by 93 publications

(43 citation statements)

References 9 publications

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“…(invA), and C. jejuni (species-specific sequence) was quantified by S-qPCR, as reported previously (7)(8)(9)(10). S-qPCR analyses were performed on DNA samples obtained using Qkit (employing 3 conditions for cell lysis: lysis at 709 C or 959 C, and disruption of cells using the Fastprep24 instrument at 4.0 m/s for 60 s subsequent to lysis at 709 C) and Ukit (cell disruption using Fastprep24 instrument at 4.0 m/s for 60 s), as described above.…”

Section: Methodsmentioning

confidence: 99%

Comparison of Two Methods of Bacterial DNA Extraction from Human Fecal Samples Contaminated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni

et al. 2014

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SUMMARY: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 realtime 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.

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Section: Methodsmentioning

confidence: 99%

Comparison of Two Methods of Bacterial DNA Extraction from Human Fecal Samples Contaminated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni

et al. 2014

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“…One can simultaneously analyze eight pathogenic or specific genes of food-or waterborne pathogens in five stool specimens by using duplex LC-PCR and 32 capillary tubes. Single or multiple real-time PCR assays were reported for detection of one species among foodor waterborne pathogens, such as EHEC O157 (1,11,14,29), Salmonella (5,10,17), Y. enterocolitica (30,35,36), Campylobacter jejuni (19,25,39), V. cholerae (21), V. parahaemolyticus (13), P. shigelloides (20), and S. aureus (28,32). Nineteen pairs of primers for food-or waterborne pathogens were selected from earlier publications (Table 2), and one pair of primers for Y. enterocolitica and Y. pseudotuberculosis was constructed.…”

Section: Discussionmentioning

confidence: 99%

Duplex Real-Time SYBR Green PCR Assays for Detection of 17 Species of Food- or Waterborne Pathogens in Stools

2003

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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.The traditional cultural techniques for the direct isolation and identification of food-or waterborne pathogens from stool specimens in food poisoning outbreaks are time-consuming and laborious. Efforts have been made in diagnostic laboratories to reduce the time required for identification of food-or waterborne pathogens. Real-time PCR is currently used for the diagnosis of infectious agents, and there are few reports of the application of real-time PCR for the direct detection of Escherichia coli strain O157:H7 (11) and Plesiomonas shigelloides (20) in stool specimens. However, traditional PCR is often used for the detection of infectious agents from stool specimens and food samples, and there are a number of previous reports of PCR use for detection of food-or waterborne pathogens, including E. coli (6,14,12,27,37,38,40), Salmonella spp. (26) (38). The published PCR protocols used for detection of food-or waterborne pathogens differ and include the use of different PCR cycler machines, annealing temperatures, and buffer systems. For routine laboratory analysis, the development of streamlined PCR methods would be ideal. In addition, not all of the published PCR methods are so sensitive or specific. Therefore, careful choice of PCR primers, modification of some existing PCR primers, and development of new PCR methods for detection and identification of a wide range of food-or waterborne pathogens are all necessary for the development of a standardized PCR protocol (38). Moreover, interference and inhibitors should be considered...

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“…Although confirmation of the presence of Salmonella directly in chicken in remaining food items using the RTi-PCR assay for an outbreak of acute gastroenteritis among attendees of a church picnic illustrated the feasibility of using RTi-PCR assays during real-world outbreaks, the levels of contaminating organisms in the food samples remained unclear (2). In this study, the RTi-qPCR assay and viable-cell counting after rapid separation and concentration were used for the first time for rapid quantification (within 3 h) of S. aureus in food items suspected to be the source of poisoning.…”

Section: Discussionmentioning

confidence: 99%

Rapid Separation and Concentration of Food-Borne Pathogens in Food Samples Prior to Quantification by Viable-Cell Counting and Real-Time PCR

Fukushima

Katsube

Hata

et al. 2007

Appl Environ Microbiol

146

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Buoyant density gradient centrifugation has been used to separate bacteria from complex food matrices, as well as to remove compounds that inhibit rapid detection methods, such as PCR, and to prevent false-positive results due to DNA originating from dead cells. Applying a principle of buoyant density gradient centrifugation, we developed a method for rapid separation and concentration following filtration and low-and highspeed centrifugation, as well as flotation and sedimentation buoyant density centrifugation, for 12 food-borne pathogens (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Vibrio cholerae O139, Vibrio parahaemolyticus O3K6, Vibrio vulnificus, Providencia alcalifaciens, Aeromonas hydrophila, Bacillus cereus, Staphylococcus aureus, and Clostridium perfringens) in 13 different food homogenates. This method can be used prior to real-time quantitative PCR (RTi-qPCR) and viable-cell counting. Using this combined method, the target organisms in the food samples theoretically could be concentrated 250-fold and detected at cell concentrations as low as 10 1 to 10 3 CFU/g using the RTi-qPCR assay, and amounts as small as 10 0 to 10 1 CFU/g could be isolated using plate counting. The combined separation and concentration methods and RTi-qPCR confirmed within 3 h the presence of 10 1 to 10 2 CFU/g of Salmonella and C. jejuni directly in naturally contaminated chicken and the presence of S. aureus directly in remaining food items in a poisoning outbreak. These results illustrated the feasibility of using these assays for rapid inspection of bacterial food contamination during a real-world outbreak.

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Real-Time PCR Detection of Salmonella in Suspect Foods from a Gastroenteritis Outbreak in Kerr County, Texas

Cited by 93 publications

References 9 publications

Comparison of Two Methods of Bacterial DNA Extraction from Human Fecal Samples Contaminated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni

Comparison of Two Methods of Bacterial DNA Extraction from Human Fecal Samples Contaminated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni

Duplex Real-Time SYBR Green PCR Assays for Detection of 17 Species of Food- or Waterborne Pathogens in Stools

Rapid Separation and Concentration of Food-Borne Pathogens in Food Samples Prior to Quantification by Viable-Cell Counting and Real-Time PCR

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