Evaluation of a 5′-Nuclease (TaqMan) Assay for the Detection of Virulent Strains of Yersinia enterocolitica in Raw Meat and Tofu Samples

Vishnubhatla, A.; Oberst, Richard D.; Fung, Daniel Y. C.; Wonglumsom, Wijit; Hays, Michael P.; Nagaraja, T. G.

doi:10.4315/0362-028x-64.3.355

Cited by 38 publications

(20 citation statements)

References 27 publications

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“…However, the detection limit of the PCR reaction was 1-10 cfu per PCR. This is in consistent with earlier studies (Boyapalle et al, 2001;Vishnubhatla et al, 2001).…”

Section: Resultssupporting

confidence: 83%

Detection of Pathogenic Yersinia enterocolitica in Meat using Real-Time PCR

et al. 2007

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Yersinia enterocolitica is an important zoonosis, which can cause disease in humans and animals. The culture methods available for detection of Y. enterocolitica in food samples are time-consuming and seldom successful. Using DNA-based methods, like PCR, this pathogen can be detected more rapidly and with greater sensitivity. The aim of this study was to establish a rapid and accurate real-time PCR method to detect pathogenic Y. enterocolitica in pork. The chromosomal ail-gene, which is only present in pathogenic Y. enterocolitica strains, was used as DNA target for the 5' nuclease PCR assay. The probe was labelled at the 5' end with the fluorescent reporter dye (FAM) and at the 3' end with the quencher dye (TAMRA). A 2-step protocol with 45 cycles was used in a multicolour real-time PCR detection system. A Ct value over 40 indicated a negative result. The DNA extraction procedure for the natural samples was rapid and simply. This qualitative real-time PCR method was shown to be specific and sensitive. Detection rate of ail-positive Y. enterocolitica in 200 pig tonsils was 88 % and 35 % with PCR and culture methods, respectively. When 100 raw pork samples were studied, 7 were positive with PCR and all were culture negative.Zusammenfassung (Redaktion): Yersinia enterocolitica ist als Pathogen von Bedeutung für Mensch und Tier. Die Kultur-Methoden, die für den Nachweis von Y. enterocolitica in Lebensmittel-Proben zur Verfügung stehen, sind Zeit-aufwendig und selten erfolgreich. Wenn DNA-basierte Methoden wie zum Beispiel die PCR angewandt werden, kann dieser pathogene Mikroorganismus schneller und besser nachgewiesen werden. In diesem Bericht wird eine schnelle und sehr spezifische real-time PCR vorgestellt, um Y. enterocolitica in Schweinefleisch nachzuweisen. Das chromosomale ail-Gen, das nur in pathogener Y. enterocolitica vorkommt, wurde als DNA-Sonde für den 5`Nuklease-PCR-Assay eingesetzt. Die Probe wurde am 5`-Ende mit dem fluoreszierenden FAM und am 3`-Ende mit dem Quencher TAMRA markiert. Ein Verfahren -bestehend aus zwei Abschnitten -mit 45 Zyklen wurde in einem Multicoulor real-time-PCR NachweisSystem durchgeführt. Ct-Werte größer als 40 stehen für negative Ergebnisse. Die DNA-Extraktion aus den Proben war schnell und einfach. Die hier beschriebene real-time PCR ist spezifisch und sensitiv. Die Nachweisrate für ail-positive Y. enterocolitica in 200 Schweinemandeln lag bei 88 % bei der PCR-Methode und bei 35 % bei Kulturmethoden. Bei 100 Proben rohem Schweinefleisch waren mit Hilfe der PCR 7 Proben positiv, aber mit Hilfe der Kulturmethoden alle negativ.

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“…However, the detection limit of the PCR reaction was 1-10 cfu per PCR. This is in consistent with earlier studies (Boyapalle et al, 2001;Vishnubhatla et al, 2001).…”

Section: Resultssupporting

confidence: 83%

Detection of Pathogenic Yersinia enterocolitica in Meat using Real-Time PCR

et al. 2007

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“…In the same study, the isolation rates of Y. enterocolitica in ground beef and pork were also elevated. The contamination level of these products was obviously very high, because the culture method used was able to identify Y. enterocolitica only when 10 6 CFU or more organisms per g were present (152). The occurrence of pathogenic Y. enterocolitica was also shown to be clearly higher by the PCR assay than by culturing (Table 5).…”

Section: Food Samplesmentioning

confidence: 85%

“…For artificially contaminated pork, Boyapalle et al (20) have shown that the TaqMan assay targeting the ail gene was 100 to 1,000 times more sensitive than the traditional PCR assay with gel-based detection and 10,000 times more sensitive than the culture method. Visnubhatla et al (152) used the same TaqMan assay, but instead of ail, they targeted the yst gene. This was the first time when a high occurrence of yst-positive Y. enterocolitica was detected in retail ground beef.…”

Section: Food Samplesmentioning

confidence: 99%

Low Occurrence of Pathogenic Yersinia enterocolitica in Clinical, Food, and Environmental Samples: a Methodological Problem

2003

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While Yersinia enterocolitica is an important pathogen, which can cause yersiniosis in humans and animals, its epidemiology remains obscure. The pig is the major reservoir of pathogenic Y. enterocolitica of bioserotype 4/O:3, the most common type found in humans. Y. enterocolitica is thought to be a significant food-borne pathogen, although pathogenic isolates have seldom been recovered from foods. The low isolation rate of this pathogenic bacterium in natural samples, including clinical, food, and environmental samples, may be due to the limited sensitivity of culture methods. During the last decade, numerous DNA-based methods, such as PCR and colony hybridization assays, have been designed to detect pathogenic Y. enterocolitica in natural samples more rapidly and with better sensitivity than can be achieved by culture methods. In addition, the occurrence of pathogenic Y. enterocolitica in natural samples is clearly higher with PCR than with culture methods. The methods available for detection of pathogenic Y. enterocolitica in natural samples are reviewed in this article

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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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Evaluation of a 5′-Nuclease (TaqMan) Assay for the Detection of Virulent Strains of Yersinia enterocolitica in Raw Meat and Tofu Samples

Cited by 38 publications

References 27 publications

Detection of Pathogenic Yersinia enterocolitica in Meat using Real-Time PCR

Detection of Pathogenic Yersinia enterocolitica in Meat using Real-Time PCR

Low Occurrence of Pathogenic Yersinia enterocolitica in Clinical, Food, and Environmental Samples: a Methodological Problem

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

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