Rapid PCR with nested primers for direct detection ofCampylobacter jejuniin chicken washes

Winters, Debra K.; O'leary, Awilda; Slavik, Michael F.

doi:10.1006/mcpr.1997.0116

Cited by 29 publications

(26 citation statements)

References 13 publications

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“…In this study, the compatibility of primers and annealing temperatures were examined for multiplex PCR, which differentiated C. jejuni, C. coli, and C. lari. Various primers specific to each species of campylobacters have been developed since now [22,25,29,30,36]. We chose such primers that could amplify different targets of the gene and different sizes of amplicon to avoid non-specific amplification and competition.…”

Section: Discussionmentioning

confidence: 99%

“…Polymerase chain reaction (PCR) can idenfity the bacteria on a gene level. Some primers specific to each of C. jejuni, C. coli, and C. lari have already been developed [22,25,29,30,36] and their functions verified. Harmon et al [14] have established a PCR method that can differentiate C. coli from C. jejuni and Wesley et al [35] have succeeded in identifying C. jejuni and C. coli in pigs by the method, but detection of thermophilic campylobacters from birds has not been achieved until now.…”

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confidence: 99%

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Detection of Thermophilic Campylobacter from Sparrows by Multiplex PCR: The Role of Sparrows as a Source of Contamination of Broilers with Campylobacter.

2000

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ABSTRACT. The best combination of primers and the annealing temperature of multiplex PCR for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari were examined. The multiplex PCR was able to detect type strains of the three species. All results of identification of wild strains (30 strains of C. jejuni, 20 strains of C. coli, and 4 strains of C. lari) by the multiplex PCR coincided with those of the conventional biochemical identification tests, suggesting that the multiplex PCR can simultaneously differentiate C. jejuni, C. coli, and C. lari from wild strains of campylobacters easily and rapidly. Campylobacters were detected from sparrow feces by the multiplex PCR and antimicrobial sensitivities of the strains were determined to discuss the role of sparrows in contamination of broilers with C. jejuni. Three out of 13 strains of C. jejuni isolated from sparrow feces showed quinolone resistance. From the frequent use of quinolones for treatment of industrial animals like chickens, pigs, and cows, the three strains of quinolone-resistant C. jejuni in sparrows must have been originated from those industrial animals. Sparrows that have quinolone-resistant C. jejuni were considered to have contacted with industrial animals or thier feed. It may be presumed, on the contrary, that C. jejuni in sparrows could be a potential source of contamination of broilers.

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Section: Discussionmentioning

confidence: 99%

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Detection of Thermophilic Campylobacter from Sparrows by Multiplex PCR: The Role of Sparrows as a Source of Contamination of Broilers with Campylobacter.

2000

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“…The biochemical tests used oxidase, hippurate hydrolysis, indoxyl acetate hydrolysis, and susceptibility to disks containing cephalothin (30 g) and nalidixic acid (30 g). Species-specific multiplex PCR were done with combined C. jejuni-and C. coli-specific primer sets (18,29).…”

Section: Stools Of Gbs and Fs Patientsmentioning

confidence: 99%

Epidemiology ofCampylobacter jejuniIsolated from Patients with Guillain-Barré and Fisher Syndromes in Japan

et al. 2005

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Campylobacter jejuni isolation is the standard for the diagnosis of this type of bacterial infection, but there have been no epidemiological studies of a large number of C. jejuni isolates from patients with Guillain-Barré syndrome (GBS) and Fisher syndrome (FS). For 13 years, stool specimens from GBS/FS patients have been sent from 378 hospitals throughout Japan to the Tokyo Metropolitan Institute of Public Health. A total of 113 strains (11%) were isolated from the stool specimens from 1,049 patients. The isolation rate did not differ by region. The rates were 22% for 449 patients with a history of diarrhea and 2% for the others. An additional 18 isolates were provided by various hospitals. There was no noticeable seasonal distribution in the onset of C. jejuni isolated from patients with GBS/FS. The male/female ratios were 1.7:1 for GBS and 2.2:1 for FS. The patient age range showed a peak in 10-to 30-year-old subjects who had GBS and in 10-to 20-year-old subjects who had FS. The predominance of young adults and male patients who had C. jejuni-associated GBS/FS may be related to the preponderance of young adults and male patients who had C. jejuni enteritis. The median interval from diarrhea onset to neurologic symptom onset was 10 days for GBS/FS. Penner's C. jejuni serotype HS:19 was more frequently present in GBS (67%) than in enteritis (6%) patients. HS:2 was more frequent in FS (41%) than in enteritis (14%) patients. These findings suggest that certain C. jejuni strains specifically trigger GBS and that others specifically trigger FS.

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“…Due to the aforementioned problems with Campylobacter detection, PCR and real-time PCR have been increasingly used for detection (12,17,21,25). While this technology has tremendous potential for sensitive and simple identification, further studies addressing the quantification aspect of this technology are required before it can be widely accepted.…”

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Quantification of Campylobacter spp. in Chicken Rinse Samples by Using Flotation prior to Real-Time PCR

Wolffs

Norling²,

Hoorfar

et al. 2005

Appl Environ Microbiol

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Real-time PCR is fast, sensitive, specific, and can deliver quantitative data; however, two disadvantages are that this technology is sensitive to inhibition by food and that it does not distinguish between DNA originating from viable, viable nonculturable (VNC), and dead cells. For this reason, real-time PCR has been combined with a novel discontinuous buoyant density gradient method, called flotation, in order to allow detection of only viable and VNC cells of thermotolerant campylobacters in chicken rinse samples. Studying the buoyant densities of different Campylobacter spp. showed that densities changed at different time points during growth; however, all varied between 1.065 and 1.109 g/ml. These data were then used to develop a flotation assay. Results showed that after flotation and real-time PCR, cell concentrations as low as 8.6 ؋ 10 2 CFU/ml could be detected without culture enrichment and amounts as low as 2.6 ؋ 10 3 CFU/ml could be quantified. Furthermore, subjecting viable cells and dead cells to flotation showed that viable cells were recovered after flotation treatment but that dead cells and/or their DNA was not detected. Also, when samples containing VNC cells mixed with dead cells were treated with flotation after storage at 4 or 20°C for 21 days, a similar percentage resembling the VNC cell fraction was detected using real-time PCR and 5-cyano-2,3-ditolyl tetrazolium chloride-4,6-diamidino-2-phenylindole staining (20% ؎ 9% and 23% ؎ 4%, respectively, at 4°C; 11% ؎ 4% and 10% ؎ 2%, respectively, at 20°C). This indicated that viable and VNC Campylobacter cells could be positively selected and quantified using the flotation method.

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Rapid PCR with nested primers for direct detection ofCampylobacter jejuniin chicken washes

Cited by 29 publications

References 13 publications

Detection of Thermophilic Campylobacter from Sparrows by Multiplex PCR: The Role of Sparrows as a Source of Contamination of Broilers with Campylobacter.

Detection of Thermophilic Campylobacter from Sparrows by Multiplex PCR: The Role of Sparrows as a Source of Contamination of Broilers with Campylobacter.

Epidemiology ofCampylobacter jejuniIsolated from Patients with Guillain-Barré and Fisher Syndromes in Japan

Quantification of Campylobacter spp. in Chicken Rinse Samples by Using Flotation prior to Real-Time PCR

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