A PCR assay for the detection of Campylobacter jejuni and Campylobacter coli in water

Kirk, R.B.; Rowe, M.T.

doi:10.1111/j.1472-765x.1994.tb00460.x

Cited by 52 publications

(23 citation statements)

References 9 publications

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“…High recovery rates were also found after vortexing for 1 min, sonicating, or performing direct lysis from the filter (P ϭ 0.83, P ϭ 0.51, and P ϭ 0.84, respectively, compared to vortexing for 15 seconds). The results obtained following sonication were similar to those of previous studies on Campylobacter (7). Nonetheless, due to the b Two independent experiments using purified DNA were run, and the duplicate data were used to create one standard curve.…”

Section: Resultssupporting

confidence: 73%

Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR

Wolffs

Glencross

Thibaudeau

et al. 2006

Appl Environ Microbiol

119

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A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the invA gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 m to remove large particles and of 0.22 m to recover the Salmonella cells. After this, the Salmonella cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 ؋ 10 2 CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.Salmonella is one of the most common causes of food-borne disease (27), with 40,000 reported annual cases of salmonellosis and an even higher number of estimated cases in the United States (data available at www.cdc.gov). In order to minimize risks for the consumer, microbial auditing of food is increasingly being applied. For this reason, the number of rapid test methods for Salmonella has grown rapidly in the last decade. PCR and real-time PCR have become powerful tools for the detection of pathogens in food. Many different PCR assays have been developed for Salmonella, all with different specificities, accuracies, and detection limits (13,15,35). The most recent assays, with detection in 12 to 20 h, have drastically improved the speed of the detection process compared to that of culture-based methods (3, 10). However, due to the potential for very low levels of salmonellae in foods and standards requiring detection of 1 CFU of salmonellae in food, all these methods include a significant enrichment time that limits the ability for same-day analysis. Also, even though real-time PCR allows for quantitation of targets, after enrichment the number of cells present has generally been changed in an unpredictable manner, making quantitation of the initial amount of target difficult. For these reasons, it would be considered an improvement to be able to detect and, if possible, quantitate (low) levels of salmonellae in foods without the need for enrichment.Aside from concentrating the target, sample treatments are performed prior to PCR in order to remove PCR inhibitors or to improve the homogeneity of samples (19). There are currently several methods for detect...

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Section: Resultssupporting

confidence: 73%

Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR

Wolffs

Glencross

Thibaudeau

et al. 2006

Appl Environ Microbiol

119

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“…A number of PCR assays have been developed for the detection of campylobacters in water samples (10,15,18,27). Oyofo and Rollins (15) attempted to detect campylobacters by PCR directly in filter-concentrated samples.…”

Section: Discussionmentioning

confidence: 99%

“…A number of PCR methods have been reported for the detection of campylobacters in water samples (7,8,10,15,18,27); however, only three have been applied to the detection of campylobacters in naturally contaminated waters (8,18,27). In these PCR assays, the amplification products were detected by gel electrophoresis, which may lack sensitivity or specificity; there are also limits to the numbers of samples that can be tested by this approach.…”

mentioning

confidence: 99%

Detection of Campylobacter jejuni and Campylobacter coli in Environmental Waters by PCR Enzyme-Linked Immunosorbent Assay

Sails

Bolton

Fox

et al. 2002

Appl Environ Microbiol

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A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELISA assay. A total of 51 samples were positive by either PCR ELISA or culture; of these, 43 were found to be positive by PCR ELISA and 43 were found to be positive by culture. Overall, including positive and negative results, 59 samples were concordant in both methods. Several samples were positive in the PCR ELISA assay but were culture negative; therefore, this assay may be able to detect sublethally damaged or viable nonculturable forms of campylobacters. The method is rapid and sensitive, and it significantly reduces the time needed for the detection of these important pathogens by 2 to 3 days.

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“…in water and wastewater were successfully achieved employing various PCR protocols, using diverse primers [18,[85][86][87][88][89][90]. Most of these protocols aimed at the detection of the presence or absence of campylobacters; whereas other protocols [91] used real-time PCR for obtaining quantitative results.…”

Section: Detection In Water and Wastewatermentioning

confidence: 99%

Campylobacter in the environment: A major threat to public health

Abulreesh¹,

Organji²,

Elbanna³

et al. 2017

APJTD

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A PCR assay for the detection of Campylobacter jejuni and Campylobacter coli in water

Cited by 52 publications

References 9 publications

Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR

Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR

Detection of Campylobacter jejuni and Campylobacter coli in Environmental Waters by PCR Enzyme-Linked Immunosorbent Assay

Campylobacter in the environment: A major threat to public health

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