Development of a real-time fluorescence resonance energy transfer PCR to identify the main pathogenic Campylobacter spp.

Ménard, Armelle; Dachet, F.; Prouzet‐Mauléon, Valérie; Oleastro, Mónica; Mégraud, Françis

doi:10.1111/j.1469-0691.2005.01072.x

Cited by 54 publications

(35 citation statements)

References 21 publications

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“…Bessede et al [4] compared molecular methods in comparison to culture methods. In their study, two different PCR, a real-time PCR targeting gyrA gene [21] and Seeplex Diarrhea-B1 ACE Detection (Seegene Inc., South Korea), produced 78% and 87% sensitivity and three distinct immunoassays (RidaScreen, Premier CAMPY, ImmunoCard STAT! CAMPY) gave 91, 96 and 91% sensitivity, respectively.…”

Section: Discussionmentioning

confidence: 99%

A Cytolethal Distending Toxin Gene-Based Multiplex PCR Assay for Detection of <i>Campylobacter</i> spp. in Stool Specimens and Comparison with Culture Method

Shiramaru

Asakura

Inoue

et al. 2012

The Journal of Veterinary Medical Science

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ABSTRACT. In this study, we evaluated the applicability of cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR for the direct detection and identification of Campylobacter jejuni, C. coli and C. fetus from stool specimens of patients with gastroenteritis in comparison to culture methods. A total of 711 stool specimens were examined for the isolation or detection of campylobacters by using Skirrow's selective agar culture plates, a filtration method and the multiplex PCR assay. Forty-one and 36 C. jejuni strains were isolated by culture and filtration methods, respectively. In addition, 2 and 3 C. coli strains were isolated by Skirrow and the filtration methods, respectively. However, when the multiplex PCR was employed, the cdtB genes of C. jejuni and C. coli were detected in 45 and 4 stool samples, respectively, and 9 C. jejuni PCR-positive samples by multiplex PCR were negative by culture method. Sequence analysis of the PCR products obtained from 8 stool specimens from which campylobacters were not isolated by culture method but the sequences exactly matched with that of the cdtB gene of C. jejuni strain 81-176. None of the remaining stool samples which were culture negative for campylobacters produced any amplicon. Stool samples were defined as Campylobacterpositive if detected by any method. The sensitivity of the multiplex PCR was 83%, which was higher than Skirrow (74%) and filtration method (66%). These data indicate that cdtB gene-based multiplex PCR is a rapid and more sensitive method to identify the most important species of Campylobacter for human diseases. (248)

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Section: Discussionmentioning

confidence: 99%

A Cytolethal Distending Toxin Gene-Based Multiplex PCR Assay for Detection of <i>Campylobacter</i> spp. in Stool Specimens and Comparison with Culture Method

Shiramaru

Asakura

Inoue

et al. 2012

The Journal of Veterinary Medical Science

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“…This was followed by a melting program of 95°C for 60 s and 38°C for 50 s at a temperature transition rate of 20°C/s and 80°C for 0 s (hold time) at a rate of 0.1°C/s, with continuous monitoring of the fluorescence. The final step consisted of cooling at 20°C/s to 40°C with a 30-s hold (3).…”

Section: Methodsmentioning

confidence: 99%

“…Two different PCRs were used for molecular diagnosis: (i) the in-house PCR routinely performed in the laboratory, which is a real-time fluorescence resonance energy transfer (FRET) PCR specific for C. jejuni and C. coli, targeting the gyrA gene, followed by a melting-curve analysis to differentiate C. jejuni and C. coli (3), and (ii) Seeplex Diarrhea-B1 ACE Detection (Seegene Inc., South Korea), which is a multiplex PCR based on dual-priming oligonucleotides (DPO) (10). The latter assay also permits the simultaneous amplification of target DNA of Salmonella spp.…”

Section: Methodsmentioning

confidence: 99%

New Methods for Detection of Campylobacters in Stool Samples in Comparison to Culture

Bessède

Delcamp²,

Sifré³

et al. 2011

J Clin Microbiol

120

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Campylobacter species, especially Campylobacter jejuni and Campylobacter coli, are a major cause of human bacterial enteritis. Current detection in stools is done essentially by culture on selective and nonselective media with filtration. These methods were compared to 2 molecular biology methods, an in-house real-time PCR and a multiplex PCR named Seeplex Diarrhea ACE Detection, and 3 immunoenzymatic methods, Premier Campy, RidaScreen Campylobacter, and ImmunoCard Stat!Campy. Out of 242 stool specimens tested, 23 (9.5%) fulfilled the positivity criteria, i.e., they were positive by one or both culture methods or, in case of a negative culture, by a positive molecular method and a positive immunoenzymatic method. The striking feature of this study is the low sensitivity of culture, in the range of 60%, in contrast to immunoenzymatic and molecular tests.The incidence of Campylobacter-associated food poisoning has gradually increased, and the organism is now considered to be the leading cause of bacterial gastroenteritis worldwide (4). These infections can also lead to extraintestinal diseases and severe long-term complications (9). Campylobacter jejuni and Campylobacter coli are the most frequently isolated species in this context and in our experience account for 80% and 16%, respectively, of all the isolates received in our laboratory every year (2a). Campylobacter species are bacteria with a special culture requirement, i.e., a microaerobic environment. During stool processing, the bacteria may have long contact with a normal atmosphere, and in addition, the progressive decrease in oxygen tension when gas-generating kits are used may not favor adequate growth. Furthermore, selective media are commonly used, and the antibiotics incorporated may inhibit certain Campylobacter strains. Anecdotal data have shown that spiral or curved bacteria are sometimes observed on stool smears while Campylobacter growth does not occur. In a recent study, DNA sequences of the Campylobacter genome were detected by a metagenomic analysis, while the standard culture methods were negative (5). In a pilot study using real-time PCR as a diagnostic tool, we also detected more campylobacters than with culture, but without being able to confirm that true positives were detected, since, at that time, we did not use multiple detection methods to establish positivity when culture was negative. Some immunoenzymatic tests have already been commercialized for several years, such as the ProSpecT Campylobacter Microplate Assay (Remel) (8) and the RidaScreen Campylobacter (R-Biopharm, Darmstadt, Germany) evaluated in our study. In this study, we took advantage of the availability of several kits to compare Campylobacter detection by molecular methods (2 PCRs) and by 3 immunoenzymatic methods to the standard culture methods. MATERIALS AND METHODS Materials.From 15 June to 30 October 2009, every stool specimen obtained from a symptomatic patient, i.e., a patient with a gastrointestinal illness, who was hospitalized for less than 48 h at Pellegrin H...

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“…All of the Campylobacter isolates were identified by phenotypic methods at the genus level. From 2003 to 2009, identification to species level was performed both by standard phenotypic methods and a real-time PCR targeting the gyrA gene (accuracy, 99.9%) to differentiate Campylobacter jejuni and Campylobacter coli (1). In the case of negative results, primers specific for C. fetus were used.…”

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confidence: 99%

Comparison of Characteristics of Patients Infected by Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus

et al. 2014

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A large database of Campylobacter isolates precisely identified at the species level was used to compare patients' characteristics. In a multivariate analysis, Campylobacter coli was found more often in older patients and in patients having traveled abroad and less often in summertime than Campylobacter jejuni. Campylobacter fetus infection occurred in much older patients and in hospitalized patients with a systemic disease.T hermotolerant Campylobacter spp. are recognized as the leading cause of bacterial enteric infections worldwide, while Campylobacter fetus often causes systemic infection.A subset of French clinical laboratories participates in the surveillance of Campylobacter infection, carried out by the National Reference Centre (NRC) for Campylobacters and Helicobacters under the leadership of the Institut de Veille Sanitaire (InVS), sending approximately a quarter of campylobacters isolated in France to the NRC.Our aim was to use this large database (Ͼ22,000 strains) to look for differences in the characteristics of the patients and the circumstances under which these Campylobacter species were isolated.(The results of this study were presented as posters at the European Congress of Clinical Microbiology and Infectious Diseases, London, United Kingdom, 2012, and at the Réunion Interdisciplinaire de Chimiothérapie Anti-infectieuse, Paris, France, November 2012.)Network of laboratories. The laboratories sending strains were not randomly selected but were contacted from the list of laboratories already sending isolated Salmonella species to the corresponding NRC. A survey performed in 2009 showed that equal proportions of all strains isolated came from the different regions, with a few exceptions (InVS-NRC, unpublished data). The methods used by these laboratories to isolate Campylobacter species were fairly uniform, including the use of Karmali medium or Campylosel (bioMérieux, Marcy l'Etoile, France) but no filtration techniques.Identification of campylobacters at the NRC. All of the Campylobacter isolates were identified by phenotypic methods at the genus level. From 2003 to 2009, identification to species level was performed both by standard phenotypic methods and a real-time PCR targeting the gyrA gene (accuracy, 99.9%) to differentiate Campylobacter jejuni and Campylobacter coli (1). In the case of negative results, primers specific for C. fetus were used. If no result was obtained, a PCR specific for Arcobacter species was carried out (2). These PCRs allowed the identification of approximately 99% of the isolates. For the remaining 1%, the 16S rRNA gene was sequenced. In 2010, these methods were replaced by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry identification, which has an accuracy of 99.4% for C. jejuni and 100% for the other Campylobacter species (3).Susceptibility testing was performed by disk diffusion with the same methods and cutoffs from the Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM) that have been used durin...

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Development of a real-time fluorescence resonance energy transfer PCR to identify the main pathogenic Campylobacter spp.

Cited by 54 publications

References 21 publications

A Cytolethal Distending Toxin Gene-Based Multiplex PCR Assay for Detection of <i>Campylobacter</i> spp. in Stool Specimens and Comparison with Culture Method

A Cytolethal Distending Toxin Gene-Based Multiplex PCR Assay for Detection of <i>Campylobacter</i> spp. in Stool Specimens and Comparison with Culture Method

New Methods for Detection of Campylobacters in Stool Samples in Comparison to Culture

Comparison of Characteristics of Patients Infected by Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus

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