BACKGROUND: Thermotolerant Campylobacter jejuni, coli and lari are recognized as leading food-borne pathogens causing an acute bacterial enteritis worldwide. Due to narrow spectrum of their biochemical activity, it is very complicated to distinguish between individual species. For reliable risk assessment, proper incidence evaluation or swift sample analysis regarding individual species, a demand for simple and rapid method for their distinguishing is reasonable. In this study, we evaluated a reliable and simple approach for their simultaneous detection, species identification and quantification using multiplex qPCR. RESULTS: Species specific primers and hydrolysis probes are directed to hippuricase gene of C. jejuni, serine hydroxymethyltransferase gene of C. coli and peptidase T gene of C. lari. Efficiencies of reactions were 90.85% for C. jejuni, 96.97% for C. coli and 92.89% for C. lari. At 95.00% confidence level and when cut off is set to 38 cycles, limits of detection are in all cases under 10 genome copies per reaction which is very appreciated since it is known that infectious doses are very low. CONCLUSIONS: Proposed assay was positively validated on different food matrices (chicken wing rinses, chicken juice and homogenized fried chicken strips). No inhibition of PCR reaction occurred. Assay was evaluated in accordance with MIQE handbook.

• Klíčová slova
• MIQE, Multiplex qPCR, Quantification, Thermotolerant Campylobacter spp,
• Publikační typ
• časopisecké články MeSH

The Campylobacter species strains (n = 42; isolated from clinical samples and deposited in Czech National Collection of Type Cultures, Prague) originally phenotypically (and biochemically) identified as Campylobacter jejuni were re-classified using molecular biological and mass spectrometric methods. Whole-cell MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) separated the isolates into two genetically related strains--C. jejuni (n = 26) and C. coli (n = 16) and, moreover, distinguished the intimate details in the group of tested strains. It also made it possible to create the MALDI-TOF MS dendrogram; similarly, the spectral characteristics were used for the 3D cluster analysis. Polymerase chain reaction (PCR) confirmed the results obtained by mass spectrometry. Both methods (PCR and MALDI-TOF MS) gave the same results which supports their suitability in the rapid and accurate Campylobacter-species determination.

• MeSH
• Campylobacter coli klasifikace   genetika   MeSH
• Campylobacter jejuni klasifikace   genetika   MeSH
• DNA bakterií analýza   izolace a purifikace   MeSH
• druhová specificita MeSH
• feces mikrobiologie   MeSH
• kočky MeSH
• lidé MeSH
• polymerázová řetězová reakce * MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• techniky typizace bakterií * MeSH
• zvířata MeSH
• Check Tag
• kočky MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• Názvy látek
• DNA bakterií MeSH

High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) were used to analyze the phospholipids and fatty acids of four Arcobacter species (becoming routinely isolated from a wide variety of food sources, especially of animal origin) to provide information for the identification within these species. Phospholipid differences were observed in the HPLC profiles. GC-MS analysis provided a complete fatty acid composition for each arcobacter that after pattern recognition analysis allows taxonomic classification of each species.

• MeSH
• analýza hlavních komponent MeSH
• Arcobacter klasifikace   metabolismus   MeSH
• fosfolipidy analýza   MeSH
• klasifikace MeSH
• mastné kyseliny analýza   MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfolipidy MeSH
• mastné kyseliny MeSH

The PCR amplicons (about 1450 bp in length) of flaA gene fragments of 11 isolates of urease-positive thermophilic Campylobacter (UPTC) isolated from the natural environment not including wild birds in Northern Ireland were demonstrated to be shorter than those of C. jejuni 81116 and six isolates of C. jejuni and C. coli (about 1700 bp) isolated in Northern Ireland and Japan. When the nucleotide lengths of the possible open reading frame (ORF) of the flaA genes were determined, those from the 11 UPTC isolates were estimated to be 1464-1503 bp, and those from the six C. jejuni and C. coli isolates and C. jejuni 81116 strain to be 1716-1728 bp. Nucleotide sequence and deduced amino acid sequence alignments of the possible ORFs demonstrated that the ORFs from the 11 UPTC isolates lack about 80 amino acid residues, mainly from the approximate residue numbers 390-470 of the large variable region in the flaA protein of the seven isolates of C. jejuni and C. coli, and do not have any internal termination codons. High amino acid sequence similarity of both amino- and carboxy-termini of the ORFs of the flaA gene was demonstrated between the 11 isolates of UPTC and the 7 isolates of C. jejuni and C. coli. The 11 UPTC isolates examined were strongly suggested to possess a shorter flaA gene without any internal termination codons.

• MeSH
• Campylobacter coli genetika   izolace a purifikace   MeSH
• Campylobacter jejuni genetika   izolace a purifikace   MeSH
• flagelin genetika   MeSH
• molekulární sekvence - údaje MeSH
• otevřené čtecí rámce MeSH
• polymerázová řetězová reakce MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza proteinů MeSH
• sekvenční seřazení MeSH
• ureasa metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Japonsko MeSH
• Severní Irsko MeSH
• Názvy látek
• flaA protein, bacteria MeSH Prohlížeč
• flagelin MeSH
• ureasa MeSH