Multiplex PCR for identification of Campylobacter coli and Campylobacter jejuni from pure cultures and directly on stool samples.
A multiplex-PCR method, specifically designed for application in routine diagnostic laboratories, was developed for the detection of Campylobacter coli and Campylobacter jejuni. Primers were directed towards the following loci: the hippuricase gene (hipO) characteristic of C. jejuni, a sequence partly covering an aspartokinase gene characteristic of C. coli, and a universal 16S rDNA gene sequence serving as an internal positive control for the PCR. The method was tested on 47 C. coli strains and 88 C. jejuni strains, and found to be almost 100% in concordance with biochemical analyses (all except for one C. coli strain), regardless of whether the DNA was prepared from colonies by a simple boiling procedure or by DNeasy Tissue Kit. Pure cultures of C. coli and C. jejuni were identified at 10-100 cells per PCR. When the multiplex-PCR method was used on spiked human stool samples, both strains were identified at 10(5) cells per ml stool. This sensitivity limit was the same whether the DNA was purified by the method of KingFisher mL or QIAamp DNA Stool Kit. When the same spiked stools were grown on modified charcoal cefoperazone deoxycholate agar (mCCDA) plates before PCR, the sensitivity was 100 cells per ml stool, indicating that culturing of campylobacters on mCCDA plates is superior to direct DNA extraction at least when fresh stool samples are analysed by PCR.
Persson
Søren
S
Department of Bacteriology, Mycology and Parasitology, National Reference Laboratory for Enteropathogenic Bacteria, Unit of Gastrointestinal Infections, Statens Serum Institut, Artillerivej 5, Building 37B, 2300 Copenhagen S, Denmark.
Olsen
Katharina Ep
KE
Department of Bacteriology, Mycology and Parasitology, National Reference Laboratory for Enteropathogenic Bacteria, Unit of Gastrointestinal Infections, Statens Serum Institut, Artillerivej 5, Building 37B, 2300 Copenhagen S, Denmark.
D000581:Amidohydrolases / Q000235:genetics; D001222:Aspartate Kinase / Q000235:genetics; D002169:Campylobacter Infections / Q000175:diagnosis* / Q000382:microbiology; D017000:Campylobacter coli / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016123:Campylobacter jejuni / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D001222:Aspartate Kinase; D000581:Amidohydrolases; C019672:aminoacylase I