Isolation of DNA for PCR assays from noncultivable Campylobacter jejuni isolates.
Isolates of Campylobacter jejuni shipped internationally often arrive in a noncultivable state. We describe a PCR-based methodology whereby phylogenetic information can be recovered from noncultivable C. jejuni stored in Wang's transport medium. The robustness of this methodology was initially tested using 5 previously characterized strains of C. jejuni isolated from various sources associated with poultry production. These isolates were stored in Wang's transport medium before being subjected to 1 of 5 treatments designed to render the stored cells noncultivable: prolonged storage at room temperature, prolonged incubation at 42 degrees C, multiple rounds of freezing and thawing, boiling, or contamination with Pseudomonas aeruginosa (ATCC 27853). This method resulted in DNA appropriate for PCR. An approximately 400-nucleotide amplicon from the flaA gene and an approximately 800-nucleotide amplicon from 16S rDNA were readily obtained, and a 1.5-kb section of the flaA locus was amplified from about half of the samples. These results indicate that this method may be useful for isolate typing schemes based on PCR amplification of Campylobacter DNA, including flaA short variable region (flaA SVR) sequencing, multilocus sequence typing (MLST), and flaA PCR-RFLP. By using this method, isolates unrecoverable from transport medium can still be used to provide phylogenetic information for epidemiological studies.
Callicott
K A
KA
United States Department of Agriculture, Agricultural Research Service, Poultry Microbiological Safety Research Unit, Athens, Georgia 30605, USA. kcallicott@saa.ars.usda.gov
Stern
N J
NJ
Hiett
K L
KL
D016123:Campylobacter jejuni / Q000235:genetics* / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D005819:Genetic Markers / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
D004269:DNA, Bacterial; D005819:Genetic Markers