title: Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase and novel internal amplification controls for rapid and specific detection.
abstract: Although Campylobacter is an important food-borne human pathogen, there remains a lack of molecular diagnostic assays that are simple to use, cost-effective, and provide rapid results in research, clinical, or regulatory laboratories. Of the numerous Campylobacter assays that do exist, to our knowledge none has been empirically tested for specificity using high-throughput sequencing. Here we demonstrate the power of next-generation sequencing to determine the specificity of a widely cited Campylobacter-specific polymerase chain reaction (PCR) assay and describe a rapid method for direct cell suspension PCR to quickly and easily screen samples for Campylobacter. We present a specific protocol which eliminates the need for time-consuming and expensive genomic DNA extractions and, using a high-processivity polymerase, demonstrate conclusive screening of samples in <1 h. Pyrosequencing results show the assay to be extremely (>99%) sensitive, and spike-back experiments demonstrated a detection threshold of <10(2) CFU mL(-1). Additionally, we present 2 newly designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls. Empirical testing of putative taxon-specific assays using high-throughput sequencing is an important validation step that is now financially feasible for research, regulatory, or clinical applications.
authors.affiliation: Poultry Microbiological Safety Research Unit, USDA Agricultural Research Service, Richard B. Russell Agricultural Research Center, Athens, GA 30605, USA. brian.oakley@ars.usda.gov
authors.forename: Brian B
authors.initials: BB
authors.lastname: Oakley
authors.forename: J Eric
authors.initials: JE
authors.lastname: Line
authors.forename: Mark E
authors.initials: ME
authors.lastname: Berrang
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Johnson
authors.forename: R Jeff
authors.initials: RJ
authors.lastname: Buhr
authors.forename: Nelson A
authors.initials: NA
authors.lastname: Cox
authors.forename: Kelli L
authors.initials: KL
authors.lastname: Hiett
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Seal
mesh_terms: D002167:Campylobacter / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004259:DNA-Directed DNA Polymerase / Q000145:classification*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D004259:DNA-Directed DNA Polymerase