ARS | Publication request: Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter... of high-processivity polymerase with novel internal amplification controls for rapid and specific detection.

Submitted to: Diagnostic Microbiology and Infectious Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 3, 2011
Publication Date: February 1, 2012
Citation: Oakley, B., Line, J.E., Berrang, M.E., Johnson, J., Buhr, R.J., Cox Jr, N.A., Hiett, K.L., Seal, B.S. 2012. Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase with novel internal amplification controls for rapid and specific detection. Diagnostic Microbiology and Infectious Disease. 72(2):131-138.

Interpretive Summary: Campylobacters are the leading cause of human food-borne bacterial diseases and commonly associated as a commensal organism with poultry. There still remains a lack of reliable diagnostic assays that are simple to use, cost-effective and provide rapid results in research, clinical, or regulatory laboratories. Our research unit demonstrated the power of next-generation pyrosequencing to determine the specificity of a Campylobacter-specific PCR assay and an internal PCR control was developed to provide confidence in the assay results. The assay system is fast (less than one hour), cheap and simple because it can be used with direct cell suspension PCR to screen a variety of different sample types for Campylobacter. This avoids the time-consuming step of having to isolate nucleic acids as genomic DNA prior to testing for the presence of these important disease-causing bacteria. Using the next-generation pyrosequencing technology to examine the metagenomic bacterial community by non-cultural methods we were able to demonstrate that the PCR assay was extremely sensitive and highly specific for Campylobacter when assaying a complex mixed community of bacteria in a sample.

Technical 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 have been emperically 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 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 hr. 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 two newly-designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls (IAC).