Submitted to: FEMS Microbiology Ecology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/24/2011
Publication Date: 7/1/2011
Citation: Oakley, B., Dowd, S.E., Purdy, K. 2011. Pyrosequencing-based validation of a simple cell-suspension PCR assay for Campylobacter with application of high-processivity polymerase with novel internal amplification controls for rapid and specific detection.. FEMS Microbiology Ecology. 77(1):17-27. Interpretive Summary: Molecular diagnostics relies on the ability to target a particular gene or organism in a sample containing many different types of organisms. Recently, it has been recognized that most sample types, whether meat, produce, human, or environmental samples, harbor so many different types of microbes, that designing a diagnostic assay to target a particular pathogen can be overwhelming. To solve this problem, we wrote a software program to identify molecular assays which are optimally specific (i.e. only return a positive result for the targeted group) and sensitive (i.e. return a positive result for all members of the target group). The program was succesfully used to develop assays for particular genes and the specificity and sensitivity validated with high-throughput DNA sequencing. The manuscript also provides a website where the program can be downloaded.
Technical Abstract: The ability to specifically and sensitively target genotypes of interest is critical for the success of many PCR-based analyses of environmental or clinical samples that contain multiple templates. Next-generation sequence data clearly show that such samples can harbour hundreds to thousands of operational taxonomic units, a richness that precludes the manual evaluation of candidate assay specificity and sensitivity using multiple sequence alignments. To solve this problem, we have developed and validated a free software tool that automates the identification of PCR assays targeting specific genotypes in complex samples. ThermoPhyl uses user-defined target and nontarget sequence databases to assess the phylogenetic sensitivity and specificity of thermodynamically optimized candidate assays derived from primer design software packages. ThermoPhyl derives its name from its central premise of testing Thermodynamically optimal assays for Phylogenetic specificity and sensitivity and can be used for two primer (traditional PCR) or two primers with an internal probe (e.g. TaqMans qPCR) application and potentially for oligonucleotide probes. Here, we describe the use of ThermoPhyl for traditional PCR and qPCR assays. PCR assays selected using ThermoPhyl were validated using 454 pyrosequencing of a traditional specific PCR assay and with a set of four genotype-specific qPCR assays applied to estuarine sediment samples.