Creation of Universal Primers Targeting Nonconserved, Horizontally Mobile Genes: Lessons and Considerations

Effective and accurate primer design is an increasingly important skill, as the use of PCR-based diagnostics in clinical and environmental settings is on the rise. While universal primer sets have been successfully designed for highly conserved core genes, such as 16S rRNA, and characteristic genes, such as dsrAB and dnaJ, primer sets for mobile, accessory genes such as multidrug resistance efflux pumps (MDREP) have not been explored. Here, we describe an approach to create universal primer sets for select MDREP genes chosen from five superfamilies (small multidrug resistance [SMR], major facilitator superfamily [MFS], multidrug and toxic [compound] extrusion [MATE], ATP-binding cassette [ABC], resistance-nodulation-cell division [RND], and proteobacterial antimicrobial compound efflux [PACE]) identified in a model community of six members (Acetobacterium woodii, Bacillus subtilis, Desulfovibrio vulgaris, Geoalkalibacter subterraneus, Pseudomonas putida, and Thauera aromatica). Using sequence alignments and in silico PCR analyses, a new approach for creating universal primer sets targeting mobile, nonconserved genes has been developed and compared to more traditional approaches used for highly conserved genes. The potential shortfalls of the primer sets designed this way are discussed. The approach described here can be adapted to any unique gene set and can aid in creating a wider, more robust library of primer sets to detect less-conserved genes and improve the field of PCR-based screening research. IMPORTANCE Increasing use of molecular detection methods, specifically PCR and quantitative PCR (qPCR), requires utmost confidence in the results while minimizing false positives and false negatives due to poor primer designs. Frequently, these detection methods are focused on conserved core genes, which limits their applications. These screening methods are being used in various industries for specific genetic targets or key organisms, such as viral or infectious strains, or characteristic genes indicating the presence of key metabolic processes. The significance of this work is to improve primer design approaches to broaden the scope of detectable genes. The use of the techniques explored here will improve detection of nonconserved genes through unique primer design approaches. Additionally, the approaches here highlight additional, important information which can be gleaned during the in silico phase of primer design and will improve our gene annotations based on percent identities.