Guidelines in Designing a Universal Primer Mixture to Probe and Quantify Antibiotic-Resistant Genes Using the Polymerase Chain Reaction (PCR)

Multidrug resistance efflux pumps (MDREPs) in biofilm communities have become an increasingly expensive problem in clinical settings. Polymerase chain reaction (PCR)-based detection can be used to diagnose and characterize these genes, but this requires effective primer design to minimize false positives and negatives in test conclusions. A universal primer approach has previously been used to detect conserved core genes but not for accessory genes such as MDREPs. This study describes a guideline for the design of primers used in the detection of MDREP genes and an optimization approach for creating primers by using multiple sequence alignments to target conserved regions <i>in silico</i>, progressing from <i>in silico</i> to <i>in vitro</i> to generate working primers. Using this approach, this paper was able to generate primers to target <i>sugE</i>, a small multidrug resistance (SMR) protein found in microbial species. Primers were tested positively against synthetic DNA sequences but were inconsistent with DNA extracted from the organism of interest. Primer design informs the shortfalls of this detection technique and the difficulty in characterizing such genomic elements.