A feasible protocol for identifying macrolide resistant mutations in the 23S rRNA domain V sequence of Mycoplasma pneumoniae
Continuing a nationwide surveillance of pediatric Mycoplasma pneumoniae infections since 2008, our department has been analyzing macrolide-resistance conferring mutations in the pathogen’s 23S rRNA gene sequence. There are three target positions, approximately 600 bases apart. We had been reading these positions by amplifying the flanking sequences of these target positions in two different PCR reactions, followed by sequencing of each PCR product, independently. Recent advances have boosted the expected read length of Sanger sequencing using dye-terminators from ~500 bases to ~1 Kb. Owing to a constant demand to process tens of specimens, the authors sought to refine the protocols with an aim to reduce handling time, complexity, and cost. Hereby presented is our refined procedure that enhanced our laboratory’s capacity enormously.