Thermostable heptaplex pcr assay for the detection of six respiratory bacterial pathogens

Abstract

A thermostabilized, multiplex polymerase chain reaction (mPCR) assay was developed in this study for the detection of six respiratory bacterial pathogens. Specific primers were designed for an internal amplification control (IAC) and six target sequences from Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Haemophilus influenzae. The resultant seven-band positive amplification control (PAC) of this heptaplex PCR assay corresponded to 105 base pairs (bp) of IAC, 202 bp of K. pneumoniae, 293 bp of S. aureus, 349 bp of S. pneumoniae, 444 bp of P. aeruginosa, 505 bp of M. tuberculosis, and 582 bp of H. influenzae. Results found that 6% (w/v) of the stabilizer was optimum to preserve the functional conformation of Taq DNA polymerase enzyme. This assay was stable at ambient temperature for at least 6 months. The sensitivity and specificity of this assay were both 100% when testing on the intended target organisms (n = 119) and non-intended species (n = 57). The mPCR assay developed in this study enabled accurate, rapid, and simple detection of six respiratory bacteria.