Role of viral and bacterial pathogens in causing pneumonia among Western Australian children: a case–control study protocol

Bhuiyan, Mejbah Uddin; Snelling, Thomas L; West, Rachel; Lang, Jurissa; Rahman, Tasmina; Borland, Meredith L; Thornton, Ruth; Kirkham, Lea-Ann; Sikazwe, Chisha; Martin, Andrew C; Richmond, Peter C; Smith, David W; Jaffe, Adam; Blyth, Christopher C

Quantification of bacterial loads in NPS specimen is performed using quantitative real time PCR (qPCR) using methods established at UWA School of Biomedical Science (Kirkham L et al. unpublished data). NPS in STGG are centrifuged at high speed to pellet bacteria and associated cells, DNA is then extracted from the pellet using enzymatic extraction and the QIAamp DNA minikit (Qiagen, Chadstone, VIC, Australia) as previously described [65] . Real-time qPCR to detect S. pneumoniae, S. aureus, M. catarrhalis, and non-typable H. influenzae is conducted on the CFX96 real-time PCR detection system (Bio-Rad, CA, USA) using the primers and probes listed in Table 4 . A standard curve is generated for each run for each reference strain. All samples are run in duplicate and density is calculated as an average of the two measurements. The bacterial load is assessed in colony forming units (CFU)/mL which is equivalent to copies/mL assuming one copy of a bacteria would yield one colony forming unit.