The viral aetiology of influenza-like illnesses in Kampala and Entebbe, Uganda, 2008

Balinandi, Stephen; Bakamutumaho, Barnabas; Kayiwa, John T.; Ongus, Juliette; Oundo, Joseph; Awor, Anna C.; Lutwama, Julius J.

A commercially-available multiplex PCR kit (Seeplex ® RV Detection; Seegene Inc, Rockville, MD, USA) for the detection of adenovirus, influenza A and B viruses, respiratory syncytial viruses A and B, human metapneumovirus, parainfluenza viruses 1, 2 and 3, rhinovirus A, and coronaviruses 229E and OC43 was used. This kit has been used elsewhere to identify respiratory viruses in similar specimens. 20,21 The assay was performed according to the manufacturer's instructions. Briefly, total nucleic acids (both ribonucleic acid [RNA] and deoxyribonucleic acid [DNA]) were extracted from the collected specimens (Viral Gene-Spin TM , iNtRON Biotechnology, Gyeonggi-do, Korea) and target sequences present in the extracts were then reverse transcribed (for the RNA form) and amplified in two separate assays of multiplex polymerase chain reaction (PCR; mPCR) using virus-specific kit primers. One assay contained primers for adenovirus (amplicon size 534 bp), human metapneumovirus (469 bp), coronavirus 229E (375 bp), parainfluenza virus 1 (324 bp), parainfluenza virus 2 (264 bp), and parainfluenza virus 3 (219 bp), whilst the other mPCR assay had primer sets for influenza A virus (513 bp), influenza B virus (455 bp), respiratory syncytial virus B (391 bp), rhinovirus A (337 bp), respiratory syncytial virus A (273 bp) and coronavirus OC43 (231 bp). The amplified products were observed using agarose gel electrophoresis. Specimens that had matching bands corresponding to the expected amplicon sizes for each type of virus relative to the molecular weight marker (Seeplex ® ) were scored as positive for that virus. Kit positive and negative controls were also included in every assay run. An internal positive control (Seeplex ® ) was also included to evaluate the amplification efficiency of each tested specimen.