An optimisation of four SARS-CoV-2 qRT-PCR assays in a Kenyan laboratory to support the national COVID-19 rapid response teams
Said K., de Laurent ZR., Omuoyo DO., Lewa C., Gicheru E., Cheruiyot R., Bartilol B., Mutua S., Musyoki J., Gumba H., Mwacharo J., Riako D., Mwangi SJ., Gichuki BM., Nyamako L., Karani A., Karanja H., Mugo D., Gitonga JN., Njuguna S., Gumbi W., Tawa B., Tendwa M., Cheruiyot W., Sein Y., Nyambu JK., Patta SO., Thani TS., Maitha EK., Kitole B., Mwakinangu MS., Muslih BS., Otieno JO., Nyiro JU., Kiyuka P., Ndwiga Marigi LM., Wamae K., Kimani D., Makale J., Morobe JM., Osoti V., Lambisia AW., Odundo C., Mwarumba S., Mutunga M., Bejon P., Tsofa B., Agoti CN., Ochola-Oyier LI.
<ns4:p><ns4:bold>Background:</ns4:bold> The global COVID-19 outbreak relies on a quantitative real-time polymerase chain reaction (qRT-PCR) for the detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2), to facilitate the roll-out of patient care and infection control measures. There are several qRT-PCR assays with little evidence on their comparability. We report alterations to the developers’ recommendations to sustain the testing capability in our setting, where the supply of testing reagents is limited.</ns4:p><ns4:p> <ns4:bold>Methods:</ns4:bold> Standards generated from a serially-diluted positive control and previously identified positive/negative samples were used to determine the optimal volumes of the qRT-PCR reagents and to evaluate the validity and performance of four assays: Charité Berlin and European Virus Archive – GLOBAL (EVAg) primer-probe sets, and DAAN and Beijing Genomics Institute (BGI) premixed commercial kits. A multiplex and singleplex RT-PCR kit was used with the two primer-probe sets and the recommended assay volumes of the two premixed kits were altered.</ns4:p><ns4:p> <ns4:bold>Results:</ns4:bold> In comparison to the multiplex RT-PCR kit, the singleplex RT-PCR kit combined with the primer-probe sets yielded consistent cycle threshold (Ct) values across the different titrations tested. The DAAN premixed kit produced comparable Ct values across the titrations, while the BGI kit showed incomparable Ct values and inconsistent results between batches using the manufacturer’s recommended volumes.</ns4:p><ns4:p> <ns4:bold>Conclusion:</ns4:bold> We achieved a 2.5-fold and 4-fold increase in the number of tests/kit for the premixed kits and the primer-probe sets, respectively. The primer-probe set assays were reliable and consistent, and we preferred a combination of an EVAg and a Berlin target. Any inconclusive result was repeated by different individuals following the same protocol. DAAN was a consistent and reliable assay even at lower concentrations from the stated recommendations. BGI in contrast, required dilution to improve its performance and was hence an assay that was used in combination with EVAg or Berlin targets.</ns4:p>