An optimization of four SARS-CoV-2 qRT-PCR assays in a Kenyan laboratory to support the national COVID-19 rapid response teams

Wellcome Open Research

article

Philip Bejon¹  Benjamin Tsofa¹  Charles N. Agoti¹  Lynette Isabella Ochola-Oyier¹  Khadija Said Mohammed¹  Zaydah R. de Laurent¹  Donwilliams O. Omuoyo¹  Clement Lewa¹  Elijah Gicheru¹  Robinson Cheruiyot¹  Brian Bartilol¹  Shadrack Mutua¹  Jennifer Musyoki¹  Horace Gumba¹  Jedidah Mwacharo¹  Debra Riako¹  Shaban J. Mwangi¹  Bonface M. Gichuki¹  Lydia Nyamako¹  Angela Karani¹  Henry Karanja¹  Daisy Mugo¹  John N. Gitonga¹  Susan Njuguna¹  Wilson Gumbi¹  Brian Tawa¹  Metrine Tendwa¹  Wesley Cheruiyot¹  Yiakon Sein¹  John K. Nyambu³  Shem O. Patta³  Thani Suleiman Thani³  Eric K. Maitha⁴  Benson Kitole⁴  Mohamed S. Mwakinangu³  Barke S. Muslih⁵  John Ochieng Otieno⁶  Joyce U. Nyiro¹  Patience Kiyuka¹  Leonard Ndwiga¹  Kevin Wamae¹  Domtila Kimani¹  Johnstone Makale¹  John Mwita Morobe¹  Victor Osoti¹  Arnold W. Lambisia¹  Calleb Odundo¹  Salim Mwarumba¹  Martin Mutunga¹ 

[1] KEMRI-Wellcome Trust Research Programme;Nuffield Department of Medicine, Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, University of Oxford;Department Of Health Services;Kilifi County Hospital;Hola Referral Hospital;King Fahd Lamu County Referral Hospital

关键词: COVID-19;    SARS-CoV-2;    coronavirus;    qRT-PCR;    diagnosis;    optimization;   

DOI  :  10.12688/wellcomeopenres.16063.2

学科分类：内科医学

来源: Wellcome

【 摘 要 】

Background: The COVID-19 pandemic relies on real-time polymerase chain reaction (qRT-PCR) for the detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), to facilitate 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 a resource-limited setting.Methods: We used a SARS-CoV-2 positive control RNA sample to generate several 10-fold dilution series that were used for optimization and comparison of the performance of the four qRT-PCR assays: i) Charité Berlin primer-probe set, ii) European Virus Archive – GLOBAL (EVAg) primer-probe set, iii) DAAN premixed commercial kit and iv) Beijing Genomics Institute (BGI) premixed commercial kit. We adjusted the manufacturer- and protocol-recommended reaction component volumes for these assays and assessed the impact on cycle threshold (Ct) values.Results: The Berlin and EVAg E gene and RdRp assays reported mean Ct values within range of each other across the different titrations and with less than 5% difference. The DAAN premixed kit produced comparable Ct values across the titrations, while the BGI kit improved in performance following a reduction of the reaction components.Conclusion: We achieved a 2.6-fold and 4-fold increase in the number of tests per kit for the commercial kits and the primer-probe sets, respectively. All the assays had optimal performance when the primers and probes were used at 0.375X, except for the Berlin N gene assay. The DAAN kit was a reliable assay for primary screening of SARS-CoV-2 whereas the BGI kit’s performance was dependent on the volumes and concentrations of both the reaction buffer and enzyme mix. Our recommendation for SARS-CoV-2 diagnostic testing in resource-limited settings is to optimize the assays available to establish the lowest volume and suitable concentration of reagents required to produce valid results.

【 授权许可】

CC BY