tetano
Editor, Senior Moderator
Wellcome Open Res
. 2022 Mar 4;5:162.
doi: 10.12688/wellcomeopenres.16063.2. eCollection 2020.
An optimization of four SARS-CoV-2 qRT-PCR assays in a Kenyan laboratory to support the national COVID-19 rapid response teams
Khadija Said Mohammed[SUP] #[/SUP][SUP] 1 [/SUP], Zaydah R de Laurent[SUP] #[/SUP][SUP] 1 [/SUP], Donwilliams O Omuoyo[SUP] #[/SUP][SUP] 1 [/SUP], Clement Lewa[SUP] 1 [/SUP], Elijah Gicheru[SUP] 1 [/SUP], Robinson Cheruiyot[SUP] 1 [/SUP], Brian Bartilol[SUP] 1 [/SUP], Shadrack Mutua[SUP] 1 [/SUP], Jennifer Musyoki[SUP] 1 [/SUP], Horace Gumba[SUP] 1 [/SUP], Jedidah Mwacharo[SUP] 1 [/SUP], Debra Riako[SUP] 1 [/SUP], Shaban J Mwangi[SUP] 1 [/SUP], Bonface M Gichuki[SUP] 1 [/SUP], Lydia Nyamako[SUP] 1 [/SUP], Angela Karani[SUP] 1 [/SUP], Henry Karanja[SUP] 1 [/SUP], Daisy Mugo[SUP] 1 [/SUP], John N Gitonga[SUP] 1 [/SUP], Susan Njuguna[SUP] 1 [/SUP], Wilson Gumbi[SUP] 1 [/SUP], Brian Tawa[SUP] 1 [/SUP], Metrine Tendwa[SUP] 1 [/SUP], Wesley Cheruiyot[SUP] 1 [/SUP], Yiakon Sein[SUP] 1 [/SUP], John K Nyambu[SUP] 2 [/SUP], Shem O Patta[SUP] 3 [/SUP], Thani Suleiman Thani[SUP] 3 [/SUP], Eric K Maitha[SUP] 4 [/SUP], Benson Kitole[SUP] 4 [/SUP], Mohamed S Mwakinangu[SUP] 5 [/SUP], Barke S Muslih[SUP] 6 [/SUP], John Ochieng Otieno[SUP] 7 [/SUP], Joyce U Nyiro[SUP] 1 [/SUP], Patience Kiyuka[SUP] 1 [/SUP], Leonard Ndwiga[SUP] 1 [/SUP], Kevin Wamae[SUP] 1 [/SUP], Domtila Kimani[SUP] 1 [/SUP], Johnstone Makale[SUP] 1 [/SUP], John Mwita Morobe[SUP] 1 [/SUP], Victor Osoti[SUP] 1 [/SUP], Arnold W Lambisia[SUP] 1 [/SUP], Calleb Odundo[SUP] 1 [/SUP], Salim Mwarumba[SUP] 1 [/SUP], Martin Mutunga[SUP] 1 [/SUP], Philip Bejon[SUP] 1 8 [/SUP], Benjamin Tsofa[SUP] 1 [/SUP], Charles N Agoti[SUP] 1 [/SUP], Lynette Isabella Ochola-Oyier[SUP] 1 [/SUP]
Affiliations
Abstract
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.
Keywords: COVID-19; SARS-CoV-2; coronavirus; diagnosis; optimization; qRT-PCR.
. 2022 Mar 4;5:162.
doi: 10.12688/wellcomeopenres.16063.2. eCollection 2020.
An optimization of four SARS-CoV-2 qRT-PCR assays in a Kenyan laboratory to support the national COVID-19 rapid response teams
Khadija Said Mohammed[SUP] #[/SUP][SUP] 1 [/SUP], Zaydah R de Laurent[SUP] #[/SUP][SUP] 1 [/SUP], Donwilliams O Omuoyo[SUP] #[/SUP][SUP] 1 [/SUP], Clement Lewa[SUP] 1 [/SUP], Elijah Gicheru[SUP] 1 [/SUP], Robinson Cheruiyot[SUP] 1 [/SUP], Brian Bartilol[SUP] 1 [/SUP], Shadrack Mutua[SUP] 1 [/SUP], Jennifer Musyoki[SUP] 1 [/SUP], Horace Gumba[SUP] 1 [/SUP], Jedidah Mwacharo[SUP] 1 [/SUP], Debra Riako[SUP] 1 [/SUP], Shaban J Mwangi[SUP] 1 [/SUP], Bonface M Gichuki[SUP] 1 [/SUP], Lydia Nyamako[SUP] 1 [/SUP], Angela Karani[SUP] 1 [/SUP], Henry Karanja[SUP] 1 [/SUP], Daisy Mugo[SUP] 1 [/SUP], John N Gitonga[SUP] 1 [/SUP], Susan Njuguna[SUP] 1 [/SUP], Wilson Gumbi[SUP] 1 [/SUP], Brian Tawa[SUP] 1 [/SUP], Metrine Tendwa[SUP] 1 [/SUP], Wesley Cheruiyot[SUP] 1 [/SUP], Yiakon Sein[SUP] 1 [/SUP], John K Nyambu[SUP] 2 [/SUP], Shem O Patta[SUP] 3 [/SUP], Thani Suleiman Thani[SUP] 3 [/SUP], Eric K Maitha[SUP] 4 [/SUP], Benson Kitole[SUP] 4 [/SUP], Mohamed S Mwakinangu[SUP] 5 [/SUP], Barke S Muslih[SUP] 6 [/SUP], John Ochieng Otieno[SUP] 7 [/SUP], Joyce U Nyiro[SUP] 1 [/SUP], Patience Kiyuka[SUP] 1 [/SUP], Leonard Ndwiga[SUP] 1 [/SUP], Kevin Wamae[SUP] 1 [/SUP], Domtila Kimani[SUP] 1 [/SUP], Johnstone Makale[SUP] 1 [/SUP], John Mwita Morobe[SUP] 1 [/SUP], Victor Osoti[SUP] 1 [/SUP], Arnold W Lambisia[SUP] 1 [/SUP], Calleb Odundo[SUP] 1 [/SUP], Salim Mwarumba[SUP] 1 [/SUP], Martin Mutunga[SUP] 1 [/SUP], Philip Bejon[SUP] 1 8 [/SUP], Benjamin Tsofa[SUP] 1 [/SUP], Charles N Agoti[SUP] 1 [/SUP], Lynette Isabella Ochola-Oyier[SUP] 1 [/SUP]
Affiliations
- PMID: 35330938
- PMCID: PMC8921690
- DOI: 10.12688/wellcomeopenres.16063.2
Abstract
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.
Keywords: COVID-19; SARS-CoV-2; coronavirus; diagnosis; optimization; qRT-PCR.