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

Abstract

<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>

Tracking the introduction and spread of SARS-CoV-2 in coastal Kenya

“…Nasopharyngeal (NP) and oropharyngeal (OP) swabs were collected into a single tube and transported to KEMRI-Wellcome Trust Research Programme (KWTRP) for SARS-CoV-2 diagnosis. Briefly, the positive infections were RNA purified using a QIAGEN extraction kit followed by real-time reverse-transcription PCR (RT-PCR) (Agoti et al, 2020; Said et al, 2020).…”

“…The RRTs collected nasopharyngeal (NP) and oropharyngeal (OP) swabs into a single tube and transported them to the KEMRI-Wellcome Trust Research Programme (KWTRP) for SARS-CoV-2 diagnosis. The laboratory diagnostic protocol for SARS-CoV-2 at KWTRP has been described elsewhere 8,10 Briefly, the positive infections were identified using a two stage procedure, first, viral RNA purification using a QIAGEN extraction kit followed by real-time reverse-transcription PCR (RT-PCR) using one or two of four SARS-CoV-2 detection assays that we deployed at the centre since the beginning of the pandemic namely the Berlin Charite protocol, Europe Virus Archive Global (EVA-g) protocol, Beijing Genomics Institute (BGI) protocol and Da An Commercial Kit protocol (Said et al, 2020).…”

Validation of saline, PBS and a locally produced VTM at varying storage conditions to detect the SARS-CoV-2 virus by qRT-PCR

“…For the qRT-PCR assay, the European Virus Archive-GLOBAL (EVAg) primer/probe set containing 1.75μl primer probe mix, 3.75μl Nuclease free water, 2.5μl TaqMan™ Fast Virus master mix and 2.5μl of template RNA was used [12]. Cycling conditions were 50˚C for 5min, 95˚C for 20sec then 40 cycles of 95˚C for 3sec and 58˚C for 45sec on the QuantStudio 5 and 7 qRT-PCR systems (Applied Biosystems, CA).…”

“…Furthermore, data obtained from the PCR analyser were in Ct values. These were reviewed and interpreted using a Ct cut-off of 36, as determined by Mohammed et al (2020), interpreting those values of less than and greater than the cut-off as positive and negative test results, respectively [12]. The qRT-PCR machine also gave an 'undetermined' result among the Ct values, and these were interpreted as negative test results.…”

Validation of saline, PBS and a locally produced VTM at varying storage conditions to detect the SARS-CoV-2 virus by qRT-PCR

“…For the qRT-PCR assay, the European Virus Archive -GLOBAL (EVAg) primer/probe set containing 1.75μl primer probe mix, 3.75μl Nuclease free water, 2.5μl TaqMan™ Fast Virus master mix and 2.5μl of template RNA were used (12). Cycling conditions were 50°C for 5min, 95°C for 20sec then 40 cycles of 95°C for 3sec and 58°C for 45sec on the QuantStudio 5 and 7 qRT-PCR systems (Applied Biosystems, CA).…”