Verification of Three Commercial Quantitative RT-PCR Kits for COVID-19 Using Cycle Threshold Values
Keywords:
qRT-PCR, Cycle threshold, Cut off, COVID-19Abstract
Background and Objectives: During the SARS-CoV-2 pandemic, several effective and reliable qRT-PCR kits were accessible worldwide. To increase the capacity of COVID-19 detection the three commercial kits were chosen in our laboratory. Herein, we performed the verification of the three commercial qRT-PCR kits including a fully automation Cobas 6800 system, Allplex SARS-CoV2 Master Assay and Sansure Assay and finding the Ct values for interpretation and reporting. This study utilized the clinical samples and performed under our ISO 15189 certified laboratory.
Methods: Ten positive qRT-PCR for COVID-19 samples were 10-folds serially diluted with viral transport media as undilute, 1:10-1, 1:10-2, 1:10-3, 1:10-4, 1:10-5, and 1:10-6 dilution. Viral nucleic acid was extracted. The qRT-PCR was triplicate performed by using three commercial kits. The performance of the kits were considerated and the Ct values were recorded and calculated.
Results: We found that the performance of a fully automation Cobas 6800 system, Allplex SARS-CoV2
Master Assay and Sansure Assay with the maximum detectable dilution at 1:10-5, 1:10-3 and 1:10-3 respectively. In addition, we found the Ct values for precise interpretation for COVID-19 qRT-PCR testing. The grey zone Ct value of E gene is 36.67, ORF1ab gene is 35.59 and N gene is 36.09 which these values could be considered to repeat for accurate detection.
Conclusions: The result demonstrated that the best performance commercial kit was a fully automation Cobas
6800 system following by Allplex SARS-CoV2 Master Assay was equal to Sansure Assay. The Ct values of target genes (E = 36.67, ORF1ab = 35.59 and N= 36.09) were implemented for the qRT-PCR for COVID-19 guideline in our laboratory.
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