Clear or White? A RT-PCR plate comparison for SARS-CoV-2 diagnosis

Abstract

Abstract Objective: During the COVID-19 pandemic, laboratories have used various extraction and amplification kits, associated with different auxiliary materials. This study aims to investigate how different types of plates may affect RT-PCR performance. Methods: Data on the positive controls (PCs) of RT-PCR runs for SARS-CoV-2 detection between December 2020 and April 2022 was collected retrospectively in the Molecular biology department of the County Emergency Clinical Hospital of Târgu Mureș’s clinical laboratory. The materials used consisted in MOLgen SARS-CoV-2 (Adaltis) and EliGene COVID19 CONFIRM 500 R amplification kits, 96-well full-skirted white and clear plates, and clear films. Microsoft Excel was used for the database and it included information about Cycle threshold (Ct) and maximum fluorescence. Statistical analysis, performed in MedCalc, consisted of Grubbs test, Kolmogorov–Smirnov Test, F test, T student test, and Mann-Whitney test to compare central tendencies. The significance threshold was set at p<0.05. Results: The Ct comparison for MOLgen kit white plates vs clear plates: FAM channel- U=1052.5, Z=2.07, p=0.038, medians for white plates and clear plates were 22.80 and 23.25, respectively; ROX channel- U=784, Z=3.21, p=0.001, medians 21.93 and 21.43, respectively; Cy5 channel- U=1028.5, Z=1.95, p=0.518, medians 22.12, 21.75, respectively. For EliGene kit: U=848.5, Z=3.27, p=0.001, medians 28.26 (white plates) and 28.0 (clear plates). Comparison of the maximum fluorescence reached on both kits with white and clear plates computed p values <0.0001. Conclusions: Between white and clear plates there are statistically significant differences considering Ct values and maximum read fluorescence, but with no impact on test outcome.