Evaluation of the basic performance of the Ampdirect^TM 2019-nCoV detection kit without the RNA purification steps and inter-measurer reproducibility of cycle thresholds values

Abstract

This study evaluated the basic performance of the Ampdirect^TM 2019-nCoV detection kit (Shimadzu Corporation), which does not require RNA purification and inter-measurer reproducibility of cycle thresholds. Within-run imprecision (repeatability) and within-laboratory imprecision (total precision) tests were performed on samples of EDX SARS-CoV-2 Standard (Bio-Rad Laboratories, Inc.) adjusted to two concentrations. The Coefficient of Variation (CV) of the cycle thresholds was stable at less than 2%. For the limit of detection test, samples of EDX SARS-CoV-2 Standard adjusted to 6 concentrations were measured. The limit of detection was 250 copies/mL for the N1 gene and 750 copies/mL for the N2 gene, which were sufficient for laboratory diagnosis of COVID-19. Comparison with the method according to the pathogen detection manual of the National Institute of Infectious Diseases using clinical samples measured from November 2022 to February 2023 showed an overall agreement rate of 96.3% (104/108 samples). Four discrepancy samples had low viral loads with cycle thresholds of 36 or higher. Inter-measurer reproducibility of cycle thresholds showed that the CV of cycle thresholds converged between 0.79% and 1.33%, respectively. However, significant differences in cycle thresholds between measurers were found by multiple comparison test (p < 0.05). This reagent has good basic performance and is expected to be widely used in clinical laboratories as a tool for laboratory diagnosis of SARS-CoV-2. On the other hand, it was necessary to consider inter- measurer reproducibility for quantitative evaluation of the cycle threshold.