Establishment of an in‐house real‐time RT‐PCR assay for the detection of severe acute respiratory syndrome coronavirus 2 using the first World Health Organization international standard in a resource‐limited country

Abstract

Background:The COVID-19 pandemic caused by SARS-CoV-2 remains public health burdens and many unresolved issues worldwide. Molecular assays based on real-time RT-PCR are critical for the detection of SARS-CoV-2 in clinical specimens from patients suspected of COVID-19. Objective:We aimed to establish and validate an in-house real-time RT-PCR for the detection of SARS-CoV-2.Methodology: Primers and probes sets in our in-house real-time RT-PCR assay were designed in conserved regions of the N and E target genes. Optimized multiplex realtime RT-PCR assay was validated using the first WHO International Standard (NIBSC code: 20/146) and evaluated clinical performance. Results:The limit of detection validated using the first WHO International Standard was 159 IU/ml for both E and N target genes. The evaluation of clinical performance on 170 clinical samples showed a positive percent agreement of 100% and the negative percent agreement of 99.08% for both target genes. The Kappa value of 0.99 was an excellent agreement, the strong correlation of C t values observed between two tests with r 2 = 0.84 for the E gene and 0.87 for the N gene. Notably, we assessed on 60 paired saliva and nasopharyngeal samples. The overall agreement was 91.66%, and Kappa value of 0.74 showed a high agreement between two types of samples. When using nasopharyngeal swabs as the reference standard, positive percent agreement, and negative percent agreement were 91.83% and 90.90%, respectively. Conclusion:In the present study, we established and validated an in-house real-time RT-PCR for molecular detection of SARS-CoV-2 in a resource-limited country.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Risk of transfusion‐transmitted infection with severe acute respiratory syndrome coronavirus 2 from blood donors in Japan

“…The sensitivity of our rRT‐PCR test was comparable to those of other tests reported 16 . Using this test, we found an RNAemia positivity rate of 4.6% among BDs who reported SARS‐CoV‐2 infection within 2 weeks after blood donation.…”

“…The sensitivity of our rRT-PCR test was comparable to those of other tests reported. 16 Using this test, we found an RNAemia positivity rate of 4.6% among BDs who reported SARS-CoV-2 infection within 2 weeks after blood donation. French and American studies conducted in similar donor populations reported an RNAemia positivity rate of 3.4-8.7%, suggesting that the frequency of RNAemia among BDs may be at a similar level across regions.…”

“…This ratio is similar to that of all BDs in Japan (70%/30%). The median period from blood donation to COVID-19 onset was 1 day (range: 0-5 days) in 16 BDs with the available information. This could not be quantified and was calculated by inverse estimation using logistic regression analysis.…”