Reflexes

• A new RT-PCR assay based on ORF1a (Viroselect) for the detection of severe acute respiratory syndrome coronavirus-2.
• Viroselect showed high concordance with manual and automated real-time RT-PCR detection assays.
• Viroselect proved useful in resolving inconclusive samples.

Abstract

Bottom

India bears the second largest burden of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. A multitude of reverse transcription-polymerase chain reaction (RT-PCR) detection assays with disparate genetic targets are available, including high-throughput automated platforms. Varying agreement and interpretation of diagnostic results in this setting can result in significant reporting delays, leading to suboptimal disease management.

This article reports on the development of a new ORF1a-based RT-PCR assay for SARS-CoV-2, Viroselect, which shows high concordance with conventional assays and the ability to resolve inconclusive results generated during the peak of the epidemic in Mumbai, India.

Methods

A unique target region within the SARS-CoV-2 ORF1a, the non-structural protein 3 (nsp3) region, was used to design and develop the assay. This hypervariable region (1923–3956) between SARS-CoV-2, SARS-CoV-1, and the Middle East respiratory syndrome coronavirus was used to design the primers and probes for the RT-PCR assay.

The concordance of this trial was assessed with commonly used emergency use authorization manual kits (US Food and Drug Administration) and a high throughput automated test platform. In addition, a retrospective analysis was carried out using Viroselect on samples reported as “inconclusive” between April and October 2020.

Results

In total, 701 samples were analyzed. Concordance analysis of 477 samples demonstrated a high overall agreement of Viroselect with the manual (87.6%) and automated (84.7%) assays. Additionally, in a retrospective analysis of 224 additional samples reported as ‘inconclusive’, Viroselect was able to resolve 100% (19/19) and 93.7% (192/205) of the samples that had inconclusive results on manual platforms. and automated high performance. , respectively.

Conclusion

Viroselect was highly consistent with conventional assays, both manual and automated, and has the potential to resolve inconclusive samples.

Keywords

SARS-CoV 2RT-PCR Inconclusive samples Viroselect High throughput SARS-CoV-2 test Delay in reporting