Preclinical evaluation of two real-time, reverse transcription-PCR assays for detection of the severe acute respiratory syndrome coronavirus

Abstract

We verified the analytical performance characteristics of a previously described real-time reverse transcription-PCR (RT-PCR) assay targeting the open reading frame (ORF) 1b region of the severe acute respiratory syndrome coronavirus (SARS-CoV) with RNA transcripts. We then compared it to a novel nucleocapsid gene real-time RT-PCR assay with genomic RNA. The assays differed only in the primer and probe sequences and final concentrations. A commercially available armored RNA (Ambion, Austin, Tex.) was evaluated as positive control for the ORF 1b assay. The analytical sensitivity, reproducibility, amplification efficiency, and dynamic range of the assays were similar. Both were specific for SARS-CoV as determined by testing against human CoV 229E and OC43, specimens from patients without SARS, and by BLAST searches of GenBank for primer and probe sequence homology. The armored RNA was found to be a suitable positive control for the ORF 1b assay that could be reliably recovered and amplified from a variety of clinical specimens.

FIG. 1.

Standard curve for the ORF 1b assay generated with the BNI-1 RNA transcript. Each point represents the mean of the results from four determinations.

FIG. 2.

Serial log₁₀ dilutions of SARS-CoV genomic RNA tested with the ORF 1b (▴) and N gene (▪) assays. Each point represents the mean of the results from four determinations.

FIG. 3.

Amplification plots of serial log₁₀ dilutions of SARS-CoV genomic RNA in the ORF 1b (black) and N gene (gray) assays.