Real-time reverse transcription-polymerase chain reaction assay for SARS-associated coronavirus

Abstract

A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome-associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection.

Figure

Typical amplification plot derived from serial 10-fold dilutions of severe acute respiratory syndrome–associated coronavirus RNA transcripts using TaqMan reverse transcription–polymerase chain reaction primer/probe sets SARS1, SARS2, and SARS3. A PCR Base Line Subtractive Curve Fit view of the data is shown with relative fluorescence units (RFU) plotted against cycle number. The default setting of 10 times the standard deviation of fluorescence in all wells over the baseline cycles was used to calculate the threshold cycle, or C_T value, for a positive reaction (horizontal line). Inserts show standard curve analysis of the RNA amplification plots with C_T values plotted against starting copy number. Plots derived from dilutions containing 2 x 10⁶ to 20 transcript copies for SARS2 and SARS3, and 7.5 x 10⁶ to 75 copies for SARS1.