A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification

Abstract

An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 10(2)-fold higher than the standard real-time PCR assay and 10(7)-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.

Fig. 1

Comparison of enhanced and standard real-time amplification. For each sample, the ΔRn (the ratio of the amount of reporter dye emission to quenching dye emission) is plotted against the cycle number. The intensity of fluorescence is directly related to the amount of input target DNA. The fewer cycles it takes to reach a detectable level of fluorescence the greater the initial copy number. (A) Clinical samples analyzed using enhanced real-time PCR. The SARS-CoV positive samples are clearly distinguished from the SARS-CoV negative samples, indicated by arrows. (B) The same clinical samples shown in A using standard real-time PCR. SARS-CoV, positive, and negative samples are not easily differentiated. (C) Serial 10-fold dilutions of SARS-CoV (2 × 10^2.5 TCID₅₀/ml [uppermost trace] to 2 × 10^−10.5 TCID₅₀/ml) using enhanced real-time PCR. Lower traces show reverse transcription negative control and TaqMan negative control. (D) Serial 10-fold dilutions of SARS-CoV (2 × 10^2.5 TCID₅₀/ml [uppermost trace] to 2 × 10^−10.5 TCID₅₀/ml) using standard real-time PCR.