Development and evaluation of an efficient 3'-noncoding region based SARS coronavirus (SARS-CoV) RT-PCR assay for detection of SARS-CoV infections

Abstract

The severe acute respiratory syndrome (SARS) epidemic originating from China in 2002 was caused by a previously uncharacterized coronavirus that could be identified by specific RT-PCR amplification. Efforts to control future SARS outbreaks depend on the accurate and early identification of SARS-CoV infected patients. A real-time fluorogenic RT-PCR assay based on the 3'-noncoding region (3'-NCR) of SARS-CoV genome was developed as a quantitative SARS diagnostic tool. The ideal amplification efficiency of a sensitive SARS-CoV RT-PCR assay should yield an E value (PCR product concentration increase per amplification cycle) equal to 2.0. It was demonstrated that the 3'-NCR SARS-CoV based RT-PCR reactions could be formulated to reach excellent E values of 1.81, or 91% amplification efficacy. The SARS-CoV cDNA preparations derived from viral RNA extract and the cloned recombinant plasmid both exhibit the identical amplification characteristics, i.e. amplification efficacy using the same PCR formulation developed in this study. The viral genomic copy (or genomic equivalences, GE) per infectious unit (GE/pfu) of SARS-CoV used in this study was also established to be approximate 1200-1600:1. The assay's detection sensitivity could reach 0.005 pfu or 6-8 GE per assay. It was preliminarily demonstrated that the assay could efficiently detect SARS-CoV from clinical specimens of SARS probable and suspected patients identified in Taiwan. The 3'-NCR based SARS-CoV assay demonstrated 100% diagnostic specificity testing samples of patients with acute respiratory disease from a non-SARS epidemic region.

Fig. 1

(a) Real-time amplification and detection of SARS-CoV using the fluorogenic SARS-CoV 3′-NCR based RT-PCR system. SARS-CoV viral stock was serially diluted (dynamic range from 18,000 to 0.24 pfu/reaction) and used to generate viral cDNA to demonstrate that the assay is a copy number dependent reaction. Wells A1–A8 contained serially diluted viral copy numbers, i.e. pfu/reaction, to yield sequential sigmoid curves arranged from high through low copy number (left to right). B6–B8 wells were non-template controls that showed no noise, or background for this assay. (b) Real-time amplification and detection of the cloned SARS-CoV 3′-NCR, pHCV1 using the fluorogenic SARS-CoV 3′-NCR based RT-PCR system. Five-fold serial dilutions of the cloned SARS-CoV plasmid, pHCV1 (from 700,000 to 2 GE/reaction) were used to demonstrate that the assay is a copy number dependent reaction. Wells A1–A8 containing serially diluted pHCV1 yielded sequential sigmoid curves arranged from high through low copy number (left to right of the figure). B6–B8 wells were non-template controls that showed no noise, or background for this assay.

Fig. 2

Comparison of quantitative SARS-CoV standard curves derived from direct viral cDNA preparation and the cloned 3′-NCR SARS-CoV plasmid DNA, pHCV1. It was shown that both standard curves yield the same slope (two parallel lines taken from Fig. 1a and b). The GE/pfu ratio of 1:1600 could be derived from the direct readout of intercepts difference at Y-(GE/pfu). The GE/pfu ratio can also be calculated from the intercepts difference at X-(C_t cycle) of 12.0 cycles between viral cDNA (pfu) and cloned plasmid (GE) standard curves as followings: GE/pfu=E¹² or 1.81^12.0=1239.