Development of Taqman RT-nested PCR system for clinical SARS-CoV detection

Abstract

Severe acute respiratory syndrome (SARS) is an acute newly emerged infectious respiratory illness. The etiologic agent of SARS was named 'SARS-associated coronavirus' (SARS-CoV) that can be detected with reverse transcription-polymerase chain reaction (RT-PCR) assays. In this study, 12 sets of nested primers covering the SARS-CoV genome have been screened and showed sufficient sensitivity to detect SARS-CoV in RNA isolated from virus cultured in Vero 6 cells. To optimize further the reaction condition of those nested primers sets, seven sets of nested primers have been chosen to compare their reverse transcribed efficiency with specific and random primers, which is useful to combine RT with the first round of PCR into a one-step RT-PCR. Based on the sensitivity and simplicity of results, the no. 73 primer set was chosen as the candidate primer set for clinical diagnoses. To specify the amplicon to minimize false positive results, a Taqman RT-nested PCR system of no. 73 nested primer set was developed. Through investigations on a test panel of whole blood obtained from 30 SARS patients and 9 control persons, the specificity and sensitivity of the Taqman RT-nested PCR system was found to be 100 and 83%, respectively, which suggests that the method is a promising one to diagnose SARS in early stages.

Fig. 1

The distribution of 12 sets of nested primers across the genome of BJ01 (AY278488). The black bars represent known genes, the blue bars represent predicted genes. The 12 sets of nested primers are also shown in the map. The black or longer line represents the region of outer amplicon, whereas the red or shorter line represents the inner amplicon. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

The schematic map of sensitivities of 12 sets of nested primers tested on serial 10-fold diluted templates. The initial concentration of total RNA from virus and Vero 6 cells is 50 ng/μl, which corresponds to <10¹³ molecules of SARS-CoV per microliter. The asterisk represents positive signal. The sensitivities of nos. 73, 79, and 80 are 100–1000-fold greater than other nested primers pairs.

Fig. 3

Comparative efficiencies of reverse transcription primed by the random primer or a specific primer on 10-fold diluted series of BJ01 genome. The lower margin of each bar shows the level of sensitivity of these outer primers. The nos. 14, 62, and 73 have higher reverse transcription efficiency when transcribed with specific primer, whereas those primers of nos. 79, 80, 81, and 87 have higher reverse transcription efficiency when transcribed with the random primer. The values on vertical axis represent the diluted concentration of RNA compared with that of initial RNA (50 ng/μl).