gms | German Medical Science

sBackground. In November 2002, an outbreak of severe atypical pneumonia was first reported in Guangdong Province of China. The disease quickly expanded to over 25 countries, resulting in 812 deaths from 8439 reported cases. The World Health Organisation named it Severe Acute Respiratory Syndrome (SARS) and the ethiologic agent of SARS is a coronavirus (SARS-CoV) which was identified in March 2003. Serological testing is possible only on specimens obtained more than 21 days after the onset of the illness and, consequently, they can not be used for rapid diagnosis. Genome sequences of numerous SARS-CoV isolates have been established and provide the basis for the development of specific molecular tests. However, viral excretion is low during the initial phase of a SARS-CoV infection. Therefore, it is crucial to develop highly sensitive molecular assays.

Objective. The aim of the work was to develop and to evaluate the performance of a qualitative SARS-CoV real-time NASBA assay used in combination with magnetic silica-based RNA extraction.

Methods. The study was performed on an External Quality Assurance (EQA) panel and on different viral strains spiked in lysis buffer or in negative clinical specimens. Co-extraction of SARS-CoV RNA and an Internal Control (IC) RNA was performed using the NucliSens® MiniMagä and the NucliSens Magnetic Extraction Reagents. For NASBA amplification, reagents from the NucliSens Basic Kit were used. The viral and the IC RNAs were co-amplified and co-detected using primers and molecular probes derived from ORF 1b of the replicase gene (POL 1) and from the nucleocapsid (NUC) gene. Reactions were monitored in real-time in a Nuclisens EasyQ Analyzer. Assay sensitivity was investigated on viral strains spiked in lysis buffer and compared to real-time quantitative RT-PCR methods (based on LightCyclerâ technology) from Roche (LightCycler SARS-CoV Quantification Kit) and Artus (RealArtä HPA-Coronavirus LC RT PCR Reagents).

Results. A 100% correct score was obtained on the EQA panel. The SARS-CoV EasyQ assay was at least 10 fold more sensitive than the kit from Roche. It showed at least similar performances to the one from Artus, depending on the primer / beacon combination and the viral strain used. In spiked specimens (gargarism, nasopharyngeal swabs transport medium, urine, plasma and stool) the limit of detection was approximately one copy of input RNA. A separate study performed by the team of C. C. Ginocchio (LIJ Health System Laboratories, NY), showed the absence of cross reactivity with other relevant viruses. More studies are currently in progress in our laboratory to confirm this observation and also to perform testing on clinical SARS specimens.

Conclusions. The results of this study demonstrate that our real-time NASBA assay provides a valuable rapid and sensitive alternative to RT-PCR for qualitative SARS-CoV RNA detection. Moreover, the data indicate that our new sample preparation system, which combines the advantages of the Boom method for RNA extraction with the convenience of magnetic beads technology, can successfully be used in combination with real-time NASBA assays.