Real-time NASBA detection of SARS-associated coronavirus and comparison with real-time reverse transcription-PCR.
Severe acute respiratory syndrome (SARS) exhibits a high mortality rate and the potential for rapid epidemic spread. Additionally, it has a poorly defined clinical presentation, and no known treatment or prevention methods. Collectively, these factors underscore the need for early diagnosis. Molecular tests have been developed to detect SARS coronavirus (SARS-CoV) RNA using real time reverse transcription polymerase chain reaction (RT-PCR) with varying levels of sensitivity. However, RNA amplification methods have been demonstrated to be more sensitive for the detection of some RNA viruses. We therefore developed a real-time nucleic acid sequence-based amplification (NASBA) test for SARS-CoV. A number of primer/beacon sets were designed to target different regions of the SARS-CoV genome, and were tested for sensitivity and specificity. The performance of the assays was compared with RT-PCR assays. A multi-target real-time NASBA application was developed for detection of SARS-CoV polymerase (Pol) and nucleocapsid (N) genes. The N targets were found to be consistently more sensitive than the Pol targets, and the real-time NASBA assay demonstrates equivalent sensitivity when compared to testing by real-time RT-PCR. A multi-target real-time NASBA assay has been successfully developed for the sensitive detection of SARS-CoV.
Keightley
Maria Cristina
MC
Clinical Virology Laboratory, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213, USA.
Sillekens
Peter
P
Schippers
Wim
W
Rinaldo
Charles
C
George
Kirsten St
KS
D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D045473:Severe acute respiratory syndrome-related coronavirus / Q000302:isolation & purification*; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology
D012367:RNA, Viral