Preclinical evaluation of two real-time, reverse transcription-PCR assays for detection of the severe acute respiratory syndrome coronavirus.
We verified the analytical performance characteristics of a previously described real-time reverse transcription-PCR (RT-PCR) assay targeting the open reading frame (ORF) 1b region of the severe acute respiratory syndrome coronavirus (SARS-CoV) with RNA transcripts. We then compared it to a novel nucleocapsid gene real-time RT-PCR assay with genomic RNA. The assays differed only in the primer and probe sequences and final concentrations. A commercially available armored RNA (Ambion, Austin, Tex.) was evaluated as positive control for the ORF 1b assay. The analytical sensitivity, reproducibility, amplification efficiency, and dynamic range of the assays were similar. Both were specific for SARS-CoV as determined by testing against human CoV 229E and OC43, specimens from patients without SARS, and by BLAST searches of GenBank for primer and probe sequence homology. The armored RNA was found to be a suitable positive control for the ORF 1b assay that could be reliably recovered and amplified from a variety of clinical specimens.
Department of Medicine (Infectious Diseases), and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.
Adam M
AM
Bressler
Frederick S
FS
Nolte
D001992:Bronchoalveolar Lavage Fluid / Q000821:virology; D005243:Feces / Q000821:virology; D006801:Humans; D016366:Open Reading Frames / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012044:Regression Analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000302:isolation & purification*; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis*; D014871:Water Microbiology
D012367:RNA, Viral