title: Evaluation of advanced reverse transcription-PCR assays and an alternative PCR target region for detection of severe acute respiratory syndrome-associated coronavirus.
abstract: First-generation reverse transcription-PCR (RT-PCR) assays for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) gave false-negative results in a considerable fraction of patients. In the present study, we evaluated two second-generation, replicase (R) gene-based, real-time RT-PCR test kits--the RealArt HPA coronavirus LC kit (Artus, Hamburg, Germany) and the LightCycler SARS-CoV quantification kit (Roche, Penzberg, Germany)--and a real-time RT-PCR assay for the nucleocapsid (N) gene. Detecting the N-gene RNA might be advantageous due to its high abundance in cells. The kits achieved sensitivities of 70.8% (Artus) and 67.1% (Roche) in 66 specimens from patients with confirmed SARS (samples primarily from the upper and lower respiratory tract and stool). The sensitivity of the N-gene assay was 74.2%. The differences in all of the sensitivities were not statistically significant (P = 0.680 [analysis of variance]). Culture cells initially contained five times more N- than R-gene RNA, but the respective levels converged during 4 days of virus replication. In clinical samples the median concentrations of R- and N-gene RNA, respectively, were 1.2 x 10(6) and 2.8 x 10(6) copies/ml (sputum and endotracheal aspirates), 4.3 x 10(4) and 5.5 x 10(4) copies/ml (stool), and 5.5 x 10(2) and 5.2 x 10(2) copies/sample (throat swabs and saliva). Differences between the samples types were significant but not between the types of target RNA. All (n = 12) samples from the lower respiratory tract tested positive in all tests. In conclusion, the novel assays are more sensitive than the first-generation tests, but they still do not allow a comprehensive ruling out of SARS. Methods for the routine sampling of sputum without infection risk are needed to improve SARS RT-PCR.
authors.affiliation: Department of Virology, Bernhard-Nocht Institute for Tropical Medicine, Hamburg, Germany. drosten@bni-hamburg.de
authors.forename: Christian
authors.initials: C
authors.lastname: Drosten
authors.forename: Lily-Lily
authors.initials: LL
authors.lastname: Chiu
authors.forename: Marcus
authors.initials: M
authors.lastname: Panning
authors.forename: Hoe Nam
authors.initials: HN
authors.lastname: Leong
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Preiser
authors.forename: John S
authors.initials: JS
authors.lastname: Tam
authors.forename: Stephan
authors.initials: S
authors.lastname: Günther
authors.forename: Stefanie
authors.initials: S
authors.lastname: Kramme
authors.forename: Petra
authors.initials: P
authors.lastname: Emmerich
authors.forename: Wooi Loon
authors.initials: WL
authors.lastname: Ng
authors.forename: Herbert
authors.initials: H
authors.lastname: Schmitz
authors.forename: Evelyn S C
authors.initials: ES
authors.lastname: Koay
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005188:False Negative Reactions; D005814:Genes, Viral; D006801:Humans; D019251:Nucleocapsid / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000302:isolation & purification*; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis / Q000821:virology
chemical_list: D017931:DNA Primers; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase