Virologica Sinica

, Volume 24, Issue 3, pp 187–193 | Cite as

Evaluation of sensitivities and specificities of SARS-CoV detection by real-time quantitative reverse transcription-PCR assays

  • Li-li Xu
  • Zhi-hong Hu
  • Hua-lin Wang
  • Xiao Han
  • Fei Deng
Article
  • 30 Downloads

Abstract

The etiological agent of severe acute respiratory syndrome (SARS) was identified as a new coronavirus, termed SARS-CoV. Establishment of an efficient and sensitive diagnostic system of SARS-CoV genetic materials is crucial for SARS control. In this study, we quantified SARS-CoV mRNAs in both infected cell culture lysate and in supernatant by using Real-time quantitative revere transcription-PCR based on EvaGreen™ dye and Taqman-MGB probes. For extensive evaluation of sensitivities and specificities, 13 pairs of primers and 4 probes were designed based on different genes of SARS-CoV. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was selected as the internal control gene. Results showed that S-gene-specific PCR was the most sensitive for detection, but because of its sequence variability in the different viral strains, primers and a probe based on the N gene were suitable substitutions. Meanwhile, we found the mRNA concentrations in cell culture lysates were much higher than in cell supernatant and facilited more sensitive detection of the SARS-CoV.

Key words

SARS-CoV Sensitivities Specificities Evaluation 

CLC number

R373 

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References

  1. 1.
    Bressler A M, Nolte F S. 2004. Preclinical evaluation of two real-time, reverse transcription-PCR assays for detection of the severe acute respiratory syndrome coronavirus. J Clin Microbiol, 42: 987–991.PubMedCrossRefGoogle Scholar
  2. 2.
    Drosten C, Chiu L L, Panning M M, et al. 2003. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med, 348: 1967–1976.PubMedCrossRefGoogle Scholar
  3. 3.
    Drosten C, Gunther S, Preiser W, et al. 2004. Evaluation of advanced reverse transcription-PCR assays and an alternative PCR target region for detection of severe acute respiratory syndrome-associated coronavirus. J Clin Microbiol, 42: 2043–2047.PubMedCrossRefGoogle Scholar
  4. 4.
    Emery S L, Erdman D D, Bowen M D, et al. 2004. Real-time reverse transcription -polymerase chain reaction assay for SARS-associated coronavirus. Emerg Infect Dis, 10: 311–316.PubMedGoogle Scholar
  5. 5.
    Gueudin M, Vabret A, Petitjean J, et al. 2003. Quantitation of respiratory syncytial virus RNA in nasal aspirates of children by real-time RT-PCR assay. J Virol Methods, 109: 39–45.PubMedCrossRefGoogle Scholar
  6. 6.
    Hui R K, Zeng F, Chan C M, et al. 2004. Reverse transcriptase PCR diagnostic assay for the coronavirus associated with severe acute respiratory syndrome. J Clin Microbiol, 42: 1994–1999.PubMedCrossRefGoogle Scholar
  7. 7.
    Ksiazek T G, Erdman D, Goldsmith C S, et al. 2003. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med, 348: 1953–1966.PubMedCrossRefGoogle Scholar
  8. 8.
    Laurendeau I, Bahuau M, Vodovar N, et al. 1999. Taq-Man PCR-based gene dosage assay for predictive testing in individuals from a cancer family with INK4 locus haploinsufficiency. Clin Chem, 45: 982–986.PubMedGoogle Scholar
  9. 9.
    Peiris J S, Chu C M, Cheng V C, et al. 2003. Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study. Lancet, 361: 1767–1772.PubMedCrossRefGoogle Scholar
  10. 10.
    Peiris J S, Lai S T, Poon L L, et al. 2003. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet, 361: 1319–1325.PubMedCrossRefGoogle Scholar
  11. 11.
    Weidmann M, Zanotto P M, Weber F, et al. 2004. High-efficiency detection of severe acute respiratory syndrome virus genetic material. J Clin Microbiol, 42: 2771–2773.PubMedCrossRefGoogle Scholar
  12. 12.
    Wong S C, Chan J K, Lee K C, et al. 2005. Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens. J Clin Pathol, 58: 276–280.PubMedCrossRefGoogle Scholar

Copyright information

© Wuhan Institute of Virology, CAS and Springer-Verlag GmbH 2009

Authors and Affiliations

  • Li-li Xu
    • 1
  • Zhi-hong Hu
    • 1
  • Hua-lin Wang
    • 1
  • Xiao Han
    • 1
  • Fei Deng
    • 1
  1. 1.State Key Laboratory of Virology, Wuhan Institute of VirologyChinese Academy of SciencesWuhanChina

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