Advertisement

Molecular Diagnosis of Severe Acute Respiratory Syndrome

  • Enders K. O. Ng
  • Y. M. Dennis Lo
Part of the Methods in Molecular Biology™ book series (MIMB, volume 336)

Abstract

The etiologic agent of severe acute respiratory syndrome (SARS) has been identified as a new type of coronavirus, known as SARS-coronavirus (SARS-CoV). Although the SARS epidemic has subsided, many authorities, including the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC), have warned of the possible re-emergence of this highly infectious disease. Although antibody-based diagnosis of SARS has been demonstrated to be a reliable proof of SARS infection, it is not sensitive enough for detection during the early phase of the disease. To date, based on the publicly released full genomic sequences of SARS-CoV, various molecular detection methods based on reverse-transcription polymerase chain reaction (RT-PCR) have been developed. Although most of the assays have initially been focused on RNA extracted from nasopharyngeal aspirates, urine, and stools, several of the more recently developed assays have been based on the analysis of RNA extracted from plasma and serum. Such assays allow the more standardized quantitative expression of viral loads and are potentially useful for early SARS diagnosis. In this chapter, two real-time quantitative RT-PCR systems for the quantification of SARS-CoV RNA in serum are discussed. The two RT-PCR systems, one aimed toward the nucleocapsid region and the other toward the polymerase region of the virus genome, have a detection rate of up to 80% during the first week of illness. These quantitative systems are potentially useful for the early diagnosis of SARS and can also provide viral load information that might assist clinicians in making a prognostic evaluation of an infected individual.

Key Words

Serum RNA SARS-CoV RNA viral RNA extraction RNA quantification real-time quantitative reverse-transcription PCR 

Notes

Acknowledgments

This work is supported by a Special Grant for SARS Research (CUHK 4508/ 03M) from the Research Grants Council of the Hong Kong Special Administrative Region (China).

References

  1. 1.
    Peiris, J. S., Guan, Y., and Yuen, K. Y. (2004) Severe acute respiratory syndrome. Nat. Med. 10, S88–S97.PubMedCrossRefGoogle Scholar
  2. 2.
    Drosten, C., Gunther, S., Preiser, W., 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.
    Peiris, J. S., Lai, S. T., Poon, L. L. M., et al. (2003) Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361, 1319–1325.PubMedCrossRefGoogle Scholar
  4. 4.
    Poutanen, S. M., Low, D. E., Henry, B., et al. (2003) Identification of severe acute respiratory syndrome in Canada. N. Engl. J. Med. 348, 1995–2005.PubMedCrossRefGoogle Scholar
  5. 5.
    Fouchier, R. A., Kuiken, T., Schutten, M., et al. (2003) Aetiology: Koch’s postulates fulfilled for SARS virus. Nature 423, 240.PubMedCrossRefGoogle Scholar
  6. 6.
    Kuiken, T., Fouchier, R. A. M., Schutten, M., et al. (2003) Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 362, 263–270.PubMedCrossRefGoogle Scholar
  7. 7.
    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
  8. 8.
    Tsui, S. K. W., Chim, S. S. C., Lo, Y. M. D., and The Chinese University of Hong Kong Molecular SARS Research Group. (2003) Coronavirus genomic-sequence variations and the epidemiology of the severe acute respiratory syndrome. N. Engl. J. Med. 349, 187–188.PubMedCrossRefGoogle Scholar
  9. 9.
    Rota, P. A., Oberste, M. S., Monroe, S. S., et al. (2003) Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300, 1394–1399.PubMedCrossRefGoogle Scholar
  10. 10.
    Marra, M. A., Jones, S. J., Astell, C. R., et al. (2003) The genome sequence of the SARS-associated coronavirus. Science 300, 1399–1404.PubMedCrossRefGoogle Scholar
  11. 11.
    Poon, L. L. M., Wong, O. K., Luk, W., Yuen, K. Y., Peiris, J. S., and Guan, Y. (2003) Rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (SARS). Clin. Chem. 49, 953–955.PubMedCrossRefGoogle Scholar
  12. 12.
    Poon, L. L. M., Chan, K. H., Wong, O. K., et al. (2003) Early diagnosis of SARS coronavirus infection by real time RT-PCR. J. Clin. Virol. 28, 233–238.PubMedCrossRefGoogle Scholar
  13. 13.
    Ng, E. K. O., Hui, D. S. C., Chan, K. C. A., et al. (2003) Quantitative analysis and prognostic implication of SARS coronavirus RNA in the plasma and serum of patients with severe acute respiratory syndrome. Clin. Chem. 49, 1976–1980.PubMedCrossRefGoogle Scholar
  14. 14.
    Ng, E. K. O., Ng, P. C., Hon, K. L., et al. (2003) Serial analysis of the plasma concentration of SARS coronavirus RNA in pediatric patients with severe acute respiratory syndrome. Clin. Chem. 49, 2085–2088.PubMedCrossRefGoogle Scholar
  15. 15.
    Hung, E. C. W., Chim, S. S. C., Chan, P. K., et al. (2003) Detection of SARS coronavirus RNA in the cerebrospinal fluid of a patient with severe acute respiratory syndrome. Clin. Chem. 49, 2108–2109.PubMedCrossRefGoogle Scholar
  16. 16.
    Zhao, J. R., Bai, Y. J., Zhang, Q. H., Wan, Y., Li, D., and Yan, X. J. (2005) Detection of hepatitis B virus DNA by real-time PCR using TaqMan-MGB probe technology. World J. Gastroenterol. 11, 508–510.Google Scholar
  17. 17.
    Castelain, S., Descamps, V., Thibault, V., et al. (2004) TaqMan amplification system with an internal positive control for HCV RNA quantitation. J. Clin. Virol. 31, 227–234.PubMedCrossRefGoogle Scholar
  18. 18.
    Bustin, S. A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169–193.PubMedCrossRefGoogle Scholar
  19. 19.
    Ng, E. K. O., Tsui, N. B. Y., Lau, T. K., et al. (2003) mRNA of placental origin is readily detectable in maternal plasma. Proc. Natl. Acad. Sci. USA 100, 4748–4753.PubMedCrossRefGoogle Scholar
  20. 20.
    Chiu, W. K., Cheung, P. C., Ng, K. I., et al. (2003) Severe acute respiratory syndrome in children: Experience in a regional hospital in Hong Kong. Pediatr. Crit. Care Med. 4, 279–283.PubMedCrossRefGoogle Scholar
  21. 21.
    Hon, K. L., Leung, C. W., Cheng, W. T., et al. (2003) Clinical presentations and outcome of severe acute respiratory syndrome in children. Lancet 361, 1701–1703.PubMedCrossRefGoogle Scholar
  22. 22.
    Borst, A., Box, A. T. A., and Fluit, A. C. (2004) False-positive results and contamination in nucleic acid amplification assays: suggestions for a prevent and destroy strategy. Eur. J. Clin. Microbiol. Infect. Dis. 23, 289–299.PubMedCrossRefGoogle Scholar
  23. 23.
    Longo, M. C., Berninger, M. S., and Hartley, J. L. (1990) Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93, 125–128.PubMedCrossRefGoogle Scholar
  24. 24.
    Gibson, U. E., Heid, C. A., and Williams, P. M. (1996) A novel method for real time quantitative RT-PCR. Genome Res. 6, 995–1001.PubMedCrossRefGoogle Scholar
  25. 25.
    Myers, T. W. and Gelfand, D. H. (1991) Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry 30, 7661–7666.PubMedCrossRefGoogle Scholar
  26. 26.
    Tsui, N. B. Y., Ng, E. K. O., and Lo, Y. M. D. (2002) Stability of endogenous and added RNA in blood specimens, serum, and plasma. Clin. Chem. 48, 1647–1653.PubMedGoogle Scholar
  27. 27.
    Poddar, S. K., Sawyer, M. H., and Connor, J. D. (1998) Effect of inhibitors in clinical specimens on Taq and Tth DNA polymerase-based PCR amplification of influenza A virus. J. Med. Microbiol. 0, 1131–1135.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Enders K. O. Ng
  • Y. M. Dennis Lo

There are no affiliations available

Personalised recommendations