Publication: Development and comparison of the real-time amplification based methods - NASBA-Beacon, RT-PCR TaqMan and RT-PCR hybridization probe assays - For the qualitative detection of sars coronavirus
1
Issued Date
2004-09-01
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ISSN
01251562
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2-s2.0-8444228607
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Mahidol University
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SCOPUS
Bibliographic Citation
Southeast Asian Journal of Tropical Medicine and Public Health. Vol.35, No.3 (2004), 623-629
Suggested Citation
Wasun Chantratita, Wiroj Pongtanapisit, Wantanich Piroj, Chutatip Srichunrasmi, Somying Seesuai Development and comparison of the real-time amplification based methods - NASBA-Beacon, RT-PCR TaqMan and RT-PCR hybridization probe assays - For the qualitative detection of sars coronavirus. Southeast Asian Journal of Tropical Medicine and Public Health. Vol.35, No.3 (2004), 623-629. Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/21553
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Title
Development and comparison of the real-time amplification based methods - NASBA-Beacon, RT-PCR TaqMan and RT-PCR hybridization probe assays - For the qualitative detection of sars coronavirus
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Abstract
The aim of this study was to develop a rapid, sensitive and robust procedure for the qualitative detection of SARS coronavirus RNA. Three unique detection formats were developed for real-time RNA amplification assays: a post amplification detection step with a virus-specific internal capture probe based on Taqman (RT-PCR TaqMan assay), hybridization probe (RT-PCR hybridization probe assay) and a real-time assay with virus-specific molecular beacon probes (NASBA-Beacon assay). The analytical sensitivity or reproducibility of the test results among those three assays was compared. All assays yielded results by detecting SARS coronavirus targeting the BNI-1 region in less than 2 hours. RNA detection by all the formats was unaffected by the presence of human sputum. The limits of detection were at least 10 copies of input RNA for both RT-PCR formats (RT-PCR TaqMan and RT-PCR hybridization probe assays), while the NASBA-Beacon assay could detect as little as 1 copy per reaction, with high reproducibility of the coefficient of variation (CV) of <10. These results demonstrate that real-time NASBA provides a rapid and sensitive alternative to RT-PCR for the routine qualitative assay of sputum for SARS corona viral RNA detection.