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.
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.
Chantratita
Wasun
W
Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Bangkok, Thailand. rawct@mahidol.ac.th
Pongtanapisit
Wiroj
W
Piroj
Wantanich
W
Srichunrasmi
Chutatip
C
Seesuai
Somying
S
D003199:Computer Systems; D015342:DNA Probes; D006801:Humans; D015336:Molecular Probe Techniques; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D045473:Severe acute respiratory syndrome-related coronavirus / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology; D012984:Software; D013183:Sputum / Q000821:virology; D019914:Taq Polymerase
D015342:DNA Probes; D019914:Taq Polymerase