Rapid and sensitive detection of multiple genes from the SARS-coronavirus using quantitative RT-PCR with dual systems.
The outbreak of severe acute respiratory syndrome (SARS) was caused by a newly identified coronavirus (SARS-CoV) in 2003. To detect early SARS-CoV infection, a one-step, real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was developed that could simultaneously detect nucleocapsid (N), membrane (M), and spike (S) genes of SARS-CoV with the same PCR condition using either Applied Biosystems (ABI) Prism 7700 Sequence Detection System or Roche LightCycler. The sensitivity of this assay was evaluated using cell culture-derived viruses, in vitro transcribed viral RNA, and clinical specimens. The SARS-S, -M, and -N primer/probe sets described in this paper could detect one to ten copies of in vitro transcribed S, M, and N RNA per test using both the ABI and Roche assay systems. The relative sensitivities for detecting cell culture-derived SARS-CoV were 0.01, 0.01, and 0.001 PFU/test, respectively. It showed that SARS-N has comparable detection efficiencies to SARS2 and SARS3 which are primers sets designed by Centers for Disease Control and Prevention. In addition, SARS-S and SARS-M also demonstrated equivalent sensitivity to the commercially available RealArt HPA-Coronavirus reagents (Artus). The relative sensitivity of these primer/probe sets was also examined using human sera spiked viruses and clinical specimens from four confirmed SARS patients. Similar results as above were obtained. Specificity tests and sequence alignment showed that these primer/probe sets annealed perfectly to 31 isolates of SARS-CoV; and there was no cross detection with other coronaviruses and human respiratory tract-associated viruses. Therefore, not only is it compatible with the ABI and Roche systems, this multiple-gene detection assay also has the merit of being a rapid, safe, sensitive, and specific tool for accurate diagnosis of SARS-CoV infection.
Huang
Jau-Ling
JL
Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan. JauLingH@mail.cju.edu.tw
Lin
Hui-Tsu
HT
Wang
Yu-Ming
YM
Yeh
Yi-Chien
YC
Peck
Konan
K
Lin
Bai-Ling
BL
Liu
Huan-Wun
HW
Chen
Ann
A
Lin
Chang-Shen
CS
D005814:Genes, Viral; D006801:Humans; D012367:RNA, Viral; D020133:Reverse Transcriptase Polymerase Chain Reaction; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis / Q000821:virology
D012367:RNA, Viral