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Journal of Clinical Microbiology, May 2005, p. 2041-2046, Vol. 43, No. 5
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.5.2041-2046.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Development and Evaluation of a Multitarget Real-Time Taqman Reverse Transcription-PCR Assay for Detection of the Severe Acute Respiratory Syndrome-Associated Coronavirus and Surveillance for an Apparently Related Coronavirus Found in Masked Palm Civets

Wenqian Hu,1 Bingke Bai,1 Zhihong Hu,1 Ze Chen,1 Xuefang An,1 Lijun Tang,2 Jihong Yang,3 Hualin Wang,1 and Hanzhong Wang1*

State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences,1 Hubei Center for Diseases Control and Prevention,2 Wuhan Center for Diseases Control and Prevention, Wuhan, Hubei, People's Republic of China3

Received 18 September 2004/ Returned for modification 5 December 2004/ Accepted 14 January 2005

Severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) is the etiological agent of SARS. It is believed that SARS-CoV originates from wild animals. We have developed a multitarget real-time Taqman reverse transcription-PCR (RT-PCR) assay for the quantitative detection of SARS-CoV. The sequences of the Taqman probes with a minor groove binder and the corresponding primers were based on the sequences of the N gene, open reading frame (ORF) 3, and ORF 8. The overall linear range of this assay was from at least 101 to 106 copies per reaction, and the detection limit could reach less than 10 copies per reaction. The quantification results for SARS-CoV from cell culture correlated well with those of the RT-PCR by using any two of the three sets of primer and probe used in this assay. However, the results of quantification of SARS-CoV obtained by using a few available throat swab specimens from SARS patients and the N gene as the target were almost 10 times higher than those obtained by using ORF 3 and ORF 8. Using this assay, we also detected an apparently SARS-CoV-related coronavirus in the throat swab specimens from masked palm civets in the west part of Hubei Province, People's Republic of China.

* Corresponding author. Mailing address: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430072 Hubei, People's Republic of China. Phone: 86-27-87199239. Fax: 86-27-87198072. E-mail: wanghz{at}pentium.whiov.ac.cn.

Journal of Clinical Microbiology, May 2005, p. 2041-2046, Vol. 43, No. 5
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.5.2041-2046.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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