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Journal of Clinical Microbiology, October 2006, p. 3557-3561, Vol. 44, No. 10
0095-1137/06/$08.00+0     doi:10.1128/JCM.00713-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Preparation of His-Tagged Armored RNA Phage Particles as a Control for Real-Time Reverse Transcription-PCR Detection of Severe Acute Respiratory Syndrome Coronavirus

Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministration of Education, Molecular Diagnostics Laboratory, Department of Biomedical Sciences and School of Life Sciences, Xiamen University,¹ Xiamen Center for Disease Control and Prevention,² Key Laboratory of Chemical Biology of Fujian, Xiamen, Fujian 361005, China³

Received 5 April 2006/ Returned for modification 2 June 2006/ Accepted 24 July 2006

Armored RNA has been increasingly used as both an external and internal positive control in nucleic acid-based assays for RNA virus. In order to facilitate armored RNA purification, a His₆ tag was introduced into the loop region of the MS2 coat protein, which allows the exposure of multiple His tags on the surface during armored RNA assembly. The His-tagged armored RNA particles were purified to homogeneity and verified to be free of DNA contamination in a single run of affinity chromatography. A fragment of severe acute respiratory syndrome coronavirus (SARS-CoV) genome targeted for SARS-CoV detection was chosen for an external positive control preparation. A plant-specific gene sequence was chosen for a universal noncompetitive internal positive control preparation. Both controls were purified by Co²⁺ affinity chromatography and were included in a real-time reverse transcription-PCR assay for SARS-CoV. The noncompetitive internal positive control can be added to clinical samples before RNA extraction and enables the identification of potential inhibitive effects without interfering with target amplification. The external control could be used for the quantification of viral loads in clinical samples.

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