Preparation of His-tagged armored RNA phage particles as a control for real-time reverse transcription-PCR detection of severe acute respiratory syndrome coronavirus.
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.
Cheng
Yangjian
Y
Department of Biomedical Sciences, Xiamen University, South Road of Siming 422, Xiamen 361005, China.
Niu
Jianjun
J
Zhang
Yongyou
Y
Huang
Jianwei
J
Li
Qingge
Q
D006639:Histidine / Q000737:chemistry*; D012317:RNA Phages; D012367:RNA, Viral; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D012367:RNA, Viral; D006639:Histidine