Preparation of armored RNA as a control for multiplex real-time reverse transcription-PCR detection of influenza virus and severe acute respiratory syndrome coronavirus.
The common respiratory viruses, including influenza A, influenza B, and newly emerging severe acute respiratory syndrome (SARS) viruses, may cause similar clinical symptoms. Therefore, differential diagnosis of these virus pathogens is frequently required for single clinical samples. In addition, there is an urgent need for noninfectious and stable RNA standards and controls for multivirus detection. In this study, reverse transcription-PCR (RT-PCR) targeting of the RNAs of influenza A and influenza B viruses and SARS coronavirus was performed, and the resulting products were spliced into a fragment which was packaged into armored RNA for use as a noninfectious, quantifiable synthetic substitute. Furthermore, in the present study we developed a multiplex real-time RT-PCR assay in which the armored RNA was used as an external positive control and the three RNA viruses could be detected simultaneously in a single reaction mix. The detection limit of the multiplex real-time PCR was 10 copies/microl of armored RNA.
Yu
Xin-Fen
XF
Microbiology Laboratory, Hangzhou Center for Disease Control and Prevention, No. 277, Wu Lin Road, Hangzhou, People's Republic of China. yuxinfen@hotmail.com
Pan
Jing-Cao
JC
Ye
Rong
R
Xiang
Hai-Qing
HQ
Kou
Yu
Y
Huang
Zhi-Cheng
ZC
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000821:virology; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis; D017909:Levivirus / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000302:isolation & purification*; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis; D019065:Virus Assembly
D012367:RNA, Viral