Development of a Taqman RT-PCR assay for the detection and quantification of negatively stranded RNA of human enteroviruses: Evidence for false-priming and improvement by tagged RT-PCR

作者：Bessaud Mael^*; Autret Arnaud; Jegouic Sophie; Balanant Jean; Joffret Marie Line; Delpeyroux Francis

来源：Journal of Virological Methods, 2008, 153(2): 182-189.

DOI：10.1016/j.jviromet.2008.07.010

• Human enterovirus
• Minus-stranded RNA
• Real-time PCR
• Replication
• Silica-based purification
• Tagged RT-PCR

Human enteroviruses are among the most common viruses infecting humans. These viruses are known to be able to infect a wide range of tissues and are believed to establish persistent infections. Enteroviruses are positive-sense single-stranded RNA viruses whose replication involves the synthesis of negative strand intermediates. Therefore, the specific detection of negatively stranded viral RNA in tissues or cells is a reliable marker of active enteroviral replication.
The present report presents the development of a real-time RT-PCR allowing the specific detection and quantification of negatively stranded viral RNA. Since it was known that specific amplification of single-stranded RNA can be made difficult by false-priming events leading to false-positive or overestimated results, the assay was developed by using a tagged RT primer. This tagged RT-PCR was shown to be able to amplify specifically negative RNA of enteroviruses grown in cell cultures by preventing the amplification of cDNAs generated by false-priming.

• 出版日期2008-11