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

Journal of Virological Methods
Maël BessaudFrancis Delpeyroux

Abstract

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.

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Citations

Jun 15, 2010·Archives of Virology·So Young JangChan Hee Lee
Sep 13, 2013·Journal of Virology·Xiaowei ZhangHanzhong Wang
Apr 20, 2010·Journal of Environmental Sciences (China)·Minglu ZhangBaoli Cai
Sep 25, 2014·Antiviral Research·Yan Yi CheungJustin Jang Hann Chu
Nov 27, 2009·The Journal of General Virology·A TuiskunenS Plumet
May 18, 2016·Genome Biology and Evolution·Yulia MostovoyRachel B Brem
Apr 22, 2017·Virology·Vincent Raquin, Louis Lambrechts
Dec 10, 2020·Microorganisms·Sami SalmikangasVarpu Marjomäki

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