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

• Published in: Journal of virological methods Vol. 153; no. 2; pp. 182 - 189
• Main Authors: Bessaud, Maël, Autret, Arnaud, Jegouic, Sophie, Balanant, Jean, Joffret, Marie-Line, Delpeyroux, Francis
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01.11.2008; Amsterdam Elsevier; New York, NY
• Subjects: Biological and medical sciences; Cell Line; DNA Primers; DNA, Complementary; Enterovirus; Enterovirus - genetics; Enterovirus - isolation & purification; Enterovirus - physiology; Fundamental and applied biological sciences. Psychology; Human enterovirus; Humans; Life Sciences; Microbiology; Microbiology and Parasitology; Minus-stranded RNA; Real-time PCR; Replication; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Viral - analysis; Sensitivity and Specificity; Silica-based purification; Tagged RT-PCR; Taq Polymerase; Techniques used in virology; Virology; Virus Replication; Minus-stranded RNA; Human enterovirus; Replication; Real-time PCR; Tagged RT-PCR; Silica-based purification; Human; Purification; Microbiology; Picornaviridae; Enterovirus; Method; Real time; Silica; Inorganic compound; Virology; Virus; Polymerase chain reaction; Detection; Reverse transcription polymerase chain reaction; Quantitative analysis
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2008.07.010

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.