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.
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.
Bessaud
Maël
M
Unité postulante de biologie des virus entériques, Institut Pasteur, 25 rue du Dr Roux, 75 015 Paris, France. bessaud@pasteur.fr
Autret
Arnaud
A
Jegouic
Sophie
S
Balanant
Jean
J
Joffret
Marie-Line
ML
Delpeyroux
Francis
F
D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary; D004770:Enterovirus / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology; D006801:Humans; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D014779:Virus Replication
D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral; D019914:Taq Polymerase