Broadly targeted triplex real-time PCR detection of influenza A, B and C viruses based on the nucleoprotein gene and a novel "MegaBeacon" probe strategy
2010 (English)In: Journal of Virological Methods, ISSN 0166-0934, E-ISSN 1879-0984, Vol. 163, no 2, 313-322 p.Article in journal (Refereed) Published
A PCR assay that covers animal and human influenza A, B and C viruses, i.e., most of Orthomyxoviridae, is needed. Influenza types are distinguished based on differences in the nucleoprotein (NP) present in the virus. Conserved NP regions were therefore used to design a TaqMan (R)-based triplex reverse transcription real-time PCR method. Variability of influenza A within the probe target region mandated the development of a novel molecular beacon, the "Mega" molecular beacon (MegaBeacon: MegB), for the detection of influenza A with this method. MegaBeacon is a mismatch-tolerant molecular beacon that is also a TaqMan (R) probe. The triplex method (3QPCR-MegB) was evaluated with influenza A isolates covering 18 HxNx combinations, two influenza B isolates, and five Japanese influenza C isolates, as well as influenza A, B and C synthetic DNA targets. One to ten viral RNA and cDNA genome equivalents were detected per PCR reaction for influenza A, B and C. Seventy-one human nasopharyngeal aspirates from respiratory infections yielded 30 influenza A, 11 influenza B and 0 influenza C with 3QPCR-MegB, where immunofluorescence (IF) found 28 influenza A and 10 influenza B. 3QPCR-MegB was more mismatch-tolerant than a variant PCR with an influenza A TaqMan (R) probe (3QPCR) and is a sensitive and rational method to detect influenza viruses of animal and human origin. MegaBeacon probes hold promise for variable target nucleic acids.
Place, publisher, year, edition, pages
2010. Vol. 163, no 2, 313-322 p.
Influenza, Laboratory diagnosis, Real-time PCR, Respiratory infection, Virus quantitation
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:uu:diva-97407DOI: 10.1016/j.jviromet.2009.10.017ISI: 000275074500024PubMedID: 19879296OAI: oai:DiVA.org:uu-97407DiVA: diva2:172347