Belak, Sandor
- Department of Animal Biosciences, Swedish University of Agricultural Sciences
Research article2010Peer reviewedOpen access
Muradrasoli, Shaman; Mohamed, Nahla; Belák, Sándor; Czifra, György; Berencsi, George; Herrmann, Björn; Blomberg, Jonas
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. (C) 2009 Elsevier B.V. All rights reserved.
Influenza; Respiratory infection; Real-time PCR; Laboratory diagnosis; Virus quantitation
Journal of Virological Methods
2010, Volume: 163, number: 2, pages: 313-322
Publisher: ELSEVIER SCIENCE BV
Animal and Dairy Science
Veterinary Science
DOI: https://doi.org/10.1016/j.jviromet.2009.10.017
https://res.slu.se/id/publ/48530