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Abstract

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.

Keywords

Influenza; Respiratory infection; Real-time PCR; Laboratory diagnosis; Virus quantitation

Published in

Journal of Virological Methods
2010, volume: 163, number: 2, pages: 313-322
Publisher: ELSEVIER SCIENCE BV

SLU Authors

UKÄ Subject classification

Animal and Dairy Science
Veterinary Science

Publication identifier

  • DOI: https://doi.org/10.1016/j.jviromet.2009.10.017

Permanent link to this page (URI)

https://res.slu.se/id/publ/48530