J Virol Methods. 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.

[Giuseppe]

J Virol Methods. 2009 Oct 28. [Epub ahead of print]

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.

Muradrasoli S, Mohamed N, Bel?k S, Czifra G, Herrmann B, Berencsi G, Blomberg J. - Section of Clinical Virology, Department of Medical Sciences, Uppsala University, Sweden.

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. Seventyone 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; Supplementary Material) 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.

PMID: 19879296 [PubMed - as supplied by publisher]
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[JJackson]

Re: J Virol Methods. 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.

Not that I understood most of the TaqMan MegaB stuff but it seems to work.
At least as far as sorting ILI into A or B (or C but that is rather less important). What it does not seem to do is identify the Hx and Nx for serotyping which means it can not identify pandemic candidates.

The big question. Anyone know what it might cost (per device and per test), how big it would be and how fast it is - if it was mass produced? i.e. is it something that might end up in a small Indonesian hospital or just doing some of the grunt work at the CDC.

What the world needs is a cheap lab-on-a-chip which a GP can dap a swab on and will tell him within the hour you have Tamiflu sensative H1N1 or you have Tamiflu resistant H5N1. Not quite there yet.