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.
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-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 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 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.
Muradrasoli
Shaman
S
Section of Clinical Virology, Department of Medical Sciences, Uppsala University, Sweden.
Mohamed
Nahla
N
Belák
Sándor
S
Czifra
György
G
Herrmann
Björn
B
Berencsi
George
G
Blomberg
Jonas
J
D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D009305:Nasopharynx / Q000821:virology; D009698:Nucleoproteins / Q000235:genetics*; D015345:Oligonucleotide Probes / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000235:genetics
D009698:Nucleoproteins; D015345:Oligonucleotide Probes; D014764:Viral Proteins