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

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-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.

Fig. 1

Standard curves of 10-fold dilution series of synthetic influenza A, B and C target oligonucleotides. The Ct value is the cycle number at which a positive amplification reaction was measured; the straight line is the regression line. (A) Standard curve of a 10-fold dilution series of synthetic influenza A TaqMan probe (Influ ANP). (B) Standard curve of a 10 fold dilution series of synthetic influenza B antisense probe (Influ BNP). (C) Standard curve of a 10-fold dilution series of a perfectly matching synthetic influenza C probe (InfluCNP). (D) Standard curve of a 10-fold dilution series of a perfectly matching synthetic influenza A and mega beacon probe (InfluANP-MegB).

Fig. 2

(A) MegaBeacon influenza A probe construct. The FAM fluorophore and the Dabcyl quencher are shown in orange and green, respectively. The non-viral portion, added to create a hairpin loop, is shown in a box. (B) Amplification curves from seven different subtypes of influenza A from the FAM channel of 3QPCR-MegB. (C) Raw fluorescence data from reannealing the MegaBeacon probe. The correlation of residual fluorescence (“probe consumption”) with amount of amplimer is evident and derives from cleaved probes as well as probes hybridising to amplimer. Negative samples occasionally gave an atypical amplification curve with the MegaBeacon influenza A probe. These curves could be discriminated from true positive amplification curves based on (i) curve form, if the curve started after a few cycles and remained low, (ii) if the sample was found to be negative in the probe consumption test and (iii) if the sample lacked a band of the expected size in agarose gel electrophoresis. All data were obtained using a Corbett Rotorgene^© instrument and its software.