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.

Epidemiology and Clinical Characteristics of Influenza C Virus

“…In a study comparing RT-PCR to cell culture detection of ICV, RT-PCR detection rate was nearly twice that of cell culture and samples with lower viral load were more likely to be detected with sensitive nucleic acid methods [64]. Several RT-PCR assays have been reported, with significantly increased sensitivity compared to culture [63][64][65][66][67][68][69][70]. These molecular assays have facilitated epidemiologic investigations of ICV.…”

Evaluation of a Multiplex One-Step TaqMan Real-Time Reverse Transcription- PCR Assays for the Detection of H5N1 Avian Influenza Viruses in Clinical Specimens

“…A number of scientific reports about the use of rapid detection tests for type A influenza virus in poultry have been published [19][20][21][22][23][24][25] pointing out the importance of having such tests available. Information about the application of theses assays using human samples or samples collected during an epizootic from naturally infected birds isn't published.…”

Development of one-step real-time PCR assay for titrating trivalent live attenuated influenza vaccines

“…23,24 In this study, these primers and probes were modified to normalize the Tm values for simultaneous analysis of trivalent vaccine strains. Viral RNAs were extracted with the …”