Section of Clinical Virology, Department of Medical Sciences, Uppsala University, Sweden.
J Virol Methods. 2010 Feb;163(2):313-22. doi: 10.1016/j.jviromet.2009.10.017. Epub 2009 Oct 29.
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.
需要一种能够覆盖动物和人类甲型、乙型和丙型流感病毒（即大多数正粘病毒科）的 PCR 检测方法。流感病毒的类型是根据病毒中存在的核蛋白（NP）的差异来区分的。因此，保守的 NP 区域被用于设计一种基于 TaqMan 的三重反转录实时 PCR 方法。由于甲型流感病毒在探针靶标区域内的变异性，需要开发一种新型分子信标，即“Mega”分子信标（MegaBeacon；MegB），用于该方法检测甲型流感病毒。MegaBeacon 是一种容错分子信标，也是 TaqMan 探针。该三重方法（3QPCR-MegB）使用涵盖 18 种 HxNx 组合的甲型流感病毒分离株、两种乙型流感病毒分离株和五种日本丙型流感病毒分离株以及甲型、乙型和丙型流感病毒的合成 DNA 靶标进行了评估。该方法可检测到甲型、乙型和丙型流感病毒的每个 PCR 反应中 1 到 10 个病毒 RNA 和 cDNA 基因组当量。从呼吸道感染的 71 个人鼻咽抽吸物中，3QPCR-MegB 检测到 30 种甲型流感、11 种乙型流感和 0 种丙型流感，而免疫荧光（IF）检测到 28 种甲型流感和 10 种乙型流感。3QPCR-MegB 比具有甲型流感 TaqMan 探针的变体 PCR（3QPCR）更能容忍错配，是一种敏感且合理的方法，可用于检测动物和人类来源的流感病毒。MegaBeacon 探针有望用于可变靶标核酸。
