Real-time RT-qPCR assay for the analysis of human influenza A virus transcription and replication dynamics
Real-time RT-qPCR assay for the analysis of human influenza A virus transcription and replication dynamics
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DOI：
10.1016/j.jviromet.2010.04.017
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发表时间：
2010-09-01
影响因子：
3.1
通讯作者：
Reichl, Udo
中科院分区：
文献类型：
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作者：
Vester, Diana;Lagoda, Antje;Reichl, Udo
开发了一种定量实时逆转录酶PCR（RT-qPCR）测定法，用于分析哺乳动物细胞培养物中甲型流感病毒的转录和复制动力学。该检测基于极性和序列特异性逆转录，用于特异性区分流感病毒生命周期中片段4（HA）、6（NA）、7（M）和8（NS）的病毒基因组（vRNA（-））、复制中间体（cRNA（+））和病毒信使RNA（vmRNA（+））。针对每种病毒RNA类型和片段制备用作验证和定量参考标准品的合成病毒RNA。试验验证证明线性超过5个数量级，病毒RNA分子的灵敏度为1.0 x 10（3）至8.9 x 10（3），重复性和再现性小于0.8-3.1% CV（变异系数）。分析了甲型流感病毒在贴壁MOCK细胞（一种用于人流感疫苗生产的底物）中感染的动力学。一般而言，在感染的早期阶段在约0.6 hpi主要合成vmRNA（+），随后立即合成cRNA（+），并且在约1.9 hpi的短暂延迟后可以检测到病毒基因组复制。vRNA（-）以等摩尔量和相似的动力学合成，而在早期转录阶段优先合成NS 1 vmRNA（+），并发现M1 vmRNA（+）的延迟。（C）2010 Elsevier B. V.保留所有权利。
A quantitative real-time reverse transcriptase PCR (RT-qPCR) assay was developed for the analysis of influenza A virus transcription and replication dynamics in mammalian cell culture. The assay is based on a polarity- and sequence-specific reverse transcription used to distinguish specifically between viral genomes (vRNA(-)), replicative intermediates (cRNA(+)) and viral messenger RNAs (vmRNA(+)) of segments 4 (HA), 6 (NA), 7 (M) and 8 (NS) during the life cycle of influenza virus. Synthetic viral RNAs used as reference standards for validation and quantitation were prepared for each viral RNA type and segment. Assay validation demonstrated linearity over five orders of magnitude, sensitivity of 1.0 x 10(3) to 8.9 x 10(3) of viral RNA molecules, repeatability and reproducibility of less than 0.8-3.1% CV (coefficient of variation). Dynamics of influenza A virus infection in adherent MOCK cells, a substrate considered for human influenza vaccine manufacturing, were analyzed. In general, mainly vmRNA(+) were synthesized during early phases of infection at about 0.6 hpi, followed immediately by cRNA(+) synthesis and after a short delay of about 1.9 hpi viral genome replication could be detected. The vRNA(-)s were synthesized in equimolar amounts and similar dynamics whereas preferential synthesis of NS1 vmRNA(+) in early transcription phases and a delay for M1 vmRNA(+) was found. (C) 2010 Elsevier B.V. All rights reserved.