Quick assessment of influenza a virus infectivity with a long-range reverse-transcription quantitative polymerase chain reaction assay
Quick assessment of influenza a virus infectivity with a long-range reverse-transcription quantitative polymerase chain reaction assay
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DOI：
10.1186/s12879-020-05317-8
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发表时间：
2020-08-06
影响因子：
3.7
通讯作者：
Fujimaki, Makoto
中科院分区：
文献类型：
--
作者：
Nakaya, Yuki;Fukuda, Takashi;Fujimaki, Makoto
背景聚合酶链反应（PCR）因其高度的敏感性和特异性而被广泛应用于病毒病原体的检测。然而，常规PCR方法不能确定病毒感染性。病毒感染性通常用噬斑试验等方法进行检查，尽管这种试验需要几天时间。远程逆转录定量PCR（RT-qPCR）以前已建议用于快速评估RNA病毒的感染性，其中感染性的损失是由于基因组fragmentation.MethodsIAV照射与253.7nm的紫外线（UV）射线诱导基因组链断裂，证实了全长RT-PCR测定。然后对IAV进行空斑试验、常规RT-qPCR和远程RT-qPCR，以检查感染性滴度与拷贝数之间的关系。一个简单的线性回归分析进行检查这些assays.ResultsA远程RT-qPCR检测的结果之间的相关性，开发和验证甲型流感病毒（IAV）。尽管仅需要几分钟的UV照射就可完全灭活IAV，但通过常规RT-qPCR和灭活后病毒基因组NS的全长RT-PCR仍可检测到基因组RNA。然后使用在每个基因组区段的3个末端处的RT引发和随后的5 '区域的qPCR设计长范围RT-qPCR测定。通过远程RT-qPCR测定成功分析了UV介导的IAV灭活，特别是当靶向病毒基因组的PA时。结论pid =Par本研究表明，在不进行感染性检测的情况下，可以预测IAV的感染性。因此，利用这种传感技术实现了对致病性IAV的快速检测。
BackgroundThe polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity. However, conventional PCR methods cannot determine virus infectivity. Virus infectivity is conventionally examined with methods such as the plaque assay, even though such assays require several days. Long-range reverse-transcription quantitative PCR (RT-qPCR) has previously been suggested for the rapid assessment of RNA virus infectivity where the loss of infectivity is attributable to genomic fragmentation.MethodsIAV was irradiated with 253.7nm ultraviolet (UV) rays to induce genomic strand breaks that were confirmed by a full-length RT-PCR assay. The IAV was then subjected to plaque assay, conventional RT-qPCR and long-range RT-qPCR to examine the relationship between infectious titer and copy number. A simple linear regression analysis was performed to examine the correlation between the results of these assays.ResultsA long-range RT-qPCR assay was developed and validated for influenza A virus (IAV). Although only a few minutes of UV irradiation was required to completely inactivate IAV, genomic RNA remained detectable by the conventional RT-qPCR and the full-length RT-PCR for NS of viral genome following inactivation. A long-range RT-qPCR assay was then designed using RT-priming at the 3 termini of each genomic segment and subsequent qPCR of the 5 ' regions. UV-mediated IAV inactivation was successfully analyzed by the long-range RT-qPCR assay especially when targeting PA of the viral genome. This was also supported by the regression analysis that the long-range RT-qPCR is highly correlated with plaque assay (Adjusted R-2=0.931, P=0.000066).Conclusions p id=Par This study suggests that IAV infectivity can be predicted without the infectivity assays. The rapid detection of pathogenic IAV has, therefore, been achieved with this sensing technology.