Development of strand-specific real-time RT-PCR to distinguish viral RNAs during Newcastle disease virus infection.
Development of strand-specific real-time RT-PCR to distinguish viral RNAs during Newcastle disease virus infection.
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开发链特异性实时 RT-PCR 来区分新城疫病毒感染期间的病毒 RNA。
DOI：
10.1155/2014/934851
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发表时间：
2014
影响因子：
--
通讯作者：
Ding C
中科院分区：
文献类型：
--
作者：
Qiu X;Yu Y;Yu S;Zhan Y;Wei N;Song C;Sun Y;Tan L;Ding C
新城疫病毒(NDV)在全球养鸡业造成巨大损失。为了更好地了解新城疫病毒的复制和转录周期，定量检测新城疫病毒感染细胞中新城疫病毒基因组RNA(GRNA)、抗基因组RNA(CRNA)和信使RNA(MRNAs)是必不可少的。针对新城疫病毒感染细胞中gRNA、cRNA和NP基因的核蛋白(NP)区设计了3条逆转录引物，并建立了相应的实时定量RT-PCR方法。该方法具有很好的特异性、敏感性和重现性。方法的检测范围为5.5×10 2~1.1×10 9拷贝/μL。应用这些方法研究了新城疫病毒La Sota感染的DF-1细胞中gRNA、cRNA和mRNA合成的动态变化。结果表明，病毒gRNA、cRNA和NP基因的拷贝数在开始时均呈指数级增加。病毒拷贝数在感染后10‘h趋于平稳，16 h后逐渐下降。在新城疫病毒感染过程中，复制(gRNA和cRNA)和转录(mRNA量)之间不存在合成优先的关系。然而，从感染后2 h开始，cRNA的积累速度比gRNA更快，因为cRNA的拷贝数比gRNA高3~10倍。结论。首次建立了用于NDV感染细胞中特异性病毒RNA片段绝对定量的实时RT-PCR方法。该方法的建立为进一步研究新城疫病毒的致病机理和控制策略奠定了基础。
Newcastle disease virus (NDV) causes large losses in the global fowl industry. To better understand NDV replication and transcription cycle, quantitative detection methods for distinguishing NDV genomic RNA (gRNA), antigenomic RNA (cRNA), and messenger RNA (mRNA) in NDV-infected cells are indispensible. Three reverse transcription primers were designed to specifically target the nucleoprotein (NP) region of gRNA, cRNA, and NP mRNA, and a corresponding real-time RT-PCR assay was developed to simultaneously quantify the three types of RNAs in NDV-infected cells. This method showed very good specificity, sensitivity, and reproducibility. The detection range of the assay was between 5.5 × 102 and 1.1 × 109 copies/μL of the target gene. These methods were applied to investigate the dynamics of the gRNA, cRNA, and mRNA synthesis in NDV La Sota infected DF-1 cells. The results showed that the copy numbers of viral gRNA, cRNA, and NP mRNA all exponentially increased in the beginning. The viral RNA copy number then plateaued at 10'h postinfection and gradually decreased from 16 h postinfection. No synthesis priority was observed between replication (gRNA and cRNA amounts) and transcription (mRNA amounts) during NDV infection. However, the cRNA accumulated more rapidly than gRNA, as the cRNA copy number was three- to tenfold higher than gRNA starting from 2 h postinfection. Conclusion. A real-time RT-PCR for absolute quantitation of specific viral RNA fragments in NDV-infected cells was developed for the first time. The development of this assay will be helpful for further studies on the pathogenesis and control strategies of NDV.