Comprehensive multiplex one-step real-time TaqMan qRT-PCR assays for detection and quantification of hemorrhagic fever viruses.
Comprehensive multiplex one-step real-time TaqMan qRT-PCR assays for detection and quantification of hemorrhagic fever viruses.
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DOI：
10.1371/journal.pone.0095635
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Li D
中科院分区：
文献类型：
--
作者：
Pang Z;Li A;Li J;Qu J;He C;Zhang S;Li C;Zhang Q;Liang M;Li D
病毒性出血热(VHFs)是一组动物和人类疾病，主要由几个不同的病毒家族引起，包括布尼亚病毒、黄病毒、丝状病毒和阿雷纳病毒。虽然具体的体征和症状因VHF的类型而异，但最初的体征和症状非常相似。因此，用于鉴别诊断出血热病毒(HFV)的快速免疫学和分子工具对于有效的病例管理和控制VHFs的传播是重要的。实时定量逆转录聚合酶链式反应(qRT-PCR)是检测和定量病毒载量的可靠和理想的方法之一。多重聚合酶链式反应有可能在实验室检测中节省大量的时间和资源。根据28个HFV的特异性基因设计了相应的引物/探针组，对几乎覆盖所有HFV的28个HFV进行了单链分析，具有良好的特异性和敏感性。然后在一个通用的实验系统中建立了7组多重一步实时QRT-PCR方法，将所有的引物/探针组合成四重反应，并以合成的病毒RNA的系列稀释度进行评估。在所有多重检测中，均未观察到与其他HFV的交叉反应，检测下限主要在45-150拷贝/聚合酶链式反应。重复性令人满意，因为无论是批内还是批间，每次稀释合成的病毒RNA的Ct值的变异系数都小于5%。用HFRS患者、SFTS患者和登革热患者的临床血清样本对该方法进行评价，结果表明，相关的多重检测方法对临床样本具有较高的敏感性和特异性。综上所述，本研究建立了全面的多重一步实时荧光定量聚合酶链式反应检测方法，该方法具有特异性强、灵敏度高、稳定性好等特点，可作为快速检测HFV的有效工具。
Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection. Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens. Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be specific, sensitive, stable and easy to serve as a useful tool for rapid detection of HFVs.
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