Genecology Research Centre, School of Science and Engineering, University of the Sunshine Coast, Sippy Downs, QLD, Australia.
Inflammation and Healing Research Cluster, School of Science and Engineering, University of the Sunshine Coast, Sippy Downs, QLD, Australia.
Virol J. 2018 Apr 23;15(1):75. doi: 10.1186/s12985-018-0985-8.
The 2014/2015 Ebolavirus outbreak resulted in more than 28,000 cases and 11,323 reported deaths, as of March 2016. Domestic transmission of the Guinea strain associated with the outbreak occurred mainly in six African countries, and international transmission was reported in four countries. Outbreak management was limited by the inability to rapidly diagnose infected cases. A further fifteen countries in Africa are predicted to be at risk of Ebolavirus outbreaks in the future as a consequence of climate change and urbanization. Early detection of cases and reduction of transmission rates is critical to prevent and manage future severe outbreaks. We designed a rapid assay for detection of Ebolavirus using recombinase polymerase amplification, a rapid isothermal amplification technology that can be combined with portable lateral flow detection technology. The developed rapid assay operates in 30 min and was comparable with real-time TaqMan™ PCR.
Designed, screened, selected and optimized oligonucleotides using the NP coding region from Ebola Zaire virus (Guinea strain). We determined the analytical sensitivity of our Ebola rapid molecular test by testing selected primers and probe with tenfold serial dilutions (1.34 × 10 1.34 × 10 copies/μL) of cloned NP gene from Mayinga strain of Zaire ebolavirus in pCAGGS vector, and serially diluted cultured Ebolavirus as established by real-time TaqMan™ PCR that was performed using ABI7500 in Fast Mode. We tested extracted and reverse transcribed RNA from cultured Zaire ebolavirus strains - Mayinga, Gueckedou C05, Gueckedou C07, Makona, Kissidougou and Kiwit. We determined the analytical specificity of our assay with related viruses: Marburg, Ebola Reston and Ebola Sudan. We further tested for Dengue virus 1-4, Plasmodium falciparum and West Nile Virus (Kunjin strain).
The assay had a detection limit of 134 copies per μL of plasmid containing the NP gene of Ebolavirus Mayinga, and cultured Ebolavirus and was highly specific for the Zaire ebolavirus species, including the Guinea strain responsible for the 2014/2015 outbreak. The assay did not detect related viruses like Marburg, Reston, or Sudan viruses, and other pathogens likely to be isolated from clinical samples.
Our assay could be suitable for implementation in district and primary health laboratories, as only a heating block and centrifuge is required for operation. The technique could provide a pathway for rapid screening of patients and animals for improved management of outbreaks.
截至 2016 年 3 月，2014-2015 年埃博拉病毒爆发导致超过 28000 例病例和 11323 例报告死亡。与疫情相关的几内亚株在国内的主要传播发生在六个非洲国家，国际传播发生在四个国家。由于无法快速诊断感染病例，疫情管理受到限制。由于气候变化和城市化，未来预计另外十五个非洲国家将面临埃博拉病毒爆发的风险。早期发现病例和降低传播率对于预防和管理未来的严重疫情至关重要。我们设计了一种使用重组酶聚合酶扩增（一种快速等温扩增技术）检测埃博拉病毒的快速检测方法，该技术可与便携式侧流检测技术结合使用。该快速检测方法在 30 分钟内运行，与实时 TaqMan™PCR 相当。
使用来自扎伊尔埃博拉病毒（几内亚株）的 NP 编码区设计、筛选、选择和优化寡核苷酸。我们通过使用克隆的 NP 基因的十倍系列稀释液（1.34×10 1.34×10 拷贝/μL）来测试选定的引物和探针，从 Mayinga 株的扎伊尔埃博拉病毒中测试我们的埃博拉快速分子检测的分析灵敏度，扎伊尔埃博拉病毒在 pCAGGS 载体中，并用实时 TaqMan™PCR 进行连续稀释培养，该 PCR 使用 ABI7500 在快速模式下进行。我们从培养的扎伊尔埃博拉病毒株中提取和逆转录 RNA-Mayinga、Gueckedou C05、Gueckedou C07、Makona、Kissidougou 和 Kiwit。我们使用相关病毒：马尔堡、埃博拉雷斯顿和苏丹埃博拉病毒来确定检测方法的分析特异性。我们进一步测试了登革热病毒 1-4、恶性疟原虫和西尼罗河病毒（Kunjin 株）。
该检测方法对含有埃博拉病毒 Mayinga NP 基因的质粒的检测限为 134 拷贝/μL，对培养的埃博拉病毒具有很高的特异性，包括导致 2014-2015 年疫情的几内亚株。该检测方法不能检测到类似马尔堡、雷斯顿或苏丹病毒的相关病毒，也不能检测到可能从临床样本中分离出来的其他病原体。
我们的检测方法可能适合在区和初级卫生实验室实施，因为仅需加热块和离心机即可运行。该技术可以为快速筛选患者和动物提供途径，以改善疫情管理。
