Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay.
Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay.
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DOI：
10.3390/v13091731
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发表时间：
2021-08-31
期刊：
影响因子：
--
通讯作者：
Truyen U
中科院分区：
文献类型：
--
作者：
Ceruti A;Kobialka RM;Ssekitoleko J;Okuni JB;Blome S;Abd El Wahed A;Truyen U
非洲猪瘟病毒（ASFV）是猪的致命疾病的病原体，并迅速跨越国界传播。从疑似病例中采集的样本必须送往参考实验室，利用聚合酶链反应进行诊断。在这项研究中，我们的目的是建立一个简单的DNA提取步骤和实时重组酶聚合酶扩增（RPA）检测ASFV的快速检测。建立了以ASFV B646 L基因编码区p72为靶基因的RPA检测方法。研究了检测限和交叉反应性。使用73份血液和血清样本检查诊断性能。测试了两种提取方法：基于硅胶柱的提取方法和简单的非纯化DNA分离（裂解缓冲液和加热，70 °C持续20分钟）。所有结果与成熟的实时PCR进行比较。在乌干达疾病暴发事件期间的一次实地部署中，对20份全血样本进行了检测。通过8次独立检测运行的概率单位分析确定，该检测试剂盒的分析灵敏度为每微升分子标准品的3.5个DNA拷贝。ASFV RPA检测仅检测ASFV基因型。与荧光定量PCR相比，RPA诊断的敏感性和特异性均为100%。使用加热/裂解缓冲液提取程序，ASFV-RPA显示出比实时PCR更好的抑制剂耐受性（阳性率分别为97%和38%）。在乌干达，受感染的动物在出现发烧之前就被发现了。ASFV-RPA检测显示与实时PCR一样敏感和特异。此外，简单提取方案的组合允许其在需要时用于改进控制措施。
African swine fever virus (ASFV) is the causative agent of a deadly disease in pigs and is spread rapidly across borders. Samples collected from suspected cases must be sent to the reference laboratory for diagnosis using polymerase chain reaction (PCR). In this study, we aimed to develop a simple DNA isolation step and real-time recombinase polymerase amplification (RPA) assay for rapid detection of ASFV. RPA assay based on the p72 encoding B646L gene of ASFV was established. The assays limit of detection and cross-reactivity were investigated. Diagnostic performance was examined using 73 blood and serum samples. Two extraction approaches were tested: silica-column-based extraction method and simple non-purification DNA isolation (lysis buffer and heating, 70 °C for 20 min). All results were compared with well-established real-time PCR. In a field deployment during a disease outbreak event in Uganda, 20 whole blood samples were tested. The assay’s analytical sensitivity was 3.5 DNA copies of molecular standard per µL as determined by probit analysis on eight independent assay runs. The ASFV RPA assay only detected ASFV genotypes. Compared to real-time PCR, RPA diagnostic sensitivity and specificity were 100%. Using the heating/lysis buffer extraction procedure, ASFV-RPA revealed better tolerance to inhibitors than real-time PCR (97% and 38% positivity rate, respectively). In Uganda, infected animals were identified before the appearance of fever. The ASFV-RPA assay is shown to be as sensitive and specific as real-time PCR. Moreover, the combination of the simple extraction protocol allows its use at the point of need to improve control measures.
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影响因子：
8.8
作者：
Abd El Wahed, Ahmed;Weidmann, Manfred;Hufert, Frank T.
通讯作者：
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影响因子：
3.7
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3
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3.7
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通讯作者：
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影响因子：
4.3
作者：
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