Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Subgroup J Avian Leukosis Virus
Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Subgroup J Avian Leukosis Virus
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DOI：
10.1128/jcm.02530-09
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发表时间：
2010-06-01
影响因子：
9.4
通讯作者：
Cao, Weisheng
中科院分区：
文献类型：
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作者：
Zhang, Xiaotao;Liao, Ming;Cao, Weisheng
近年来，中国种鸡感染J亚群禽白血病病毒（ALV-J）呈上升趋势。在这项研究中，我们开发了一种环介导的等温扩增（LAMP）方法，用于快速检测培养分离物和临床样品中的ALV-J。alv - j特异性LAMP实验在63℃条件下仅需简单的实验室水浴即可在45分钟内有效扩增目标基因。为了确定LAMP试验的特异性，检测了不同亚群的alv和其他相关病毒。在凝胶电泳上，ALV-J分离株呈阶梯状，其他病毒未见阶梯状。为评价LAMP法和常规PCR法对NX0101分离物质粒DNA扩增的敏感性。LAMP法的检出限为5个靶基因拷贝/反应，比传统PCR的检出限提高了20倍。为评价LAMP法在临床标本中检测ALV- j的应用价值，采用LAMP法和PCR法对49份疑似ALV感染种鸡标本进行检测。此外，还利用细胞培养从这些样品中分离病毒。常规PCR阳性率为21/49 (43%)，LAMP法为26/49(53%)，病毒分离法为19/46（41%）。此外，加入SYBR绿I染料后，通过观察浊度或颜色变化，可以直观地确定LAMP反应是否为阳性。因此，LAMP检测是一种简单、快速、灵敏的诊断方法，可用于现场快速检测ALV-J感染。
Infection of breeder flocks in China with subgroup J avian leukosis virus (ALV-J) has increased recently. In this study, we have developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of ALV-J from culture isolates and clinical samples. The ALV-J-specific LAMP assay efficiently amplified the target gene within 45 min at 63 degrees C using only a simple laboratory water bath. To determine the specificity of the LAMP assay, various subgroup ALVs and other related viruses were detected. A ladder pattern on gel electrophoresis was observed for ALV-J isolates but not for other viruses. To evaluate the sensitivities of the LAMP assay and conventional PCR, the NX0101 isolate plasmid DNA was amplified by them. The detection limit of the LAMP assay was 5 target gene copies/reaction, which was up to 20 times higher than that of conventional PCR. To evaluate the application of the LAMP assay for detection of ALV-J in clinical samples, 49 samples suspected of ALV infection from breeder flocks were tested by the LAMP assay and PCR. Moreover, virus isolation from these samples was also performed using cell culture. The positive-sample ratios were 21/49 (43%) by conventional PCR, 26/49 (53%) by the LAMP assay, and 19/46 (41%) by virus isolation. Additionally, a positive LAMP reaction can be visually ascertained by the observation of turbidity or a color change after addition of SYBR green I dye. Consequently, the LAMP assay is a simple, rapid, and sensitive diagnostic method and can potentially be developed for rapid detection of ALV-J infection in the field.