Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification
Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification
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逆转录环介导等温扩增快速检测鸭甲型肝炎病毒C基因型
DOI：
10.1016/j.jviromet.2013.11.009
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发表时间：
2014-02-01
影响因子：
3.1
通讯作者：
Liu, Guangqing
中科院分区：
文献类型：
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作者：
Li, Chuanfeng;Chen, Zongyan;Liu, Guangqing
采用一步逆转录环介导等温扩增 (RI-LAMP) 测定法并对其进行优化，开发了一种快速、灵敏的鸭甲型肝炎病毒 C 基因型 (DHAV-C) RNA 检测系统。针对 DHAV（GX1201 株）3D 基因内高度保守的序列设计了一组四个特异性引物。在最佳反应条件下，DHAV-C特异性RI-LAMP的灵敏度比逆转录酶-聚合酶链式反应（RT-PCR）高100倍，每次反应的检测限为0.3 pg（6.59 x 104拷贝）。其他鸭病毒样本中未观察到交叉反应，这与RT-PCR结果吻合良好。此外，可以通过观察添加SYBR green I染料后的浊度或颜色变化来目视检查阳性反应。对样品应用 DHAV-C 特异性 RT-LAMP 检测，并与 RT-PCR 进行比较。 RT-LAMP 检测的阳性样本率为 26.7%（45 例中的 12 例），RT-PCR 检测的阳性样本率为 20%（45 例中的 9 例）。因此，新开发的RT-LAMP检测是一种快速、特异、灵敏且经济有效的DHAV-C检测方法。该测定在 DHAV-C 感染的临床诊断和现场监测方面具有潜在的应用。 (C) 2013 年由 Elsevier B.V. 出版
A one-step reverse transcription loop-mediated isothermal amplification (RI-LAMP) assay was used and optimized to develop a rapid and sensitive detection system for duck hepatitis A virus genotype C (DHAV-C) RNA. A set of four specific primers was designed against highly conserved sequences located within the 3D gene from DHAV (strain GX1201). Under optimal reaction conditions, the sensitivity of DHAV-C-specific RI-LAMP was 100-fold higher than that of reverse transcriptase-polymerase chain reaction (RT-PCR), with a detection limit of 0.3 pg (6.59 x 104 copies) per reaction. No cross-reactivity was observed from the samples of other duck viruses, which is in good accordance with RT-PCR. Furthermore, a positive reaction can be visually inspected by observing turbidity or color change after the addition of SYBR green I dye. The DHAV-C-specific RT-LAMP assay was applied to the samples and compared with RT-PCR. The positive-sample ratios were 26.7% (12 of 45) by RT-LAMP and 20% (9 of 45) by RT-PCR. Therefore, the newly developed RT-LAMP assay is a rapid, specific, sensitive, and cost-effective method of DHAV-C detection. This assay has potential applications in both clinical diagnosis and field surveillance of DHAV-C infection. (C) 2013 Published by Elsevier B.V.