环介导等温扩增技术快速检测传染性喉气管炎病毒分离株。
Rapid detection of infectious laryngotracheitis virus isolates by loop-mediated isothermal amplification.
机构信息
College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
出版信息
J Virol Methods. 2010 Apr;165(1):71-5. doi: 10.1016/j.jviromet.2010.01.006. Epub 2010 Jan 25.
The objective of this study was to develop and evaluate a loop-mediated isothermal amplification (LAMP) method to detect infectious laryngotracheitis virus (ILTV) from commercial broiler and layer flocks in southern China. A set of six specific primers was designed to recognize six distinct genomic sequences of thymidine kinase (TK) from ILTV. The entire assay duration was recorded at 40 min under isothermal condition at 63.5 degrees C. The amplified products were analyzed by electrophoresis and visual judgment by the SYBR Green I dyeing. LAMP assay was 10-fold more sensitive than the routine PCR assay, with a detection limit of 46 copies per reaction. In detecting ILTV, the LAMP assay detected all 5 strains previously isolated, did not cross-react with other avian pathogens, and obtained a 100% sensitivity in 43 positive clinical samples with reference to virus isolation. Therefore, the LAMP assay may be a good alternative method for specific diagnosis of ILTV infection in primary care facilities, and in less well-equipped laboratories.
本研究旨在开发和评估一种环介导等温扩增（LAMP）方法，以检测来自中国南方商业肉鸡和蛋鸡群中的传染性喉气管炎病毒（ILTV）。设计了六对特异性引物，以识别 ILTV 的胸苷激酶（TK）的六个独特基因组序列。整个检测过程在 63.5°C 的等温条件下持续 40 分钟。扩增产物通过电泳进行分析，并通过 SYBR Green I 染色进行目视判断。LAMP 检测比常规 PCR 检测灵敏 10 倍，检测限为每个反应 46 个拷贝。在检测 ILTV 时，LAMP 检测方法可检测到之前分离的 5 株病毒，与其他禽病病原体无交叉反应，在参考病毒分离的 43 份阳性临床样本中，灵敏度达到 100%。因此，LAMP 检测方法可能是基层医疗机构和设备条件较差的实验室中针对 ILTV 感染的特异性诊断的一种良好替代方法。
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