Abstract

This study was aimed to establish a highly specific and sensitive loop-mediated isothermal amplification (LAMP) method for diagnosing avian infectious laryngotracheitis (AILT). DNA was extracted from isolated infectious laryngotracheitis virus (ILTV) strains and control samples, followed by PCR using three sets of six specific primers. The detection efficiency of the LAMP assay was evaluated by the turbidity and calcein methods. The sensitivity of LAMP was then assessed using a concentration gradient followed by a specificity analysis. Furthermore, the detection efficiency of LAMP and PCR was compared. Finally, a clinical test was performed to evaluate the value of the LAMP assay. The optimal temperature for the LAMP reaction was 66 °C. Meanwhile, the primers selected for the LAMP assay were highly specific for the target virus. The sensitivity of the turbidity and calcein methods for LAMP was consistent. The minimum detection concentration of LAMP was 0.06 pg/μL, which was 100-fold higher than that of PCR. Furthermore, the results from clinical samples showed that the LAMP method could identify AILT from many samples. The newly designed LAMP assay was an effective method for AILT detection at an optimal temperature of 66 °C with a minimum detection concentration of 0.06 pg/µL.

摘要

本研究旨在建立一种用于诊断禽传染性喉气管炎 (AILT) 的高度特异性和灵敏的环介导等温扩增 (LAMP) 方法。从分离的传染性喉气管炎病毒 (ILTV) 菌株和对照样本中提取 DNA，然后使用三组六种特异性引物进行 PCR。LAMP 检测的检测效率通过浊度和钙黄绿素方法进行评估。然后使用浓度梯度和特异性分析评估 LAMP 的敏感性。此外，比较了LAMP和PCR的检测效率。最后，进行了临床测试以评估 LAMP 检测的价值。LAMP 反应的最佳温度为 66 °C。同时，为 LAMP 检测选择的引物对目标病毒具有高度特异性。浊度法和钙黄绿素法对 LAMP 的灵敏度是一致的。LAMP的最低检测浓度为0.06 pg/μL，比PCR高100倍。此外，临床样本的结果表明，LAMP 方法可以从许多样本中识别 AILT。新设计的 LAMP 检测是在 66 °C 的最佳温度下检测 AILT 的有效方法，最低检测浓度为 0.06 pg/µL。