Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang 150001, China.
J Virol Methods. 2012 Feb;179(2):402-8. doi: 10.1016/j.jviromet.2011.12.001. Epub 2011 Dec 9.
A highly sensitive real-time PCR method was developed in this study for reticuloendotheliosis virus (REV) detection and quantitation. The real-time PCR method, with a minimum detection limit of 10 proviral DNA copies, was 100 times more sensitive than the conventional PCR. It was also shown to be highly specific, as no positive signals were detected for other common avian DNA viruses. The coefficients of variation of intra- and inter-assay reproducibility were both less than 2%. The chicken β-actin gene was co-amplified and used as the internal control to monitor the efficiency of DNA extraction and PCR amplification. Specific pathogen free chickens were infected with REV at different ages and the blood was detected with the real-time PCR method. High levels of proviral DNA were detected in the blood of REV-infected chickens during the experiment and chickens infected early had higher proviral loads from 2 weeks post-infection compared with late infected chickens. This study provides an excellent research and diagnostic tool that can be used for REV detection and quantitation.
本研究建立了一种用于检测和定量网状内皮组织增生症病毒 (REV) 的高度敏感实时 PCR 方法。该实时 PCR 方法的最小检测限为 10 个前病毒 DNA 拷贝，比常规 PCR 灵敏 100 倍。它还表现出高度的特异性，因为没有检测到其他常见禽 DNA 病毒的阳性信号。内和间分析重复性的变异系数均小于 2%。鸡 β-肌动蛋白基因被共扩增，并用作内部对照，以监测 DNA 提取和 PCR 扩增的效率。将无特定病原体鸡用 REV 在不同年龄感染，并使用实时 PCR 方法检测血液。在实验过程中，REV 感染鸡的血液中检测到高水平的前病毒 DNA，与晚期感染鸡相比，早期感染鸡的前病毒载量从感染后 2 周开始更高。本研究提供了一种出色的研究和诊断工具，可用于 REV 的检测和定量。
