Adhikary Arun Kumar, Hanaoka Nozomu, Fujimoto Tsuguto
Unit of Microbiology, Faculty of Medicine, AIMST University, Semeling, 08100 Bedong, Kedah Darul Aman, Malaysia ; Infectious Disease Surveillance Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
Infectious Disease Surveillance Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
Biomed Res Int. 2014;2014:363790. doi: 10.1155/2014/363790. Epub 2014 Mar 9.
Restriction endonuclease analyses (REAs) constitute the only inexpensive molecular approach capable of typing and characterizing human adenovirus (HAdV) strains based on the entire genome. However, the application of this method is limited by the need for time-consuming and labor-intensive procedures. We herein developed a simple and cost-effective REA for assessing HAdV. The method consists of (1) simple and cost-effective DNA extraction, (2) fast restriction endonuclease (RE) digestion, and (3) speedy mini agarose gel electrophoresis. In this study, DNA was isolated according to the kit-based method and 21.0 to 28.0 μg of viral DNA was extracted from prototypes (HAdV-1, HAdV-3, HAdV-4, and HAdV-37) in each flask. The amount of DNA ranged from 11.4 to 57.0 μg among the HAdV-3 (n=73) isolates. The obtained viral DNA was found to be applicable to more than 10 types of REAs. Fast-cut restriction endonucleases (REs) were able to digest the DNA within 15 minutes, and restriction fragments were easily separated via horizontal mini agarose gel electrophoresis. The whole procedure for 10 samples can be completed within approximately six hours (the conventional method requires at least two days). These results show that our REA is potentially applicable in many laboratories in which HAdVs are isolated.
限制性内切酶分析（REAs）是唯一一种基于全基因组对人腺病毒（HAdV）毒株进行分型和特征描述的低成本分子方法。然而，该方法的应用受到耗时且 labor-intensive procedures（劳动密集型程序）的限制。我们在此开发了一种用于评估HAdV的简单且经济高效的REA。该方法包括：（1）简单且经济高效的DNA提取，（2）快速限制性内切酶（RE）消化，以及（3）快速小型琼脂糖凝胶电泳。在本研究中，根据基于试剂盒的方法分离DNA，每个培养瓶中从原型毒株（HAdV-1、HAdV-3、HAdV-4和HAdV-37）中提取21.0至28.0μg病毒DNA。在HAdV-3（n = 73）分离株中，DNA量在11.4至57.0μg之间。发现所获得的病毒DNA适用于10多种类型的REAs。快速切割限制性内切酶能够在15分钟内消化DNA，并且通过水平小型琼脂糖凝胶电泳可轻松分离限制性片段。10个样品的整个过程可在大约6小时内完成（传统方法至少需要两天）。这些结果表明，我们的REA可能适用于许多分离HAdV的实验室。
