Hu Xi, Jiang Nan, Li Yiqun, Zhou Yong, Fan Yuding, Xue Mingyang, Zeng Lingbing, Liu Wenzhi, Meng Yan
National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China.
Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China.
Animals (Basel). 2022 Aug 8;12(15):1999. doi: 10.3390/ani12151999.
Molecular diagnostic testing for viral pathogens is crucial in aquaculture. The efficient and convenient preparation of pathogenic microbial nucleic acids is the basis of molecular diagnosis. Here, we developed a simplified deoxyribonucleic acid (DNA) extraction method from aquatic animal DNA viruses using the Chelex 100 resin. The nucleic acid was extracted from infected tissues and cell culture for the detection of three common aquatic viral pathogens (CEV, CyHV-2, and GSIV). We compared the extraction effects of a current commercial kit extraction method and the Chelex 100 resin extraction method according to nucleic acid concentration, conventional polymerase chain reaction (PCR), and digital droplet PCR (ddPCR). The results indicated that both extraction procedures could obtain high-quality nucleotide samples. Extracting DNA using the Chelex 100 resin led to better detective efficiency for ddPCR molecular diagnostic testing. The whole process took less than 20 min, and only Chelex 100 resin solution was added to the tissues or cells without multiple tubes being transferred several times. The extracted DNA concentration and the detection sensitivity were high. These results indicated that the Chelex 100 resin solution has the advantages of speed, efficiency, and economy compared to the commercial kit. In addition, the higher pH value (10-11) of the Chelex 100 resin solution markedly improved the detection sensitivity compared to a lower pH value (9-10). In conclusion, the comparison of the Chelex 100 Resin and commercial viral DNA extraction kits revealed the good performance of the Chelex 100 resin solution at pH 10-11 in DNA extraction for PCR amplification from aquatic animal viral samples of tissues and cells in molecular diagnostic testing. It is both rapid and cost-effective.
病毒病原体的分子诊断检测在水产养殖中至关重要。高效便捷地制备病原微生物核酸是分子诊断的基础。在此，我们开发了一种使用Chelex 100树脂从水生动物DNA病毒中提取脱氧核糖核酸（DNA）的简化方法。从感染组织和细胞培养物中提取核酸，用于检测三种常见的水生病毒病原体（CEV、CyHV - 2和GSIV）。我们根据核酸浓度、常规聚合酶链反应（PCR）和数字液滴PCR（ddPCR）比较了当前商业试剂盒提取方法和Chelex 100树脂提取方法的提取效果。结果表明，两种提取方法都能获得高质量的核苷酸样本。使用Chelex 100树脂提取DNA对ddPCR分子诊断检测具有更好的检测效率。整个过程耗时不到20分钟，只需将Chelex 100树脂溶液添加到组织或细胞中，无需多次转移多个管子。提取的DNA浓度和检测灵敏度都很高。这些结果表明，与商业试剂盒相比，Chelex 100树脂溶液具有速度快、效率高和经济的优点。此外，与较低pH值（9 - 10）相比，Chelex 100树脂溶液较高的pH值（10 - 11）显著提高了检测灵敏度。总之，Chelex 100树脂与商业病毒DNA提取试剂盒的比较表明，在分子诊断检测中，pH值为10 - 11的Chelex 100树脂溶液在从水生动物病毒组织和细胞样本中提取DNA用于PCR扩增方面表现良好。它既快速又具有成本效益。
