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通过人工模拟即在养殖池塘底泥和水样中加入阳性组织处理样品,对人工模拟的底泥样品分别采用磁珠法、酚氯仿法以及吸附柱法进行核酸提取,结果发现底泥含量对磁珠法和吸附柱法病毒DNA提取效果影响较小,而对酚氯仿法病毒DNA提取效果影响较大,且底泥含量较少提取相对较好。3种病毒DNA提取方法中采用混样提取核酸的效果均好于用上清液提取的效果。吸附柱法和磁珠法对病毒DNA提取效果较接近且明显好于酚氯仿法。通过对不同浓度的PEG6000进行浓缩后提取核酸,结果发现原始水样中未检测到Cy HV-2或其含量极低未达到检测限。PEG6000浓度为13%时,核酸浓缩提取效果最佳。本研究为实时监测养殖池塘中的底泥和水体中是否存在病毒提供了一种参考方法,从而更好地维护水产养殖环境的生态安全和促进我国水产养殖业健康可持续发展。
By artificial simulation that in the ponds and aquaculture ponds to join the positive samples of tissue samples were simulated artificial sediment samples using magnetic beads method, phenol chloroform method and adsorption column method for nucleic acid extraction and found that the sediment content of magnetic Bead method and adsorption column DNA had little effect on DNA extraction, but had a significant effect on DNA extraction of phenol-chloroform method, and the sediment content was relatively less. The results of three kinds of virus DNA extraction methods using mixed samples to extract nucleic acid were better than that of the supernatant. Adsorption column method and magnetic beads method for the extraction of viral DNA closer to and significantly better than the phenol chloroform method. Nucleic acids were extracted by concentration of PEG6000 at different concentrations. As a result, Cy HV-2 was undetectable in the original water sample or the detection limit was very low. When the concentration of PEG6000 is 13%, the best concentration of nucleic acid extraction. This study provides a reference method for real-time monitoring of sediment in ponds and the existence of viruses in aquatic ponds so as to better safeguard the ecological safety of aquaculture environment and promote the healthy and sustainable development of aquaculture in China.