| 引用本文: | 赵启宇,陈振帆,刘超,孙璐,甄毓,张清春.四种核酸快提法提取藻华微藻核酸效果的对比研究[J].海洋科学,2024,48(11):4-. |
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| 四种核酸快提法提取藻华微藻核酸效果的对比研究 |
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赵启宇1,2, 陈振帆1,3, 刘超1,2, 孙璐1,2, 甄毓4, 张清春1,3,5
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1.中国科学院海洋研究所海洋生态与环境科学重点实验室, 山东 青岛 266071;2.中国科学院大学, 北京 100049;3.青岛海洋科技中心海洋生态与环境科学功能实验室, 山东 青岛 266237;4.中国海洋大学环境科学与工程学院海洋环境与生态教育部重点实验室, 山东 青岛 266100;5.中国科学院海洋大科学研究中心, 山东 青岛 266071
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| 摘要: |
| 近年来,世界近海海域微藻藻华频发,藻华藻种多样化,应用特异性的分子生物学方法进行藻华现场检测和监测迫在眉睫。快速、简便和高效地提取藻华微藻核酸物质是实现藻华现场分子检测的前提。本研究采用重组酶聚合酶扩增(recombinase polymerase amplification,RPA)和聚合酶链式反应(polymerase chain reaction,PCR)作为检测方法,对比分析了4种核酸快速提取方法对藻华微藻核酸的提取效果。实验发现本研究研制的二甲基亚砜(dimethyl sulfoxide,DMSO)-曲拉通(Triton X100)核酸快提液及其提取的DNA溶液对RPA和PCR反应均无抑制效应,该核酸快提液在37℃条件中裂解10 min可实现对多种藻华微藻DNA的有效提取,通过模拟野外实验发现该核酸快提液具有不错的抗环境杂质干扰能力。总体而言,DMSO-Triton X100核酸快提法具有对分子反应无抑制、操作简单、裂解条件温和、核酸提取效率高等优点,可快速提取藻华微藻核酸,有助于藻华现场分子检测方法的研发和应用,也有望推广至其他研究领域核酸的快速提取。 |
| 关键词: 有害藻华 核酸快速提取 分子检测 重组酶聚合酶扩增 聚合酶链式反应 |
| DOI：10.11759/hykx20240321001 |
| 分类号:X145 |
| 基金项目:崂山实验室山东省专项经费(LSKJ202203700);国家自然科学基金项目(42076140);国家重点研发项目(2022YFC3105200) |
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| Comparative study on the effects of four rapid extraction methods on extracting harmful microalgal nucleic acids |
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ZHAO Qiyu1,2, CHEN Zhenfan1,3, LIU Chao1,2, SUN Lu1,2, Zhen Yu4, ZHANG Qingchun1,3,5
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1.Key Laboratory of Marine Ecology and Environment Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;2.University of Chinese Academy of Sciences, Beijing 100049, China;3.Laboratory for Marine Ecology and Environmental Science, Qingdao Marine Science and Technology Center, Qingdao 266237, China;4.Key Laboratory of Marine Environment and Ecology, Ministry of Education, College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China;5.Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China
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| Abstract: |
| In recent years, harmful algal bloom (HAB) events caused by diverse microalgae have become increasingly common in coastal waters worldwide. This trend underscores the pressing need for developing field and in situ molecular methods for detecting and monitoring HAB events. Rapid and efficient DNA extraction from HAB microalgae is crucial in these molecular detection techniques. This study employed recombinase polymerase amplification (RPA) and polymerase chain reaction (PCR) as detection methods to compare the effectiveness of four rapid extraction methods of nucleic acids from HAB microalgae. Among these, the dimethyl sulfoxide (DMSO)-Triton X100 rapid nucleic-acid extraction method was identified as the most effective. Its extraction solution did not inhibit RPA and PCR reactions and effectively extracted DNA from various HAB microalgae at 37 ℃ in just 10 min. Additionally, this assay demonstrated strong resistance to environmental impurities and consistently extracted DNA from marine phytoplankton. Overall, the DMSO-Triton X100-based method offers several advantages for nucleic-acid extraction from HAB microalgae, including minimal inhibition of molecular reactions as well as easy application, gentle lysis conditions, and high efficiency. This method can facilitate the development and application of field and in situ detection assays for HABs, making it an optimal choice for researchers in this field. Furthermore, its potential application for nucleic acid extraction in other research fields enhances its versatility and usefulness. |
| Key words: Harmful Algal Bloom(HAB) Rapid nucleic acid extraction Molecular detection Recombinase Polymerase Amplification(RPA) Polymerase Chain Reaction(PCR) |
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