磁珠快速核酸提取法在南美白对虾病原检测中的应用及其性能评价
PDF下载 (395)徐胜威,葛明峰,卢先东,刘艳红,黄一哲,王雯琼,王雪磊,王建平,柳 海.磁珠快速核酸提取法在南美白对虾病原检测中的应用及其性能评价[J].宁波大学学报(理工版),2023,36(3):10-15.DOI:
Xu Shengwei,Ge Mingfeng,Lu Xiandong,Liu Yanhong,Huang Yizhe,Wang Wenqiong,Wang Xuelei,Wang Jianping,Liu Hai.Application and performance evaluation of magnetic beads method for rapid nucleic acid extraction in pathogen detection of Litopenaeus vannamei[J].Journal of Ningbo University(Natural Science & Engineering Edition),2023,36(3):10-15.DOI:
| Title: | Application and performance evaluation of magnetic beads method for rapid nucleic acid extraction in pathogen detection of Litopenaeus vannamei |
| 作者: | 徐胜威, 葛明峰, 卢先东, 刘艳红, 黄一哲, 王雯琼, 王雪磊, 王建平, 柳 海 |
| Author(s): | Xu Shengwei, Ge Mingfeng, Lu Xiandong, Liu Yanhong, Huang Yizhe, Wang Wenqiong, Wang Xuelei, Wang Jianping, Liu Hai |
| 关键词: | 磁珠法; 快速; 核酸提取; 评价 |
| Keywords: | magnetic beads method; rapid; nucleic acid extraction; evaluation |
| 分类号: | S945.4 |
| 文献标识码: | A |
| 摘要: | 为实现南美白对虾相关病原核酸快速、便捷提取, 并实现原材料国产化, 降低成本, 建立了一种基于国产磁珠的核酸快速提取方法(以下简称磁珠法). 以携带虾肝肠胞虫的南美白对虾为实验样本, 通过微流控芯片对3种磁珠提取的核酸进行测试, 并对裂解液中盐酸胍浓度及洗涤液2中乙醇浓度进行了优化. 通过优选实验确定 奥润磁珠提取效果优于其他两种磁珠; 裂解液中盐酸胍的最佳浓度为4mol·L-1; 洗涤液2中乙醇的最佳质量分数为75%. 在此基础上, 分析评价了优化后的磁珠法核酸提取线性范围、灵敏度、精密度、特异性及抗干扰能力等技术性能. 结果显示 稀释104倍后的低浓度病原核酸仍保持较好的扩增效果, 变异系数为2.03%, 表明建立的磁珠法具有较宽的检测范围, 灵敏度高, 特异性和抗干扰能力强. 本文建立的磁珠法可实现快捷、通用、高纯度、低成本的南美白对虾主要病原的核酸提取. |
| Abstract: | In order to promote the rapid and convenient extraction of pathogenic nucleic acid related to Litopenaeus vannamei, and to enable the localization of raw materials and reduce costs, a rapid nucleic acid extraction method based on domestic magnetic beads (magnetic beads method) was developed. In the present study, L. vannamei carrying Enterocytozoon hepatopenaei was used as the experimental sample. The nucleic acid extracted from three magnetic beads was tested by microfluidic chip, and the concentration of guanidine hydrochloride in the lysate and the concentration of ethanol in the washing solution 2 were optimized. Through the optimization test, it was determined that the extraction effect of Allrun magnetic beads was better than the other two kinds of magnetic beads. The optimum concentration of guanidine hydrochloride in the lysate was 4mol·L-1. The optimum concentration of ethanol in washing solution 2 was 75%. On this basis, the linear range, sensitivity, precision, specificity, and anti-interference ability of optimized magnetic beads method were analyzed and evaluated. The results showed that the low concentration of pathogenic nucleic acid which was diluted 104 times still maintained good amplification effect. The coefficient of variation was 2.03%, indicating that the established magnetic beads method produced a wide detection range, high sensitivity, strong specificity and anti-interference ability. The established magnetic beads method can achieve rapid, universal, high-purity and low-cost nucleic acid extraction of the main pathogens of L. vannamei. |
| 参考文献 /References: | [1] 农业农村部渔业渔政管理局, 全国水产技术推广总站. 2022中国水生动物卫生状况报告[M]. 北京: 中国农业出版社, 2022. [2] 刘洪波, 刘晓雷, 罗小铭. 核酸提取方法进展[J]. 现代生物医学进展, 2011, 11(16):3187-3190. [3] 查瑶, 王小灵, 朱诗艳, 等. 煮沸裂解法与磁珠法在HBV DNA荧光定量检测中的核酸提取效果[J]. 浙江预防医学, 2015, 27(12):1292-1293. [4] 卢瑛, 刘照关, 徐宏, 等. 一种快速提取基因组DNA的方法[J]. 中国生物工程杂志, 2008, 28(3):69-73. [5] 高秋月, 景奉香, 李海燕, 等. 基于磁珠的细菌基因组DNA快速提取方法[J]. 安徽农业科学, 2010, 38(21): 11071-11074. [6] 许朋, 郑春辉, 孙智勇, 等. 磁珠法快速提取基因组DNA的实验研究[J]. 生物信息学, 2018, 16(3):190-195. [7] Wang J H, Ali Z, Wang N Y, et al. Simultaneous extraction of DNA and RNA from Escherichia coli BL 21 based on silica-coated magnetic nanoparticles [J]. Science China Chemistry, 2015, 58(11):1774-1778. [8] 曹飞扬, 王娉, 赵晓美, 等. 常见病原菌基因组DNA快速提取试剂盒研制[J]. 东北农业大学学报, 2016, 47(6):81-88. [9] GB/T 40171—2021. 磁珠法DNA提取纯化试剂盒检测通则[S]. [10] 陆佳飞, 周科隆, 王缦. 磁珠快速提取乙型肝炎病毒DNA方法的建立及其应用研究[J]. 中华检验医学杂志, 2012, 35(9):843-850. [11] 王舒婷, 马振男, 李玲玉, 等. 基于功能化磁珠快速提取植物病毒RNA方法的建立与优化[J]. 中国生物化学与分子生物学报, 2021, 37(1):127-134. [12] 张翠真, 袁小龙, 陈志和, 等. 基于磁珠的核酸快速提取和纯化[J]. 生物学杂志, 2019, 36(4):97-101. [13] 陆佳飞, 周克隆, 王缦. 磁珠法快速提取乙型肝炎病毒DNA的研究及其在诊断试剂中的应用[C]//第五次全国免疫诊断暨疫苗学术研讨会论文汇编. 银川, 2011: 167-171. [14] 王瑞莲, 张婷, 龙军, 等. 国产磁珠法试剂在荧光定量PCR检测乙型肝炎病毒核酸中的应用[J]. 实用医学杂志, 2015, 31(21):3594-3596. [15] 毕昊, 吴岚, 郭秋, 等. 一种国产磁珠核酸自动提取系统对人巨细胞病毒DNA检测的性能评价[J]. 临床血液学杂志, 2020, 33(8):529-533. [16] 王军, 何长生, 王维, 等. 磁珠法和柱式法提取核酸在新城疫荧光定量检测中的比较[J]. 中国动物保健, 2021, 23(3):98; 100. [17] 张英哲, 宋英兰. 磁珠法在120例丙型肝炎病毒核酸定量检测中的应用效果观察[J]. 延边大学医学学报, 2020, 43(3):205-207. [18] 郑瑜宏, 钟凤金, 陈岩松, 等. 磁珠核酸提取方法在血浆EBV-DNA实时荧光定量PCR检测中应用的性能评价[J]. 中国医药导报, 2017, 14(24):13-16. |
| 备注/Memo: | 收稿日期:2023-02-06.宁波大学学报（理工版）网址:http://journallg.nbu.edu.cn/ 基金项目:宁波市公益类科技计划（2021S076）. 第一作者:徐胜威（1983－）,男,浙江兰溪人,工程师,主要研究方向:水生动物病害防控.E-mail:546207546@qq.com *通信作者:葛明峰（1988－）,男,浙江宁波人,硕士,主要研究方向:水生动物病害防控.E-mail:910342950@qq.com 宁波大学学报（理工版）网址：http://journallg.nbu.edu.cn/ |
