| 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 |
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| 引用本文： | 徐胜威1 葛明峰1 卢先东2 刘艳红2 黄一哲2 王雯琼1 王雪磊1 王建平1 柳 海1. 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, 0(3): 10-15 |
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| 作者姓名： | 徐胜威1 葛明峰1 卢先东2 刘艳红2 黄一哲2 王雯琼1 王雪磊1 王建平1 柳 海1 |
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| 作者单位： | 1.宁波市海洋与渔业研究院, 浙江 宁波 315012; 2.宁波爱基因科技有限公司, 浙江 宁波 315105 |
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| 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4 mol·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.
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| 关 键 词： | 磁珠法 快速 核酸提取 评价 |
Application and performance evaluation of magnetic beads method for rapid nucleic acid extraction in pathogen detection of Litopenaeus vannamei |
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| Xu Shengwei1,Ge Mingfeng1,Lu Xiandong2,Liu Yanhong2,Huang Yizhe2,Wang Wenqiong1,Wang Xuelei1,Wang Jianping1,Liu Hai1. 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 and Engineering Edition), 2023, 0(3): 10-15 |
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| Authors: | Xu Shengwei1 Ge Mingfeng1 Lu Xiandong2 Liu Yanhong2 Huang Yizhe2 Wang Wenqiong1 Wang Xuelei1 Wang Jianping1 Liu Hai1 |
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| Affiliation: | 1.Ningbo Ocean and Fisheries Research Institute, Ningbo 315012, China; 2.Ningbo iGene Technology Co., Ltd., Ningbo 315105, China |
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| 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. |
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| Keywords: | magnetic beads method rapid nucleic acid extraction evaluation |
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