[1]潘永圣,蒲 丹,汪佳婕,等.三种方法提取毛霉菌核酸效能的比较实验研究[J].现代检验医学杂志,2023,38(06):148-152.[doi:10.3969/j.issn.1671-7414.2023.06.027]
　PAN Yongsheng,PU Dan,WANG Jiajie,et al.Comparative Experimental Study on the Efficiency of Three Methods for Extracting Mucor Nucleic Acid[J].Journal of Modern Laboratory Medicine,2023,38(06):148-152.[doi:10.3969/j.issn.1671-7414.2023.06.027]
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三种方法提取毛霉菌核酸效能的比较实验研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第38卷
期数:
2023年06期
页码:
148-152
栏目:
研究简报·实验技术
出版日期:
2023-11-15

文章信息/Info

Title:
Comparative Experimental Study on the Efficiency of Three Methods for Extracting Mucor Nucleic Acid
文章编号:
1671-7414（2023）06-148-05
作者:
潘永圣; 蒲 丹; 汪佳婕; 赵云平; 徐宏忍; 白敏凤; 许小靓; 尹利民
（昆明市第一人民医院检验科，昆明 650011）
Author(s):
PAN Yongsheng;  PU Dan;  WANG Jiajie;  ZHAO Yunping;  XU Hongren;  BAI Minfeng;  XU Xiaoliang;  YIN Limin
（Department of Laboratory Medicine, the First People’s Hospital of Kunming, Kunming 650011, China）
关键词:
毛霉菌; 核酸提取; 实时荧光定量PCR
分类号:
R446.5
DOI:
10.3969/j.issn.1671-7414.2023.06.027
文献标志码:
A
摘要:
目的 比较改良碱裂解法、一步法和磁珠法提取毛霉菌核酸效能的差异，探索一种用于PCR 扩增的快速、高效、简便、经济的毛霉菌核酸提取方法。方法 分别用改良碱裂解法、一步法和磁珠法提取黑根毛霉（Rhizomucor miehei，R.miehei）、伞枝犁头霉（Lichtheimia corymbifera，L. corymbifera）和卷枝毛霉（Mucor circinelloides，M. circinelloides）3 株毛霉菌核酸，使用Qbit4 核酸浓度测定仪测定核酸浓度，并进行实时荧光定量PCR 扩增实验，比较其扩增后的循环阈值（Ct）差异和核酸提取效率。结果 改良碱裂解法、一步法及磁珠法均能提取R. miehei，L. corymbifera 和M.circinelloides 3 株毛霉菌核酸，其中一步法提取所得核酸的浓度最高，改良碱裂解法次之，磁珠法最低，差异具有统计学意义（F=38.803，2 160.759，325.175，均P<0.05）。改良碱裂解法提取毛霉菌核酸耗时最短（10.00± 1.50 min），一步法次之（25.00 ± 1.50 min），磁珠法最长（75.00 ± 1.50 min），差异有统计学意义（F=2 574.074，P<0.01）。磁珠法提取毛霉菌核酸耗费成本和设备依赖度最高，其次是一步法次之，改良碱裂解法最低。改良碱裂解法、一步法及磁珠法提取的3 株毛霉菌核酸均能扩增出阳性结果。三种方法提取R. miehei 菌株核酸扩增后Ct 值分别为18.72±0.18，17.42±0.46 和18.97±0.46，差异有统计学意义（F=22.375，P<0.01），其中改良碱裂解法和磁珠法提取的核酸扩增后Ct 值差异无统计学意义（t=1.114，P>0.05），一步法提取的核酸扩增后的Ct 值小于另外两种方法，差异有统计学意义（t=5.852，5.277，均P<0.05）。提取L. corymbifera 菌株核酸扩增后Ct 值分别为16.23 ± 0.13，21.23 ± 0.25 和14.53± 0.04，差异有统计学意义（F=2 298.548，P<0.01），其中改良碱裂解法提取的核酸扩增后的Ct 值小于一步法，差异有统计学意义（t=40.031，P<0.01），磁珠法提取的核酸扩增后的Ct 值小于另外两种方法，差异有统计学意义（t=27.953，60.265，均P<0.05）。提取M. circinelloides 菌株核酸扩增后Ct 值分别为16.77±0.28，15.38±0.88 和16.13±0.20，差异有统计学意义（F=8.152，P<0.01），其中改良碱裂解法提取的核酸产物扩增后的Ct 值大于另外两种方法，差异有统计学意义（t=3.372，4.106，均P < 0.05），一步法和磁珠法提取的核酸产物扩增后的Ct 值差异无统计学意义（t=1.866，P > 0.05）。结论 改良碱裂解法、一步法及磁珠法均可成功提取毛霉菌核酸，其中改良碱裂解法操作简便、耗时最短、耗费成本和设备依赖性最低，所提取毛霉菌核酸模板能够满足实时荧光定量PCR 扩增要求。
Abstract:
Objective To compare the difference in the extraction efficiency of Mucor Mucleic acid by using improved alkaline lysis method, one-step method and magnetic beads method, and to explore a rapid, efficient and economical Mucor nucleic acid extraction method for PCR amplification. Methods The Mucor nucleic acid was extracted from Rhizomucor miehei (R. miehei), Lichtheimia corymbifera (L. corymbifera) and Mucor circinelides (M. circinelides) by using improved alkaline lysis method, one-step method and magnetic beads method. The nucleic acid concentration was measured by Qbit4, and fluorescence quantitative PCR was performed to compare the difference of cycle threshold (Ct) values after amplification and nucleic acid extraction efficiency. Results The nucleic acid was extracted from R. miehei, L. corymbifera and M. circinelloides by improved alkaline lysis method, one-step method and magnetic beads method. Among three methods, one-step method gained the highest nucleic acid concentration, followed by improved alkaline lysis method, while magnetic beads method obtained the lowest concentration, and the differences were statistically significant (F=38.803, 2 160.759, 325.175, all P<0.05). The time of Mucor nucleic acid extraction was shortest by improved alkaline lysis method (10.00 ± 1.50 min), followed by the one-step method (25.00 ± 1.50 min), and the time of Mucor nucleic acid extracted was longest by magnetic beads method (75.00 ± 1.50 min), the difference was statistically significant (F=2 574.074, P<0.01). The cost and equipment dependency of Mucor nucleic acid extracted was highest by magnetic beads method, followed by the one-step method, and the cost and equipment dependency of Mucor nucleic acid extracted was lowest by improved alkaline lysis method. The Mucor nucleic acid extracted by improved alkaline lysis method, one-step method and magnetic beads method could amplify positive results. The Ct values of nucleic acid amplification extracted from R. miehei by improved alkaline lysis method, one-step method and magnetic beads method were 18.72 ± 0.18, 17.42 ± 0.46 and 18.97 ± 0.46，respectively, and there were significant differences (F=22.375, P<0.01). Among them, there was no significant difference between Ct value of nucleic acid amplification extracted by improved alkaline lysis method and magnetic beads method (t=1.114, P>0.05), the Ct value of nucleic acid amplification extracted by one-step method was lower than that by improved alkaline lysis method and magnetic beads method, and there were significant differences (t=5.852, 5.277, all P<0.05). The Ct values of nucleic acid amplification extracted from L. corymbifera by improved alkaline lysis method, one-step method and magnetic beads method were 16.23 ± 0.13, 21.23 ± 0.25 and 14.53 ± 0.04，respectively, and there were significant differences (F=2 298.548, P<0.01). The Ct value of nucleic acid amplification extracted by improved alkaline lysis method was lower than that by one-step method, and there were also significant differences (t=40.031, P<0.01). The Ct value of nucleic acid amplification extracted by magnetic beads method was lower than that by improved alkaline lysis method and one-step method, and there were also significant differences (t=27.953, 60.265, all P<0.05). The Ct values of nucleic acid amplification extracted from M. circinelloides by improved alkaline lysis method, one-step method and magnetic beads method were 16.77 ± 0.28, 15.38 ± 0.88 and 16.13 ± 0.20， respectively, and the difference was statistically significant (F=8.152, P<0.01). The Ct value of nucleic acid amplification extracted by improved alkaline lysis method was higher than that by one-step method and magnetic beads method, and the differences were also statistically significant (t=3.372, 4.106, all P<0.05). However, there was no significant difference between Ct value of nucleic acid amplification extracted by one-step method and magnetic beads method (t=1.866，P>0.05). Conclusion The improved alkaline lysis method, one-step method and magnetic beads method could be used for Mucor nucleic acid extraction, which is sufficient for real-time fluorescence quantitative PCR amplification. Among three method, the improved alkaline lysis method may be easy and simple to perform.

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备注/Memo

备注/Memo:
基金项目： 云南省科技厅科技基础研究计划课题项目（202201AY070001-200）：毛霉菌病多重荧光PCR 检测方法建立及临床应用研究；春城计划高层次创新创业团队专项（2022SCP002）：5G+ 分子精准医学大数据平台解锁肝胆胰密码打造云南健康生活目的地。
作者简介：潘永圣（1988-），男，硕士，初级技师，研究方向：临床生化及分子生物学检验，E-mail：panysh88@126.com。
通讯作者：尹利民（1978-），男，博士，主任技师，研究方向：临床检验诊断学研究，E-mail：ylmbj@163.com。
更新日期/Last Update: 2023-11-15