不同方法提取血培养瓶培养的抗凝血中布鲁氏菌核酸的比较

杨晓雯; 刘佳音; 张宇; 范玉; 田国忠; 赵鸿雁; 朴东日; 姜海

doi:10.3784/jbjc.202112210654

不同方法提取血培养瓶培养的抗凝血中布鲁氏菌核酸的比较

doi: 10.3784/jbjc.202112210654

1.
中国疾病预防控制中心传染病预防控制所, 传染病预防控制国家重点实验室, 北京 102206
2.
包头医学院公共卫生学院, 内蒙古包头 014040

基金项目: 国家自然科学基金（No. 81902105）；传染病预防控制国家重点实验室青年科学基金（No. 2021SKLID508）；布鲁氏菌免疫富集及多重PCR检测技术（No. KFYJ-2021053）；侯云德院士科研青年基金（No. 2019HYDQNJJ08）

详细信息

作者简介:
杨晓雯，女，山东省潍坊市人，博士，副研究员，主要从事布鲁氏菌防控及致病机制研究，Email：yangxiaowen@icdc.cn

通讯作者:
姜海，Tel: 010–58900767，Email: jianghai@icdc.cn

中图分类号: R211; R516.7
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出版历程
- 收稿日期: 2021-12-21
- 网络出版日期: 2022-05-28
- 刊出日期: 2022-08-11

Comparison of different methods for extracting Brucella DNA from anticoagulant cultured in blood culture bottle

1.
State Key Laboratory for Communicable Disease Prevention and Control, National Institute for Communicable Disease Prevention and Control, Chinese Center for Disease Control and Prevention, Beijing 102206, China
2.
School of Public Health, Baotou Medical College, Baotou 014040, Inner Mongolia, China

Funds: This study was supported by the National Natural Science Foundation of China (No. 81902105), Grant from the State Key Laboratory of Infectious Disease Prevention and Control (No. 2021SKLID508), Immuno-enrichment and Multiplex PCR Detection of Brucella (No. KFYJ-2021053) and Young Scientists Research Found of Academician Yunde Hou (No. 2019HYDQNJJ08)

More Information

Corresponding author: Jiang Hai, Email: jianghai@icdc.cn

摘要

摘要: 目的比较不同提取方法对血培养瓶培养的抗凝血中布鲁氏菌检出率的影响。方法将定量检测的灭活菌液/基因组DNA梯度添加至含抗凝血的血培养瓶中，选择不同的方法提取核酸，利用荧光定量检测其含量。结果磁珠法和裂解法最低能检测添加10² CFU/mL的灭活菌液以及10⁻³ ng/μL的基因组DNA；离心柱法无法检测未经红细胞裂解液处理的添加10⁶ CFU/mL的灭活菌液以及10 ng/μL的基因组DNA，经红细胞裂解液处理后也只能检测到添加10⁴ CFU/mL的灭活菌液以及1 ng/μL的基因组DNA的模拟样品。结论红细胞裂解液处理模拟样品能够增加核酸的提取量，裂解法提取效果最好，其次是磁珠法，再次是离心柱法。本研究为疑似布鲁氏菌病样品血培养物的核酸检测提供了方法参考，为提高布鲁氏菌病的诊断率提供了新的思路。
- 布鲁氏菌 /
- 核酸提取 /
- 血培养
Abstract: Objective To compare the effects of different extraction methods of Brucella DNA in anticoagulant cultured in blood culture. Methods In this study, the quantitatively detected inactivated Brucella spp./genomic DNA gradient was added to the blood culture bottle containing anticoagulant. Then different methods were used to extract Brucella DNA. Finally, the real time PCR was used to detect the content. Results The results showed that the magnetic nanoparticles and lysis method could detect the inactivated bacterial solution added with 10² CFU/mL and 10^-3 ng/μL genomic DNA. The centrifugal column method could not detect the inactivated bacterial solution added with 10⁶ CFU/mL and 10 ng/μL genomic DNA without treatment of erythrocyte lysate. Only 10⁴ CFU/mL inactivated bacterial solution and 1 ng/μL genomic DNA were detected after treatment with erythrocyte lysate. Conclusion The treatment of samples with erythrocyte lysate could increase the extraction amount of DNA. Among these methods, the extraction effect of lysis method was best, followed by magnetic nanoparticles method and centrifugal column method. This study provided a method as reference for the Brucella DNA extraction of blood cultures of suspected brucellosis samples, and also provided a new insight for improving the diagnosis rate of brucellosis.
- Brucella /
- DNA extraction /
- Blood culture

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表 1 不同方法提取血培养物核酸的比较

Table 1. Comparison of different methods for nucleic acid extraction form blood culture

培养瓶种类	磁珠法				离心柱法				裂解法
	灭活菌液		基因组DNA		灭活菌液		基因组DNA		灭活菌液		基因组DNA
	最低检测量（CFU/mL）	Ct值（$\bar x $±s）	最低检测量（ng/μL）	Ct值（$\bar x $±s）	最低检测量（CFU/mL）	Ct值（$\bar x $±s）	最低检测量（ng/μL）	Ct值（$\bar x $±s）	最低检测量（CFU/mL）	Ct值（$\bar x $±s）	最低检测量（ng/μL）	Ct值（$\bar x $±s）
血培养瓶A
处理组	10²	37.26± 0.12	10⁻³	37.02± 0.11	−	−	−	−	10²	36.85± 0.11	10⁻³	36.65± 0.13
未处理组	10²	37.34± 0.11	10⁻³	37.15± 0.11	−	−	−	−	10²	37.14± 0.11	10⁻³	36.80± 0.09
血培养瓶B
处理组	10²	36.85± 0.10	10⁻³	37.18± 0.07	10⁴	37.77± 0.05	1	37.54± 0.11	10²	36.73± 0.11	10⁻³	36.64± 0.12
未处理组	10²	37.05± 0.80	10⁻³	37.36± 0.16	−	−	−	−	10²	36.75± 0.11	10⁻³	37.06± 0.09
注：−. 样品中添加10⁶ CFU/mL的灭活菌液或10 ng/μL的基因组DNA利用荧光定量检测无Ct值

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