Comparison of different methods for extracting Brucella DNA from anticoagulant cultured in blood culture bottle
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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。 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. -
Key words:
- 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±
0.1210−3 37.02±
0.11− − − − 102 36.85±
0.1110−3 36.65±
0.13未处理组 102 37.34±
0.1110−3 37.15±
0.11− − − − 102 37.14±
0.1110−3 36.80±
0.09血培养瓶B 处理组 102 36.85±
0.1010−3 37.18±
0.07104 37.77±
0.051 37.54±
0.11102 36.73±
0.1110−3 36.64±
0.12未处理组 102 37.05±
0.8010−3 37.36±
0.16− − − − 102 36.75±
0.1110−3 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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