ISSN 1003-8280 CN 10-1522/R 中国疾病预防控制中心 主办
收稿日期: 2016-12-06
网络出版日期: 2017-04-20
基金资助
国家重点研发计划课题（2016YFF0203205）；国家质量监督检验检疫总局科技计划项目（2015IK067，2015IK069）；广东省省级科技计划项目（2015A050502009）；中山市科技计划项目（2014A2FC252）
Next Generation Illumina Sequencing of bacteria carried by Chrysomya megacephala
Received date: 2016-12-06
Online published: 2017-04-20
Supported by
Supported by the National Quality Infrastracture Program(No. 2016YFF0203205), Administration of Quality Supervision, Inspection and Quarantine Support Program of China(No. 2015IK067,2015IK069), Science and Technology Project of Guangdong Province(No. 2015A050502009), and Science and Technology Plan Project of Zhongshan City(No. 2014A2FC252)
目的 研究高通量测序技术在医学媒介生物携带细菌检测中的应用，建立一种操作简便、经济高效和鉴定准确的检测方法。方法 优化样本处置和细菌提取等前处理方法、优化PCR反应体系和条件；以大头金蝇为研究材料，利用Illumina高通量测序平台，以二代测序技术（NGS）检测16S rRNA的V3可变区，分别研究其体内外携带细菌的情况。结果 在大头金蝇体表和体内共有95种细菌鉴定到种，235种细菌鉴定到属，体表携带15个门的细菌，而体内仅携带6个门的细菌。但是从能确定细菌种类的序列数来看，体内（376 282条）所携带细菌的数量却是体表（241 375条）的1.56倍。厚壁菌门（Phylum Firmicutes）、变形菌门（Proteobacteria）、拟杆菌门（Bacteroidetes）的细菌总数在体内外被检测到的概率分别为99.83%和97.75%，检测到的细菌种类数远超传统方法。结论 建立的样本收集和前处理方法、PCR扩增体系和条件有效、可行，利用Illumina高通量测序平台进行医学媒介生物携带细菌的NGS检测新方法可行并可推广。
关键词： Illumina测序平台; V3可变区; 细菌; 大头金蝇
陈健, 岳巧云, 邱德义, 刘德星 . 二代测序技术检测大头金蝇携带细菌的研究[J]. 中国媒介生物学及控制杂志, 2017 , 28(2) : 124 -130 . DOI: 10.11853/j.issn.1003.8280.2017.02.007
Objective Establishment of an economic, efficient, accurate and convenient detection method for the bacteria carried by medical vector with the high-throughput sequencing platform(NGS). Methods Optimizing the pretreatment method of the samples, and then sequencing with the NGS techniques through the Illumina high-throughput sequencing platform, detecting the V3 variable regions of 16S rRNA to study bacteria carried by Chrysomya megacephala. Results Ninty-five bacteria carried by C. megacephala were identified to species and 235 species were identified to genus with the improved method, which exceeded the bacteria species identified in the previous studies with the traditional methods. Conclusion The methods of sample collection, pretreatment, and PCR conditions established in this study, sequencing with the Illumina high-throughput sequencing platform were effective and trustworthy, identification of bacteria pathogens carried by medical vector with this method is feasible.
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