二代测序技术检测不同环境下美洲大蠊携带细菌的研究

doi:10.11853/j.issn.1003.8280.2017.06.007

中国媒介生物学及控制杂志 ›› 2017, Vol. 28 ›› Issue (6): 543-549.DOI: 10.11853/j.issn.1003.8280.2017.06.007

二代测序技术检测不同环境下美洲大蠊携带细菌的研究

单振菊¹, 邱德义¹, 陈健¹, 岳巧云^1,2

1 中山出入境检验检疫局检验检疫技术中心, 广东中山 528403;
2 中山大学生命科学学院, 广东广州 510275

收稿日期:2017-07-10 出版日期:2017-12-20 发布日期:2017-12-20
通讯作者: 岳巧云,Email:yueqy@zs.gdciq.gov.cn
作者简介:单振菊,女,硕士,农艺师,主要从事媒介生物的鉴定及携带病原体的研究,Email:173465531@qq.com
基金资助:
国家质量基础项目（2016YFF0203205）；广东省国际合作项目（2015A050502009）

The detection of bacteria carried by Periplaneta americana from different environment by next generation sequencing

SHAN Zhen-ju¹, QIU De-yi¹, CHEN Jian¹, YUE Qiao-yun^1,2

1 Zhongshan Entry-Exit Inspection and Quarantine Technology Center, Zhongshan 528403, Guangdong Province, China;
2 College of Life Science Sun Yat-sen University

Received:2017-07-10 Online:2017-12-20 Published:2017-12-20
Supported by:
Supported by the National Quality Infrastructure Program(No. 2016YFF0203205)and Guangdong Province International Cooperation Project(No. 2015A050502009)

摘要/Abstract

摘要： 目的研究二代测序技术（NGS）在美洲大蠊携带细菌检测中的应用，建立一种可检测美洲大蠊体内、体表所携带未知种类细菌的方法，分析不同环境下美洲大蠊携带细菌情况及其与环境的关系。方法 2016年8－9月，以中山地区居民区、港口和医院环境中捕获的美洲大蠊为材料，对其16S rDNA V1～V9可变区分析，选取V3～V4可变区作为目的基因，利用NGS对其携带的细菌种类进行检测和分析。结果港口、居民区和医院采集的美洲大蠊分别携带1 564、1 786和1 779个运算分类单位；利用NGS分别可鉴定出细菌123属36种、131属42种和136属48种，细菌种类数超过传统方法。通过对样品进行物种多样性和聚类分析发现，同一环境下的样本无法完全聚集在一起。结论 16S rDNA V3～V4可变区适合对美洲大蠊体内、体表携带的细菌进行检测，利用NGS可一次性检出多种细菌；其检出的细菌种类远超出传统方法且操作简便；美洲大蠊携带细菌的种类与其生存环境无明显的区域特异性。

关键词: 二代测序技术, 美洲大蠊, 细菌

Abstract: Objective Establish an efficient method for detection of bacteria carried by Periplaneta americana both inside and outside with next generation sequencing (NGS) platform. The P. americana were collected from different environment, and the relationship between the P. americana-carrying bacteria to its living environment was analyzed as well. Methods During August and September in 2016, P. americana were collected from the port, hospital and residential areas of Zhongshan, 16S rDNA V3-V4 was selected as the target gene. Bacteria carried by P. americana both inside and outside was studied by NGS method. Results Periplaneta americana collected at the port area were identified 1 564 OTU, 123 genera, 36 species of bacteria; 1 779 OTU, 136 genus, 48 species of bacteria from the P. americana collected at the hospital area, 1 786 OTU, 131 genus, 42 species of bacteria from the P. americana collected at the residential area. The number of bacteria detected by NGS method was more than that of traditional methods. Through the analysis of species diversity and cluster analysis, the samples in the same environment cannot be aggregated completely. Conclusion 16S rDNA V3-V4 variable region was suitable for detection of bacteria carried by P. americana, NGS technology can be used to detect a variety of pathogenic bacteria of P. americana both inside and outside. Compared with traditional methods, the bacterial species detected by NGS were far beyond the traditional methods and were easy to operate; By cluster analysis of the samples, it has no obvious relationship between the bacteria carried by P. americana to its living environment.

Key words: Next generation sequencing, Periplaneta americana, Bacteria

中图分类号:

R384.9
R37

单振菊, 邱德义, 陈健, 岳巧云. 二代测序技术检测不同环境下美洲大蠊携带细菌的研究[J]. 中国媒介生物学及控制杂志, 2017, 28(6): 543-549.

SHAN Zhen-ju, QIU De-yi, CHEN Jian, YUE Qiao-yun. The detection of bacteria carried by Periplaneta americana from different environment by next generation sequencing[J]. Chines Journal of Vector Biology and Control, 2017, 28(6): 543-549.

参考文献

[1] 徐毅. 蜚蠊病原体携带研究进展[J]. 实用预防医学, 2015, 22(5):636-639.
[2] 邓梅葵,孙迎,韩雯晴. 细菌鉴定方法[J]. 生物医学工程学进展, 2014, 35(2):84-88.
[3] Becker K,Harmsen D,Mellmann A,et al. Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species[J]. J Clin Microbiol, 2004, 42(11):4988-4995.
[4] Bosshard PP,Zbinden R,Abels S,et al. 16S rRNA gene sequencing versus the API 20 NE system and the VITEK 2 ID-GNB card for identification of nonfermenting Gram-negative bacteria in the clinical laboratory[J]. J Clin Microbiol, 2006, 44(4):1359-1366.
[5] Capurro A,Artursson K,Waller KP,et al. Comparison of a commercialized phenotyping system,antimicrobial susceptibility testing,and tuf gene sequence-based genotyping for species-level identification of coagulase-negative Staphylococci isolated from cases of bovine mastitis[J]. Vet Microbiol, 2009, 134(3/4):327-333.
[6] Quail MA,Kozarewa I,Smith F,et al. A large genome center's improvements to the Illumina sequencing system[J]. Nat Med, 2008, 5(12):1005-1010.
[7] Meyer M,Stenzel U,Hofreiter M. Parallel tagged sequencing on the 454 platform[J]. Nat Protoc, 2008, 3(2):267-278.
[8] Mardis ER. The impact of next-generation sequencing technology on genetics[J]. Trends Genet, 2008, 24(3):133-141.
[9] Degnan PH,Ochman H. Illumina-based analysis of microbial community diversity[J]. ISME J, 2012, 6(1):183-194.
[10] Gilbert JA,Meyer F,Antonopoulos D,et al. Meeting report:the terabase metagenomics workshop and the vision of an earth microbiome project[J]. Stand Genomic Sci,2010,3(3):243-248.
[11] Bartram AK,Lynch MDJ,Stearns JC,et al. Generation of multimillion-sequence 16S rRNA gene libraries from complex microbial communities by assembling paired-end Illumina reads[J]. Appl Environ Microbiol, 2011, 77(11):3846-3852.
[12] Xiao M, Zhang ZZ, Wang JX, et al. Bacterial community diversity in a low-permeability oil reservoir and its potential for enhancing oil recovery[J]. Bioresour Technol, 2013, 147:110-116.
[13] Chakravorty S,Helb D,Burday M,et al. A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria[J]. J Microbiol Methods,2007,69(2):330-339.
[14] Moreau MM, Eades SC, Reinemeyer CR, et al. Illumina sequencing of the V4 hypervariable region 16S rRNA gene reveals extensive changes in bacterial communities in the cecum following carbohydrate oral infusion and development of early-stage acute laminitis in the horse[J]. Vet Microbiol, 2014, 168(2/4):436-441.
[15] Claesson MJ,Wang Q,O' Sullivan O,et al. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions[J]. Nucleic Acids Res, 2010, 38(22):e200.
[16] Jin CY,Zeng ZY,Fu ZW,et al. Oral imazalil exposure induces gut microbiota dysbiosis and colonic inflammation in mice[J]. Chemosphere, 2016, 160:349-358.
[17] Bennett DC,Tun HM,Ji EK,et al. Characterization of cecal microbiota of the emu (Dromaius novaehollandiae)[J]. Vet Microbiol, 2013, 166(1/2):304-310.
[18] 杨正时,房海. 人及动物病原细菌学[M]. 石家庄:河北科学技术出版社, 2003:2-180.
[19] WHO. 世卫组织发布迫切需要新型抗生素的细菌清单[EB/OL]. (2017-02-27)[2017-04-05]. http://www.who.int/mediacentre/news/releases/2017/bacteria-antibiotics-needed/zh/.

二代测序技术检测不同环境下美洲大蠊携带细菌的研究

The detection of bacteria carried by Periplaneta americana from different environment by next generation sequencing

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	向昱龙, 周敬祝, 刘英, 刘平涛, 胡勇, 梁文琴. 贵州省部分地区蜱及其携带细菌调查[J]. 中国媒介生物学及控制杂志, 2022, 33(1): 148-152.
[2]	董昊炜, 南春燕, 周秋明, 袁浩, 李翔宇, 彭恒, 马雅军. 基于16S rRNA高通量测序的现场中华按蚊及其孳生地水体菌群结构研究[J]. 中国媒介生物学及控制杂志, 2021, 32(5): 533-540.
[3]	何志伟, 董起钰, 刘锐, 李欣, 刘欣, 徐从武, 赵鑫祺, 万晴, 张忠. 白斑蛾蚋幼虫肠道可培养细菌的分离鉴定与产消化酶活性分析[J]. 中国媒介生物学及控制杂志, 2021, 32(2): 224-229.
[4]	常锐锐, 陈海敏, 陈宏敏, 万晴, 乐倩倩, 张瑞玲, 张忠. 家蝇幼虫肠道可培养非厌氧细菌对家蝇成虫取食及产卵的影响[J]. 中国媒介生物学及控制杂志, 2020, 31(3): 294-299.
[5]	李思思, 张晓雨, 张艳凯, 刘敬泽. 内蒙古地区全沟硬蜱细菌群落结构及多样性分析[J]. 中国媒介生物学及控制杂志, 2019, 30(6): 607-612.
[6]	黄振东, 万晴, 薛志静, 张瑞玲, 张忠. 德国小蠊肠道可培养非厌氧细菌的分离、鉴定与产消化酶活性分析[J]. 中国媒介生物学及控制杂志, 2019, 30(4): 409-413.
[7]	宋暖, 黄振东, 薛志静, 万晴, 庄桂芬, 张瑞玲, 许永玉, 张忠. 喂食盐酸环丙沙星对家蝇生长发育和种群繁殖的影响[J]. 中国媒介生物学及控制杂志, 2019, 30(3): 300-305.
[8]	冯娟, 达拉, 董周立, 常春, 曹晓梅, 杨朝春. 西藏口岸2017年蝇类携带病原体的检测分析[J]. 中国媒介生物学及控制杂志, 2019, 30(2): 194-196.
[9]	吕洁毅, 何振毅, 丁国允, 刘谢, 伍华驹, 张才志, 王忱. 硫酰氟熏蒸杀灭蜚蠊卵荚剂量与时间的量效学研究[J]. 中国媒介生物学及控制杂志, 2018, 29(3): 279-282.
[10]	单振菊, 邱德义, 陈佩, 岳巧云. 克隆法检测不同环境下美洲大蠊携带细菌的比较研究[J]. 中国媒介生物学及控制杂志, 2017, 28(5): 428-432.
[11]	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.
[12]	张晓, 王永明, 王东, 辛正. 德国小蠊对美洲大蠊取食过的饵料摄食行为研究[J]. 中国媒介生物学及控制杂志, 2016, 27(6): 570-572.
[13]	韩娜, 张琳, 张雯, 强裕俊, 侯学霞, 陈晨, 郝琴, 张媛媛. 长角血蜱细菌群落结构及多样性研究[J]. 中国媒介生物学及控制杂志, 2016, 27(5): 426-431.
[14]	罗成旺, 逄波, 阳波. 高通量半自动化细菌传代系统的初步构建[J]. 中国媒介生物学及控制杂志, 2016, 27(3): 302-304.
[15]	王影姣, 沈娟, 方霞, 陈至里, 金小宝. 美洲大蠊肠道内生戈登放线菌的分离与鉴定[J]. 中国媒介生物学及控制杂志, 2016, 27(2): 172-175.