[1]黄佳莹,曹林.食源性致病菌核糖体RNA高效去除体系的建立[J].南京农业大学学报,2019,42(2):358-364.[doi:10.7685/jnau.201804049]
 HUANG Jiaying,CAO Lin.Development of an efficient ribosomal RNA depletion system for foodborne pathogens[J].Journal of Nanjing Agricultural University,2019,42(2):358-364.[doi:10.7685/jnau.201804049]
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食源性致病菌核糖体RNA高效去除体系的建立()
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
42卷
期数:
2019年2期
页码:
358-364
栏目:
食品与工程
出版日期:
2019-03-19

文章信息/Info

Title:
Development of an efficient ribosomal RNA depletion system for foodborne pathogens
作者:
黄佳莹 曹林
南京农业大学食品科学技术学院, 江苏 南京 210095
Author(s):
HUANG Jiaying CAO Lin
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
关键词:
食源性致病菌核糖体RNA(rRNA)文库构建转录组
Keywords:
foodborne pathogensribosomal RNA(rRNA)library constructiontranscriptome
分类号:
TS207.4
DOI:
10.7685/jnau.201804049
摘要:
[目的] 本文旨在解决转录组技术研究食源性致病菌致病机制过程中核糖体RNA(rRNA)无法高效去除的问题。[方法] 以9种常见食源性致病菌的201条rRNA序列为参考对象,设计每条rRNA编码基因的反向互补配对DNA探针,随后将探针与细菌总RNA进行杂交,在RNase H和DNaseⅠ的作用下去除rRNA。以大肠杆菌O157:H7 Sakai、单增李斯特菌EGD-e、肠道沙门氏菌CT18以及铜绿假单胞菌PAO1这4株菌的总RNA为模板,分别使用本研究设计的rRNA去除探针以及建立的rRNA去除体系与Ribo-Zero rRNA Removal Kit(Bacteria)进行rRNA去除,随后用相同方法进行转录组文库构建,并使用Illumina HiSeq X测序平台进行相同数据量的测序,最后通过生物信息分析比较rRNA去除效果。[结果] 共设计rRNA去除探针541条。当总RNA使用量范围为1~5 μg,探针使用量为100 pmol,RNase H使用量为2 U,DNaseⅠ保持过量为10 U时,rRNA去除体系的性能达到最优,去除效率达95%以上。使用本研究构建的rRNA去除体系时,4株食源性致病菌残留rRNA平均占总数据量的1.67%,而相同条件下试剂盒残留的rRNA平均占7.61%。此外,本体系对食源性致病菌低表达丰度基因的检出数目是试剂盒的1.90倍。[结论] 该体系可以有效去除9种常见食源性致病菌的核糖体RNA,同时也大大降低了试验成本。
Abstract:
[Objectives] An efficient ribosomal RNA(rRNA) depletion system was developed to solve incomplete rRNA removal problems that exist in researching pathogenesis for foodborne pathogens by transcriptome technology.[Methods] 201 ribosomal RNA sequences from 9 common food-borne pathogens were used as references,and their coding genes reverse complementary sequences were used to design DNA probes that hybridized with bacterial total RNA,and ribosomal RNA was removed under the reaction of RNase H and DNaseⅠ. To check the working efficiency of our rRNA removal system,total RNA from Escherichia coli O157:H7 Sakai,Listeria monocytogenes EGD-e,Salmonella enterica CT18 and Paeruginosa PAO1 were used as templates,and the rRNA removal probes designed in this work and Ribo-Zero rRNA Removal Kit(Bacteria) were used to remove rRNAs from 4 different strains respectively. Then transcriptome libraries were constructed using the same method,and Illumina HiSeq X sequencing platform was used to sequence the same size of data. Finally,rRNA removal efficiencies between different rRNA removal systems were compared through bioinformatics tools.[Results] A total of 541 rRNA removal probes were designed. RT-qPCR method was used to measure the rRNA removal efficiency,and by using this method,total RNA input range was confirmed to be 1-5 μg,the using amount of rRNA removal probes was confirmed to be 100 pmol,the enzyme concentration of RNase H and DNaseⅠused in this system was confirmed to be 2 U and 10 U,respectively. At this condition,rRNA removal efficiency reached as high as 95% above. Followed by same RNA library construction pipeline and same data-output sequencing strategy,the mean rRNA ratio remaining in 4 RNA libraries prepared by the system developed in this work was 1.67%,and the mean rRNA ratio remaining in 4 RNA libraries prepared by kit was 7.61%. Besides,the number of low expression abundance genes detected in the RNA library pretreated with the system we designed was 1.90 times more than that of the library pretreated with kit.[Conclusions] Through the rRNA removal system developed in this work,we can remove rRNAs from 9 common foodborne pathogenic bacteria efficiently,and effectively reduce experiment expensive.

参考文献/References:

[1] Henao O L,Jones T F,Vugia D J,et al. Foodborne diseases active surveillance network:2 decades of achievements,1996-2015[J]. Emerging Infectious Diseases,2015,21(9):1529-1536.
[2] Martinovi? T,Andjelkovi? U,Gajdo?ik M ?,et al. Foodborne pathogens and their toxins[J]. Journal of Proteomics,2016,147:226-235.
[3] Hurd S,Patrick M,Hatch J,et al. Clinical laboratory practices for the isolation and identification of Campylobacter in Foodborne Diseases Active Surveillance Network(FoodNet) sites:baseline information for understanding changes in surveillance data[J]. Clinical Infectious Diseases,2012,54(Suppl 5):5440-5445.
[4] Neil K P,Biggerstaff G,Macdonald J K,et al. A novel vehicle for transmission of Escherichia coli O157:H7 to humans:multistate outbreak of E.coli O157:H7 infections associated with consumption of ready-to-bake commercial prepackaged cookie dough:United States,2009[J]. Clinical Infectious Diseases,2012,54(4):511-518.
[5] Swaminathan B,Gernersmidt P. The epidemiology of human listeriosis[J]. Microbes & Infection,2007,9(10):1236-1243.
[6] Klevens R M,Morrison M A,Nadle J,et al. Invasive methicillin-resistant Staphylococcus aureus infections in the United States[J]. Journal of the American Medical Association,2007,298(15):1763-1771.
[7] 宦海霞,周琼,赵李祥,等. 应用DNA芯片技术研究体外表达禽致病性大肠杆菌可能致病基因[J]. 微生物学报,2008,48(1):103-111. Huan H X,Zhou Q,Zhao L X,et al. Differential expression of virulence and potential virulence genes of avian pathogenic Escherichia coli in vitro with DNA microarray analysis[J]. Acta Microbiologica Sinica,2008,48(1):103-111(in Chinese with English abstract).
[8] Dong T,Schellhorn H E. Global effect of RpoS on gene expression in pathogenic Escherichia coli O157:H7 strain EDL933[J]. BMC Genomics,2009,10(1):349.
[9] Casey A,Fox E M,Schmitzesser S,et al. Transcriptome analysis of Listeria monocytogenes exposed to biocide stress reveals a multi-system response involving cell wall synthesis,sugar uptake,and motility[J]. Front Microbiol,2014,5(1):68.
[10] Karpinets T V. RNA:protein ratio of the unicellular organism as a characteristic of phosphorous and nitrogen stoichiometry and of the cellular requirement of ribosomes for protein synthesis[J]. BMC Biology,2006,4(1):1-10.
[11] Urich T,Anders Lanzén,Qi J,et al. Simultaneous assessment of soil microbial community structure and function through analysis of the meta-transcriptome[J]. PLoS One,2008,3(6):e2527.
[12] Vliet A H M V. Next generation sequencing of microbial transcriptomes:challenges and opportunities[J]. Fems Microbiology Letters,2010,302(1):1-7.
[13] Petrova O E,Garciaalcalde F,Zampaloni C,et al. Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes[J]. Sci Rep,2017,7:41114.
[14] 朱丛睿,周明旭,朱国强. 大肠埃希菌内参基因gapA克隆表达及抗体的制备与应用[J]. 扬州大学学报(农业与生命科学版),2015,36(2):14-18. Zhu C R,Zhou M X,Zhu G Q. Prokaryotic expression of Escherichia coli reference gene gapA,and antiserum preparation and application[J]. Journal of Yangzhou University(Agricultural and Life Science Edition),2015,36(2):14-18(in Chinese with English abstract).
[15] Amara R R,Vijaya S. Specific polyadenylation and purification of total messenger RNA from Escherichia coli[J]. Nucleic Acids Research,1997,25(17):3465-3470.
[16] Mcgrath K C,Thomas-Hall S R,Cheng C T,et al. Isolation and analysis of mRNA from environmental microbial communities[J]. J Microbiol Methods,2008,75(2):172-176.
[17] Su C,Sordillo L M. A simple method to enrich mRNA from total prokaryotic RNA[J]. Molecular Biotechnology,1998,10(1):83-85.
[18] Pang X,Zhou D,Song Y,et al. Bacterial mRNA purification by magnetic capture-hybridization method[J]. Microbiology and Immunology,2004,48(2):91-96.
[19] He S,Wurtzel O,Singh K,et al. Validation of two ribosomal RNA removal methods for microbial meta transcriptomics[J]. Nature Methods,2011,7(10):807-812.
[20] Sorek R,Cossart P. Prokaryotic transcriptomics:a new view on regulation,physiology and pathogenicity[J]. Nature Reviews Genetics,2010,11(11):9-16.
[21] Giannoukos G,Ciulla D M,Huang K,et al. Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes[J]. Genome Biology,2012,13(3):r23.

备注/Memo

备注/Memo:
收稿日期:2018-4-27。
基金项目:国家自然科学基金项目(31101223)
作者简介:黄佳莹,硕士研究生。
通信作者:曹林,副教授,研究方向为分子和高通量测序的研究,E-mail:caolin@njau.edu.cn。
更新日期/Last Update: 1900-01-01