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]





Development of an efficient ribosomal RNA depletion system for foodborne pathogens
黄佳莹 曹林
南京农业大学食品科学技术学院, 江苏 南京 210095
HUANG Jiaying CAO Lin
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
foodborne pathogensribosomal RNA(rRNA)library constructiontranscriptome
[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。
[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.


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更新日期/Last Update: 1900-01-01