<i>dndC</i>基因在脉冲场凝胶电泳基因组降解中的作用及降解处理方法

濮吉; 周娟; 赫自强; 熊衍文; 金东

doi:10.3784/jbjc.202110260558

dndC基因在脉冲场凝胶电泳基因组降解中的作用及降解处理方法

doi: 10.3784/jbjc.202110260558

中国疾病预防控制中心传染病预防控制所, 传染病预防控制国家重点实验室, 北京 102206

基金项目: 海关总署项目（No. 2019HK125）

详细信息

作者简介:
濮吉，男，北京市人，助理研究员，主要从事传染病预防控制工作，Email：puji@icdc.cn

通讯作者:
金东，Tel：010–58900749，Email：jindong@icdc.cn

中图分类号: R211; R378
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出版历程
- 收稿日期: 2021-10-26
- 网络出版日期: 2022-03-05
- 刊出日期: 2022-07-14

Role of dndC gene in DNA degeneration in pulsed-field gel electrophoresis and treatment method

State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China

Funds: This study was supported by the Project of General Administration of Customs, P.R. China (No. 2019HK125)

More Information

Corresponding author: Jin Dong, Email: jindong@icdc.cn

摘要

摘要: 目的了解大肠埃希菌和弗格森埃希菌dndC基因的携带情况，以及该基因与脉冲场凝胶电泳（PFGE）基因组降解表型之间的关系，探讨PFGE中菌株基因组降解的原因及处理方法。方法使用PFGE对450株大肠埃希菌和弗格森埃希菌进行分析，设计引物检测dndC基因的携带情况并进行序列测定。通过dndC基因序列比对和进化树的构建，获得dndC基因型别以及其在不同型别大肠埃希菌中的分布。通过添加硫脲和消毒电泳仪研究基因组降解的处理方法。结果 450株大肠埃希菌和弗格森埃希菌中，降解的菌株为40株（8.89%）。 dndC基因阳性菌株33株（7.33%），均出现了降解。大肠埃希菌携带的dndC基因可分为8个群。不同型别大肠埃希菌中均存在dndC基因阳性菌株，且阳性率存在差异。添加硫脲可以有效缓解PFGE分析中dndC基因导致的降解，电泳仪消毒可以缓解全胶降解的情况。结论 dndC基因会导致大肠埃希菌和弗格森埃希菌PFGE分析中基因组降解，添加硫脲可以缓解此降解。
- dndC基因 /
- 大肠埃希菌 /
- 弗格森埃希菌 /
- 脉冲场凝胶电泳 /
- 基因组降解 /
- 硫脲
Abstract: Objective To understand the carriage of dndC gene in Escherichia coli and E. fergusonii strains and the relationship between the dndC gene and the DNA degeneration phenotype, and explore the causes of DNA degeneration in pulsed-field gel electrophoresis (PFGE) and treatment methods. Methods A total of 450 E. coli and E. fergusonii strains were analyzed by PFGE. The primers were designed to detect and sequence the dndC gene. The phylogenetic tree was constructed based on the dndC genes obtained from this study and NCBI database to analyze the genotype of dndC gene and the distribution of dndC gene in different types of E. coli. Addition of thiourea and disinfection of electrophoresis equipment were used to study the treatment methods of DNA degeneration. Results There were 40 strains (8.89%) with the DNA degeneration phenotype by PFGE analysis and 33 strains of them were positive for dndC gene, accounting for 7.33% of 450 E. coli and E. fergusonii strains. The dndC genes of E. coli were divided into eight groups. There were positive strains carrying dndC gene in all eight types of E. coli strains, and the positive rates of dndC gene in eight types of E. coli strains were different. Addition of thiourea and disinfection of electrophoresis equipment could effectively improve the DNA degeneration phenotype. Conclusion The dndC gene can cause DNA degradation of E. coli and E. fergusonii in PFGE analysis. Addition of thiourea was an effective method to ease the DNA degradation caused by dndC.
- dndC gene /
- Escherichia coli /
- Escherichia fergusonii /
- Pulsed-field gel electrophoresis /
- DNA degeneration /
- Thiourea

HTML全文

图 1 基于dndC基因构建的Neighbor-Joining进化树

Figure 1. Neighbor-Joining phylogenetic tree based on dndC genes

下载: 全尺寸图片幻灯片

图 2 电泳液加入硫脲前后（A）及电泳仪消毒前后（B）降解菌株的脉冲场凝胶电泳情况

注：M. 分子量标准；1~11. 具有降解表型的菌株

Figure 2. PFGE for strains with DNA degradation before and after addition of thiourea (A) and before and after disinfection of electrophoresis equipment (B)

下载: 全尺寸图片幻灯片

表 1 450株菌的dndC基因携带情况及降解表型

Table 1. Carriage of dndC gene and DNA degeneration phenotype of 450 E. coli and E. fergusonii strains

菌株来源	菌种类别	菌株数（株）	降解表型		dndC基因阳性
菌株来源	菌种类别	菌株数（株）	菌株数	阳性率（%）	菌株数	阳性率（%）
环境	弗格森埃希菌	110	14	12.73	14	12.73
野生动物	大肠埃希菌	236	22	9.32	15	6.36
患者和食品	大肠埃希菌	104	4	3.85	4	3.85
合计		450	40	8.89	33	7.33

下载: 导出CSV

表 2 不同型别大肠埃希菌中dndC基因的携带情况

Table 2. Carriage of dndC gene in different types of E.coli

型别/亚型	菌株数（株）	dndC基因阳性菌株数（株）	阳性率（%）
A	2 191	96	4.38
B1	2 820	202	7.16
B2	1 907	24	1.26
B2-1	541	3	0.55
B2-2	1 366	21	1.54
C	540	10	1.85
D	616	96	15.58
D1	273	57	20.88
D2	173	31	17.92
D3	170	8	4.71
E	1 020	22	2.15
E1	270	22	8.15
E2	750	10	1.33
F	197	12	6.09
G	96	9	9.38
合计	9 387	481	5.12

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参考文献(16)

[1]	汪皓秋, 郑伟, 俞骅, 等. 2002-2013年杭州地区甲型副伤寒沙门菌分子分型研究[J]. 中华微生物学和免疫学杂志,2017,37(1):57–61. DOI:10.3760/cma.j.issn.0254−5101.2017.01.009. Wang HQ, Zheng W, Yu H, et al. Molecular typing of Salmonella paratyphi A strains isolated in Hangzhou area from 2002 to 2013[J]. Chin J Microbiol Immunol, 2017, 37(1): 57–61. DOI: 10.3760/cma.j.issn.0254−5101.2017.01.009.
[2]	吴伟元, 王辉, 陆坚, 等. 多位点串联重复序列分析和脉冲场凝胶电泳对伤寒沙门菌基因分型[J]. 中华微生物学和免疫学杂志,2014,34(4):264–268. DOI:10.3760/cma.j.issn.0254−5101.2014.04.003. Wu WY, Wang H, Lu J, et al. Comparative evaluation of multilocus variable-number tandem-repeat analysis and pulsed-field gel electrophoresis for genotyping Salmonella enterica serovar Typhi isolates[J]. Chin J Microbiol Immunol, 2014, 34(4): 264–268. DOI: 10.3760/cma.j.issn.0254−5101.2014.04.003.
[3]	Silbert S, Boyken L, Hollis RJ, et al. Improving typeability of multiple bacterial species using pulsed-field gel electrophoresis and thiourea[J]. Diagn Microbiol Infect Dis, 2003, 47(4): 619–621. DOI: 10.1016/S0732−8893(03)00164−0.
[4]	Wang LR, Chen S, Vergin KL, et al. DNA phosphorothioation is widespread and quantized in bacterial genomes[J]. Proc Natl Acad Sci USA, 2011, 108(7): 2963–2968. DOI: 10.1073/pnas.1017261108.
[5]	Wang LR, Chen S, Xu TG, et al. Phosphorothioation of DNA in bacteria by dnd genes[J]. Nat Chem Biol, 2007, 3(11): 709–710. DOI: 10.1038/nchembio.2007.39.
[6]	Liesegang A, Tschäpe H. Short communication-modified pulsed-field gel electrophoresis method for DNA degradation-sensitive Salmonella enterica and Escherichia coli strains[J]. Int J Med Microbiol, 2002, 291(8): 645–648. DOI: 10.1078/1438−4221−00180.
[7]	O'Reilly LC. A method for overcoming DNA degradation during PFGE for Serratia marcescens[J]. J Microbiol Methods, 2011, 85(2): 173–174. DOI: 10.1016/j.mimet.2011.02.014.
[8]	Corkill JE, Graham R, Hart CA, et al. Pulsed-field gel electrophoresis of degradation-sensitive DNAs from Clostridium difficile PCR ribotype 1 strains[J]. J Clin Microbiol, 2000, 38(7): 2791–2792. DOI: 10.1128/JCM.38.7.2791−2792.2000.
[9]	Römling U, Tümmler B. Achieving 100% typeability of Pseudomonas aeruginosa by pulsed-field gel electrophoresis[J]. J Clin Microbiol, 2000, 38(1): 464–465. DOI: 10.1128/JCM.38.1.464−465.2000.
[10]	Ho WS, Ou HY, Yeo CC, et al. The dnd operon for DNA phosphorothioation modification system in Escherichia coli is located in diverse genomic islands[J]. BMC Genomics, 2015, 16: 199. DOI: 10.1186/s12864−015−1421−8.
[11]	Ray T, Mills A, Dyson P. Tris-dependent oxidative DNA strand scission during electrophoresis[J]. Electrophoresis, 1995, 16(1): 888–894. DOI: 10.1002/elps.11501601149.
[12]	Ye J, Coulouris G, Zaretskaya I, et al. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction[J]. BMC Bioinformatics, 2012, 13(1): 134. DOI: 10.1186/1471−2105−13−134.
[13]	Abram K, Udaondo Z, Bleker C, et al. Mash-based analyses of Escherichia coli genomes reveal 14 distinct phylogroups[J]. Commun Biol, 2021, 4: 117. DOI: 10.1038/s42003−020−01626−5.
[14]	Xu TG, Yao F, Zhou XF, et al. A novel host-specific restriction system associated with DNA backbone S-modification in Salmonella[J]. Nucleic Acids Res, 2010, 38(20): 7133–7141. DOI: 10.1093/nar/gkq610.
[15]	Clermont O, Christenson JK, Denamur E, et al. The Clermont Escherichia coli phylo-typing method revisited: improvement of specificity and detection of new phylo-groups[J]. Environ Microbiol Rep, 2013, 5(1): 58–65. DOI: 10.1111/1758−2229.12019.
[16]	Fibke CD, Croxen MA, Geum HM, et al. Genomic epidemiology of major extraintestinal pathogenic Escherichia coli lineages causing urinary tract infections in young women across Canada[J]. Open Forum Infect Dis, 2019, 6(11): ofz431. DOI: 10.1093/ofid/ofz431.