Abstract：To compare and evaluate the discriminatory ability and potential value of pulsed field gel electrophoresis (PFGE) and multiple locus VNTRs analysis (MLVA) on the genotyping of Brucella, a total of 60 strains of Brucella and three standards (16M, 544A, 1330S) were genotyped simultaneously by PFGE and MLVA. The result indicated that the similarity coefficient among the 63 isolates was from 72.1-100.0% by PFGE, and could distinguish three species of B. melitensis, B. suis and B. abortus at the similarity level of 94.4%. There were 14 clusters and 29 PFGE types identified by PFGE with discriminatory index (DI) of 0.957 5 at the similarity level of 100%; the similarity coefficient among the 63 isolates was from 16.9-100.0% by MLVA, and could distinguish three species of Brucella at the similarity level of 52.3%. There were 8 clusters and 47 MLVA types identified by MLVA with discriminatory index (DI) of 0.985 2 at the similarity level of 100%. It's suggested that PFGE and MLVA could be used to distinguish three species of Brucella in the similarity coefficient of certain, but could not effectively distinguish the type in the same species. Both of these two methods could be used for Brucella molecular typing, but MLVA is better than PFGE for its relatively higher discriminating ability.
陈经雕, 杨杏芬, 柯昌文, 梁文佳, 柯碧霞, 刘美真, 谭海玲, 李柏生, 张万里. 两种技术在广东地区布鲁氏菌菌株分型中的应用与评价[J]. 中国人兽共患病学报, 2014, 30(7): 733-738.
CHEN Jing-diao, YANG Xing-fen, KE Chang-wen, LIANG Wen-jia, KE Bi-xia, LIU Mei-zhen, TAN Hai-ling, LI Bo-sheng, ZHANG Wan-li. Application and evaluation of PFGE and MLVA subtyping methods on Brucella genotype in Guangdong Province, China. Chinese Journal of Zoonoses, 2014, 30(7): 733-738.
[1]Al Dahouk S, Tomaso H, Prenger-Berninghoff E, et al. Identification of Brucella species and biotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)[J]. Crit Rev Microbiol, 2005, 31: 191-196.DOI: 10.1080/10408410500304041 [2]Jiang H, Cui BY, Zhao HY, et al. Use of AMOS-PCR assay for the species identification of Brucella[J].Chin J Zoonoses, 2009, 25(2): 107-109. (in Chinese) 姜海,崔步云,赵鸿雁,等.AMOS-PCR 对布鲁氏菌种型鉴定的应用[J].中国人兽共患病学报,2009,25(2):107-109. [3]Marianelli C, Ciuchini F, Tarantino M, et al. Molecular characterization of the rpoB gene in Brucella species: new potential molecular makers for genotyping[J]. Microbes Infect, 2006, 8(3): 860-865. [4]Luo LM, Cui BY, Zhao HY, et al. Analysis of Brucella reference strain s with pulsed field gel electrophoresis[J]. Lab Med, 2006, 21(5): 509-512. (in Chinese) 骆利敏,崔步云,赵鸿雁,等.脉冲场电泳分析布鲁菌标准菌株[J].检验医学,2006,21(5):509-512. [5]Hanage WP, Feil EJ, Brueggemann AB, et al. Multilocus sequence typing: strain characterization, population biology, and patterns of evolutionary descent[M]. In Persing DH, Tenover FC, Versalovic J, et al (Eds). Molecular Microbiology: Diagnostic Principles and Practice. Washington DC: American Society Press, 2004: 235-243. [6]Jiang H, Fan MG, Chen JD, et al. MLVA genotyping of Chinese human Brucella melitensis biovar 1, 2 and 3 isolates[J]. BMC Microbiol, 2011, 11: 256-262. DOI: 10.1186/1471-2180-11-256 [7]Disease Prevention and Control Bureau, the Ministry of Health of People’s Republic China. Brucellosis prevention manual[M]. Beijing: People's Medical Publishing House, 2008. (in Chinese) 卫生部疾病预防控制局.布鲁氏菌病防治手册[M].人民卫生出版社 ,2008. [8]Yang J, Zhao SL, Cui BY, et al. Establishment of standard operation procedures for multiple locus variable number tandem repeats analysis on typing of Brucella[J]. Dis Surveill, 2012, 27(2): 137-140. (in Chinese) 杨杰,赵素莲,崔步云,等.多位点可变数目串联重复序列分型方法检测布鲁氏菌分型标准化操作方法的建立和应用[J].疾病监测,2012,27(2):137-140. [9]Le Fleche P, Jacques I, Grayon M, et al. Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay[J]. BMC Microbiol, 2006, 6: 9-11. DOI: 10.1186/1471-2180-6-9 [10]Al Dahouk S, Fleche PL, Nockler K, et al. Evaluation of Brucella MLVA typing for human brucellosis[J]. J Microbiol Methods, 2007, 69(1): 137-145. DOI: 10.1016/j.mimet.2006.12.015 [11]Hunter PR, Gaston MA. Numerical index of the discriminatory ability of typing systems: an application of Simpson′s index of diversity[J]. J Clinl Microbiol, 1988, 26(11): 2465-2466. [12]Cui BY, Yin JM, Li LY, et al. Typing of Brucella isolates by repetitive element sequence-based polymerase chain reaction[J]. Dis Surveill, 2005, 20(8): 397-400. (in Chinese) 崔步云,尹继明,李兰玉,等.布鲁氏菌的 Rep-PCR 分型研究[J].疾病监测,2005,20(8):397-400. [13]Shang DQ, Li LY. Study on molecular biology of Brucella bacteria[J]. Chin J Ctrl Endem Dis, 2005, 20(5): 272-276. (in Chinese) 尚德秋,李兰玉.布氏菌分子生物学研究现况[J].中国地方病防治杂志,2005,20(5):272-276. [14]Schwartz DC, Cantor CR. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis[J]. Cell, 1984, 37(1): 67-75. [15]Annick AS, Gisele B, Michel R, et al. DNA polymorphismin strains of the genus Brucella[J].J Bacteriol,1988,10: 4603-4607. [16]Gerner-Smidt P, Hise K, Kincaid J, et al. PulseNet USA: a five-year update[J]. Foodborne Pathog Dis, 2006, 3(1): 9-19. [17]Jensen AE, Cheville NF, Ewalt DR, et al. Application of pulsed-field gel electrophoresis for differentiation of vaccine strain RB51 from field isolates of Brucella abortus from cattle, bison, and elk[J]. American J Vet Res, 1995, 56(3): 308-312. [18]Ridler AL, Leyland MJ, Fenwick SG, et al. Demonstration of polymorphism among Brucella bovis field isolates by pulsed-field gel electrophoresis[J]. Vet Microbiol, 2005, 108(1-2): 69-74. DOI: 10.1016/j.vetmic.2005.02.004 [19]Cui BY. Typing of Brucella pulse field gel electrophoresis[D]. Taiyuan: Shanxi Medical University, 2006. (in Chinese) 崔步云.布鲁菌脉冲场凝胶电泳分型研究[D].太原:山西医科大学, 2006. [20]Her M, Kang SI, Kim JY, et al. A genetic comparison of Brucella abortus isolates from animal sand human by using the MLVA assay[J]. J Microbiol Biotechnol, 2010, 20(12): 1750-1755. DOI: 10.4014/jmb.1005.05039 [21]Lukinmaa S, Takkunen E, Siitonen A. Molecular epidemiology of Clostridium perfringens related to food borne outbreaks of disease in Finland from 1984 to 1999[J]. Appl Environ Microbiol, 2002, 68(8): 3744-3749. [22]Keim P, Price LB, Klevytska AM, et al. Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis[J]. J Bacteriol, 2000, 182(10): 2928-2936. [23]Zuo TT, Duan Q. MLVA and SNP analysis in the application of Bacillus anthracis genotyping[J]. J Mil Med Sci Acad PLA, 2010, 34(3): 290-292. (in Chinese) 左庭婷,端青.MLVA和SNP分析在炭疽芽孢杆菌基因分型中的应用[J].军事医学科学院院刊,2010,34(3):290-292. [24]Klevytska AM, Price LB, Schupp JM, et al. Identification and characterization of variable number tandem repeats in the Yersinia pestis genome[J]. J Clin Microbiol, 2001, 39(9): 3179-3185. [25]Fu XP, Yu DZ, Hai R, et al. Application of tandem repeat sequence and genotyping of Yersinia pestis[J].Chin J Epidemiol, 2004, 25(3): 269-271. (in Chinese) 付秀萍,俞东征,海荣,等.串联重复序列及其在鼠疫菌基因分型中的应用[J].中华流行病学杂志,2004,25(3):269-271. [26]Sabat A, Krzyszton-Russjan J, Strzalka W, et al. New method for typing Staphylococcus aureus strains: multiple locus variable number tandem repeat analysis of polymorphism and genetic relationships of clinical isolates[J]. Clin Microbiol, 2003, 41(4): 1801-1804. [27]Supply P, Mazars E, Lesjean S, et al. Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome[J]. Mol Microbiol, 2000, 36(3): 762-771. [28]Skuce RA, McCorry TP, McCarroll JF, et al. Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets[J]. Microbiology, 2002, 148(Pt2): 519-528. [29]Deng YQ, Wang JX, Lin DH, et al. Molecular identification of the Brucella strains isolated in Fujian province[J]. Chin J Zoonoses, 2009, 25(7): 636-639. (in Chinese) 邓艳琴,王加熊,林代华,等.福建省布鲁氏菌分离株的分子生物学鉴定[J].中国人兽共患病学报,2009,25(7):636-639. [30]Bricker BJ, Ewalt DR. Evaluation of the HOOF-Print assay for typing Brucella abortus strains isolated from cattle in the United States: results with four performance criteria[J]. BMC Microbiol, 2005, 5: 37.
2014, Vol. 30 
