CAJ | 학술논문

目的优化布鲁杆菌XbaⅠ酶切脉冲场凝胶电泳基因分型方法.方法通过测试不同厂家生产的Xba I限制性内切酶,优化脉冲场凝胶电泳条件和脉冲时间,所得凝胶电泳图谱用BioNumerics V 4.0软件和UPGMA方法进行聚类分析,获得脉冲场凝胶电泳分型结果.结果优化后的条件为:XbaI限制性内切酶为美国Promega公司产品；脉冲时间为21 h,包括第一脉冲时间0.5～8.0 s,16h,第二脉冲时间10.0～56.0 s,5h;电压为6.0 V/cm;电场夹角120°；电泳温度为14℃.利用优化后的脉冲场凝胶电泳条件对布鲁杆菌6个菌种19个生物型菌株进行试验,经聚类分析能够将牛种、羊种、猪种和绵羊附睾种菌株归为不同的4大群,但是犬种菌株归在猪种群内,沙林鼠种菌株归在牛种群中.聚类分析结果与生物学分型方法具有一致性.结论布鲁杆菌XbaⅠ酶切脉冲场凝胶电泳基因分型方法得到优化,并能够反映布鲁杆菌种群间的亲缘关系,建议应用到监测网络系统.
목적 우화포로간균XbaⅠ매절맥충장응효전영기인분형방법.방법 통과측시불동엄가생산적Xba I한제성내절매,우화맥충장응효전영조건화맥충시간,소득응효전영도보용BioNumerics V 4.0연건화UPGMA방법진행취류분석,획득맥충장응효전영분형결과.결과 우화후적조건위:XbaI한제성내절매위미국Promega공사산품；맥충시간위21 h,포괄제일맥충시간0.5～8.0 s,16h,제이맥충시간10.0～56.0 s,5h;전압위6.0 V/cm;전장협각120°；전영온도위14℃.이용우화후적맥충장응효전영조건대포로간균6개균충19개생물형균주진행시험,경취류분석능구장우충、양충、저충화면양부고충균주귀위불동적4대군,단시견충균주귀재저충군내,사림서충균주귀재우충군중.취류분석결과여생물학분형방법구유일치성.결론 포로간균XbaⅠ매절맥충장응효전영기인분형방법득도우화,병능구반영포로간균충군간적친연관계,건의응용도감측망락계통.
Objective To optimize the protocol of pulsed-field gel electrophoresis(PFGE) to evaluate the applicability for molecular typing of Brucella using Xba].Methods Factors that influence PFGE including selecting restriction endonuclease from different manufacturers and pulse time were optimized.The bands were marked with their molecular weight and then analyzed with BioNumerics (Version 4.0,Applied Maths BVBA,Belgium) software.Results The Xba I from Promega Ltd was selected due to its better digestion effect.The best pulsed-field gel electrophoresis was performed in two phases for 21 h which included 16 h for pulse time of 0.5-8.0 s and 5 h for pulse time of 10.0-56.0 s; the angle of 120°,gradient of 6.0 V/cm,temperature of 14 ℃.The 6 Brucella species and 19 reference biovar strains were tested by optimized conditions of PFGE using Xba Ⅰ.The strains from four Brucella species which included Brucella abortus,Brucella melitensis,Brucella suis and Brucella ovis were clustered into four groups.But Brucella canis was classified in Brucella suis group.The Brucella neotomae was classified in Brucella abortus group.The results of clustering analysis by PFGE were similar to that of biotyping methods.Conclusions PFGE genotyping method using Xba I has good discriminatory power for molecular typing for Brucella.The optimized PFGE protocol is worthy of application.