摘要
目的应用多重聚合酶链反应(PCR)检测方法和测序研究近5年广州地区分离的霍乱弧菌的4种致病相关基因的携带情况及霍乱肠毒素基因ctxA的变异情况。方法针对霍乱肠毒素A亚单位基因(ctxA)、小带联结毒素基因(zot)、辅助霍乱肠毒素基因(ace)、毒素共调菌毛亚单位A基因(tcpA)设计引物,将多重PCR方法应用于广州地区分离的276株霍乱弧菌的致病相关基因的检测。对ctxA基因的PCR扩增产物进行核苷酸序列测定,探讨ctxA扩增产物的同源性。结果近5年广州地区分离的276株霍乱弧菌中,人源株中93．9％为致病相关基因A型(ctxA+tcpA+ace+zot+型)菌,其余6．1％为致病相关基因C型(ctxA-tcpA-ace-zot-型)菌,临床分离的致病相关基因A型菌分离自轻、中、重型病例和带菌者,其中有68．5％分离自轻型病例,21．9％分离自带菌者,临床分离的致病相关基因C型菌中有63．6％分离自轻型病例,36．4％分离自带菌者,C型菌引起带菌者的比例高于A型菌;78株环境分离株中9．0％为致病相关基因A型菌,35．9％为致病相关基因B型(ctxA-tcpA- ace+zot+型)菌,55．1％为致病相关基因C型菌。霍乱肠毒素基因ctxA变异较小。结论多重PCR方法揭示了广州地区霍乱弧菌致病相关基因模式的多态性,为深入研究人源和环境来源霍乱菌株致病相关基因的演化提供了有效手段,并对本地区霍乱的预防、控制和预警具有重要的指导意义。
Objective To apply multiplex polymerase chain reaction（MPCR） assay and sequencing in study of the carrying status of four pathogenicity-related genes of Vibrio cholerae （ V. cholerae） and the variation of ctxA. Methods Primers targeting cholera toxin sub-unit A gene（ctxA）, toxin-coregnlated pilus gene（ tcpA）, accessory cholera enterotoxin gene（ ace）, zonula occludens toxin gene （zot）were designed and the MPCR method was applied to detect the pathogenicity-related genes of 276 strains of V. cholerae isolates. The amplified fragments of ctxA gene were sequenced and the genetic homology of the amplified fragments of ctxA was analyzed. Results Of the 276 strains of V. cholerae, 93.9% strains from human sources belong to the pathogenicity-related genes type A （ ctxA^＋tcpA^＋ace^＋zot^＋ type） and 6. 1% belong to pathogenicity-related genes type C （ctxA^- tcpA^- ace^- zot^- type）. Type A strains from clinical sources were isolated from patients with mild to severe symptom and carriers, among which 68. 5% were isolated from patients with mild symptom and 21.9% from carriers. All 63.6% of type C strains from clinical sources were isolated from patients with mild symptom and 36.4% from carriers. The proportion of type C strains that caused mild symptom was higher than that of type A strains. Of the 78 strains isolated from the environment, 9. 0% strains belong to pathogenicity-related type A and 35.9% belong to the pathogenicity-related genes type B （ ctxA^ - tcpA ^- ace ^＋ zot^＋ type） , while 55.1% belong to pathogenicity-related genes type C. The sequencing results showed little genetic variation among the amplified fragments for ctxA. Conclusion MPCR disclosed the polymorphic status of pathogenicity-related gene patterns in V. cholerae isolates of Guangzhou, providing effective means for further study on evolution of pathogenicity-related genes among V. cholerae isolates from human and environmental sources. This study also offers significant guidance for effective prevention, control and warning against cholera epidemic in local area.
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2006年第4期257-261,共5页
Chinese Journal of Preventive Medicine
基金
广东省医学科学技术研究基金(A2002059)
广州市科技计划(2005J1-co191)资助项目
关键词
霍乱弧菌
霍乱
聚合酶链反应
序列分析
基因型
霍乱毒素
Vibrio cholerae, cholera
Polymerase chain reaction
Sequence analysis
Genotype
Cholera toxin
