山东省分离的霍乱弧菌毒力基因的研究

目的：了解山东省不同年代、不同地区收集的霍乱菌株携带的毒力基因情况、分布特点和变迁。方法：应用分子杂交、PCR技术对霍乱弧菌的CTX基因元件中的ctxA、zot基因，VPI毒力岛中的tcpA、tcpH、aldA、toxT、acfB基因，TLC因子中的cri、orf2—3基因，RTX基因簇中的rtxA、rtxC，以及toxR基因进行了检测。结果：用原位杂交检测，180株霍乱弧菌中有168株携带CTX遗传单元中的ctxA、zot、RS1基因，阳性率均为93．33％，表现出高度的一致性。用打点杂交检测不同时期的28株01群霍乱弧菌，所有被检菌都具有ToxR、RTX相关基因，在1980～1987年间分离的O1群霍乱弧菌流行株中，有92．86％携带编码TLC因子的基因，而1994～2000年间分离的流行株菌株中编码TLC因子的基因携带率仅有7．14％。Southern杂交结果显示，48株O1群霍乱弧菌可产生大小为9．4kb和6．5kb左右的两条ctxA杂交带，用zot基因探针进行杂交，11株O1群霍乱弧菌可产生与ctxA杂交带同样大小的条带。1株O139霍乱弧菌具有检测的所有毒力基因，但其ctxA（zot）杂交带与O1群霍乱弧菌有差异。结论：目前已知的霍乱弧菌毒力基因在山东省分离到的菌株中有广泛的分布，并且随时间的推移而发生变迁。

Study on virulence genes of Vibrio cholerae isolated in Shandong Province

Objective:To understand the distribution and characteristics of the virulence genes of V.cholerae strains isolated from different areas of Shandong Province during the past decades,to illustrate the process and mechanism of strain evolution of V.cholerae.Methods:Molecular hybridization and PCR techniques were applied to detect ctxA,zot and RS1 gene of CTX genetic element,tcpA,tcpH,aldA,toxT,acfB gene of VPI pathogenic island,cri and orf2-3 gene of TLC factor,rtxA and rtxC gene of RTX cluster,and the toxR gene.Results:Of the 180 strains of V.cholerae assayed with in situ hybridization,168 were positive for ctxA,zot,and RS1,with a positive rate of 93.33% for CTX genetic element.The three genes were of high consistency,being all positive or all negative in the strains detected.All the strains of V.cholerae O1 examined with dot hybridization were positive for the genes of ToxR and RTX.92.86% of V.cholerae O1 strains isolated from 1980 to 1987 contained the gene of TLC factor,while the strains isolated from 1994 to 2000 did not.48 strains of V.cholerae O1 could produce bands sized about 9.4Kb and 6.5Kb by Sourthern blot hybridization with ctxA gene probe,11 strains could produce similar results by Sourthern blot hybridization with zot gene probe.One strain of V.cholerae O139 harbored all the virulence genes detected,the O139 could produce two different bands sized about 6.5Kb and 4.3Kb by Sourthern blot hybridization with ctxA or zot gene probe.Conclusion:The virulence genes of V.cholerae currently known are widely distributed,and changes and evolutions in the virulence genes occurred with time in Shandong Province.