我国斑点热立克次体新种检测及其rOmpA基因序列分析

Detection and Identification of A New Spotted Fever Group Rickettisiae in Fujian Province of China

采用斑点热群立克次体特异引物扩增福建南部来源蜱标本和动物标本中的DNA ,以发现我国可能存在的新种斑点热群立克次体。以立氏立克次体rOmpA基因序列中的相关序列为引物 ,扩增标本中rOmpA基因部分片段 ,用pGEM -T载体及其宿主菌进行阳性克隆筛选 ,阳性克隆经鉴定之后提取质粒DNA ,进行序列测定。测定序列用Blast软件进行序列同源性比较并采集相关序列 ,用Bootstrip方法随机取样 10 0次 ,用PHYLIP软件包进行聚类分析。共检测采自福建宁化地区的越原血蜱 10 0只和野鼠类血标本 38份。其中从越原血蜱(不同来源 )扩增出斑点热立克次体基因片段 ,阳性率为15 0 4% ( 17/ 113) ;并从社鼠、黄胸鼠、褐家鼠扩增出阳性片段 ,以鼠类为单位计算其阳性率为 44 73%。对寄生于野鼠的越原血蜱扩增的阳性片段序列分析表明新测序列与其它斑点热群立克次体的同一基因片段不同 ,其与日本立克次体同源性最高 ,核苷酸序列同源性为 94% ,推定的氨基酸序列同源性为89 %。从病原学角度首次证实我国福建省存在日本立克次体近缘种立克次体 。

Objective\ To investigate spotted fever group rickettsiae in Fujian province,and to identify a potential new species of spotted fever group rickettsiae Fujian strain(FUJ).Methods:Using nucleotide primers derived from the 190kPa surface antigen gene of Rickettsia rickttsii to amplify a rickettssiaspecific 630bp product in different samples,including ticks and amimal blood.The productions were cloned.The plasmid DNA obtained form positive clone was sequenced.The sequence analysis was made by PHYLIP software package with computer.Results:17%(17/113) Haemaphysalis yeni ticks were found infected spotted fever group rickettsiae.44.73%(17/38)mice were positive.One of the sequence analysis showed that the DNA sequene was different from other spotted fever group rickettsiae,and close to R.japonic.PCPamplified fragment of 630bp were obtained from Haemaphysalis yeni ticks and the deletion of codon ACT(nucleotides 477 to 480) was found in the new sequence.Three fragments of 123bp,159bp and 347bp were obtaind for restriction enzyme PstI,and nearly identical to three fragments of 123bp,159bp and 350bp from R.japonica.Conclusion:These finings demonstrate that ticks and animal infect a new spotted fever group rickttsiae species in Fujin province of China.