实时荧光定量PCR检测斑点热立克次体的方法建立  

Rapid detection of spotted fever group rickettsiae with real-time quantitative PCR

作　　者：牛东升[1] 杨晓[1] 陈梅玲[1] 王锡乐[1] 温博海[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《解放军医学杂志》2008年第11期1297-1299,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家科技攻关项目(2003BA712A04-07)

摘　　要：目的建立检测斑点热立克次体的群特异性实时荧光定量PCR方法。方法根据斑点热立克次体外膜蛋白B(ompB)基因序列设计群特异性引物和探针,以克隆的斑点热立克次体ompB基因片段作为模板,在荧光定量PCR检测仪上建立实时荧光定量检测法。结果该方法定量检测标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系,最小检出量小于10个拷贝。用该方法检测斑点热立克次体(立氏立克次体、西伯利亚立克次体、西伯利亚立克次体精河株、康氏立克次体、小蛛立克次体、澳大利亚立克次体、派氏立克次体、扇头蜱立克次体、非洲立克次体、阿斯特罕立克次体、斯洛伐克立克次体、日本立克次体、马赛立克次体、猫立克次体和黑龙江立克次体)DNA样本的结果均为阳性,但检测普氏立克次体、莫氏立克次体、恙虫病东方体、贝氏柯克斯体、查菲埃立克体、汉赛巴通体以及其他9种病原菌DNA样本的结果均为阴性。用荧光定量PCR检测立氏立克次体、黑龙江立克次体、西伯利亚立克次体精河株感染BALB/C小鼠脾脏组织DNA,均检出不同水平的阳性结果,结果与感染进程相关。结论本研究建立的检测斑点热立克次体的实时荧光定量PCR具有较高的敏感性和群特异性,适合样本中各种斑点热立克次体的快速检测,可用于斑点热的实验室快速诊断和自然疫源地的流行病学研究。Objective To develop a quantitative method to quickly detect spotted fever group rickettsiae. Methods According to the DNA sequence of gene ompB encoding outer membrane protein B of spotted fever group rickettsiae, a pair of primers and one TaqMan-MGB probe were designed and a target gene fragment of ompB was cloned. A real-time quantitative polymerase chain reaction （PCR） was developed with the primers and probe as well as the ompB gene fragment. Results The relationship between the values of threshold cycle （Ct） and the DNA copy number was linear, and less than 10 copies were detected in this quantitative PCR assay. The DNA samples of spotted fever group rickettsiae, including R rickettsii, R sibrica, R sibrica Jinghe, R conorii, R akari, R australis, R parkeri, R rhipicephali, R africa, R astrakhan, R slovaca, R japonica, R massiliae, R felis, and R heilongjiangensis, were positively detected by the said quantitative PCR assay. However, The DNA samples of other rickettsial agents, R proztazekii, R mooseri, Orientia tsutsugamushi, Coxiella burnetii, Ehrichia chaffeensis and Bartonella henselae, as well as those of 9 species of bacterial pathogens were negatively detected by the said quantitative PCR assay. Using the quantitative PCR assay, the splenic DNA samples from mice experi-mentally infected with R. rickettsii, R heilongjiangensis, or R. sibrica Jinghe were positively detected in various levels that were correlative to their infectious course. Conclusions It is suggested that the quantitative PCR devised is group-specific and sensitive for specific detection of spotted fever group rickettsiae, and is useful for laboratory diagnosis and epidemiological study of spotted fever.

关 键 词：立克次体属 斑点热 聚合酶链反应 细菌外膜蛋白质类B 

分 类 号：R376.3[医药卫生—病原生物学]