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CR用于诊断人类埃立克体病方法的建立 |
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陈亮 严延生 谢显能 吴守丽
摘要： ［目的］ 建立一个快速、简便的检测埃立克体感染的实验方法，用于人类埃立克体病的临床诊断及分子流行病学调查。［方法］ 用查菲埃立克体分离株（91HE17）感染犬巨噬细胞(DH82细胞)，感染40天后收集DH82细胞。根据查菲埃立克体16S rRNA基因序列设计引物，特异性扩增查菲埃立克体DNA。运用限制性片段长度多态性分析对PCR扩增产物进行鉴定。［结果］ 扩增产物经过1.5%琼脂糖凝胶电泳和5%聚丙烯酰胺凝胶（PAGE）电泳分析，并用PGEM-3Z(+)/HaeⅢ DNA Marker作为核酸分子量参照物，证实扩增产物是查菲埃立克体16S rRNA基因序列的389 bp核酸片段。将扩增产物用HaeⅢ消化后，进行限制性片段长度多态性（RFLP）分析。产生预期338 bp和51 bp酶切片段。［结论］ 引物HE1和HE3具有高度特异性。PCR反应是具有高度敏感性和特异性，可作为对人类埃立克体病疫源地、传播媒介及储存宿主进行调查的有效方法。
关键词：聚合酶链式反应；查菲埃立克体；人类单核细胞性埃立克体病；人类埃立克体病
中图分类号：R 513 文献标识码：A
文章编号：1007-2705（2000）03-0003-03
Experimental Diagnosis of Human Ehrlichiosis by Polymerase Chain Reaction
CHEN Liang, YAN Yansheng, XIE Xianneng, et al
(Fujian Centers for Disease Control. Fuzhou 350001, China)
Abstract：［Objective］ To develop a quicker and easier method for the diagnosis of Human Ehrlichial (HE) infection and the molecular epidemiological survey. ［Methods］ DH82 cells were inoculated with Ehrlichia chaffeensis (91HE17) and collected 40 days later. Primers on the basis of 16S rRNA gene sequence of E. chaffeensis to specifically amplify E. chaffeensis DNA. PCR product was confirmed by restriction fragment length polymorphism analysis. ［Results］ The product of PCR amplification was analyzed by 1.5% agarose gel electrophoresis and 5% polyacrylamide gel electrophoresis. PGEM-3Z(+)/Hae ⅢDNA was used as nucleotide molecular weight marker. The product is 389 bp DNA fragment of 16S rRNA gene in E. chaffeensis. After PCR product digested withⅢ, the product was analyzed with restriction endonuclease, producing predicted 338 and 51 bp fragments. ［Conclusion］ Primers HE1 and HE3 are highly specific to E. chaffeensis. PCR reaction is of high sensitivity and specificity and can quickly and efficiently detect the etiologic agent of HE. PCR assay may be used to investigate the natural epidemic foci, vector and natural reservoir hosts of HE.
Key words: Polymerase Chain [1] [2] 下一页
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