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目的 :检测恙螨幼虫体内及鼠类标本中恙虫病东方体携带情况 ,预测流行趋势 ,评价套式PCR反应的敏感性和特异性 ,以进一步开展分子流行病学研究。方法 :参照文献中恙虫病东方体Karp株Sta 58群特异性抗原基因序列 ,合成两对DNA引物 ;应用套式PCR检测现场鼠体表恙螨幼虫体内及鼠类标本的恙虫病东方体DNA ;PCR扩增产物通过 2 %琼脂糖凝胶电泳分析和12 %PAGE电泳分析 ,并用PGEM - 3Zf(+) /HaeⅢDNA作为核酸分子量参照物 ,证实扩增产物是恙虫病东方体Sta 58kDa群特异性抗原基因 88bp片段。结果 :从送检的 2 9份鼠血块和 112份脾组织中均检出阳性 1份 ;从 39份恙螨幼虫体内检出阳性 8份。实验感染小鼠后 3d ,其血块及脾组织均未检出恙虫病东方体 ;而在感染后 6和 9d ,能从血块及脾组织中检出恙虫病东方体。结论 :套式PCR具有很高的敏感性和特异性 ,是疫源地及现场流行病学调查的有效手段
OBJECTIVE: To detect the carrying status of Orientia tsutsugamushi in vivo in chigger mite larvae and in rodents, predict the epidemiological trend, and evaluate the sensitivity and specificity of the nested PCR reaction in order to further carry out molecular epidemiological studies. Methods: Two pairs of DNA primers were synthesized according to the sequence of the Sta 58 gene of Orphanctacterium tsutsugamushi Strain Karp strain in the literature. The DNA of Orientia tsutsugamushi in vivo and in rodents of chigger mites were detected by nested PCR. PCR amplification products were analyzed by 2% agarose gel electrophoresis and 12% PAGE electrophoresis, and PGEM - 3Zf (+) / Hae Ⅲ DNA was used as a nucleic acid molecular weight reference. The amplified product was confirmed to be Sta 58 kDa group specific antigen Gene 88bp fragment. Results: One positive sample was detected in 29 rat blood clots and 112 spleen tissues. Eight positive samples were obtained from 39 chigger mite larvae. On the 3rd day after infection in mice, no occult body of Orientia tsutsugamushi was detected in the blood clots and spleen tissues of mice, while Orientia tsutsugamushi could be detected from blood clots and spleen tissues 6 and 9 days after infection. Conclusion: Nested PCR is highly sensitive and specific and is an effective means of epidemiological investigation in foci and field