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对广州一例未知病原体感染引起的皮肤病患者血样进行免疫学和分子生物学鉴定,以确定其病原体。取静脉血,用Leish r K39 dipstick金标试纸检测利什曼原虫抗体情况。取皮损处组织,75%乙醇固定后,提取基因组DNA。用两对利什曼原虫种特异性引物LITSR-L5.8S和NAGTL1-NAGTL4分别PCR扩增利什曼原虫核糖体DNA内转录间隔区1和N-乙酰氨基葡萄糖苷转移酶的基因片段,扩增产物进行测序和BLAST序列分析。Leish r K39 dipstick金标试纸检测结果呈弱阳性。PCR结果显示,引物LITSR-L5.8S和NAGTL1-NAGTL4分别扩增出约404 bp和1 405 bp的片段,两片段序列与硕大利什曼原虫(Leishmania major)相应序列的相似性均为99.7%。其序列Gen Bank登录号分别为KU975160和KX150476。确诊该皮肤病患者为输入性皮肤利什曼病病例,病原体为硕大利什曼原虫。
A case of skin disease caused by an unknown pathogen infection in Guangzhou was immunologically and molecularly identified to determine its pathogen. Take venous blood, Leish r K39 dipstick gold strip test Leishmania antibody situation. Take the lesion tissue, fixed with 75% ethanol, extracted genomic DNA. Two pairs of Leishmania species-specific primers LITSR-L5.8S and NAGTL1-NAGTL4 were used to amplify the gene fragment of transcribed spacer 1 and N-acetylglucosaminyltransferase in Leishmania ribosomal DNA, respectively The amplified product was sequenced and BLAST sequence analysis. Leish r K39 dipstick gold standard test strip test results were weakly positive. PCR results showed that the amplified fragments of 404 bp and 1 405 bp were amplified by LITSR-L5.8S and NAGTL1-NAGTL4 respectively. The similarity between the two fragments and the corresponding sequences of Leishmania major were 99.7% . The Gen Bank accession numbers are KU975160 and KX150476, respectively. Patients diagnosed with this skin disease are cases of imported cutaneous leishmaniasis and the pathogen is Leishmania major.