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一步法快速鉴定皮肤癣菌 被引量:3

Rapid identification of dermatophytes by One-step Method
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摘要 目的探讨一步法快速鉴定皮肤癣菌的临床价值。方法采用快速简便的一步法处理癣菌及非癣菌菌株获得菌株DNA。癣菌通用引物的敏感性测定:将获得的菌株DNA以及人DNA进行PCR扩增后跑电泳,观察是否出现特定大小的阳性条带。PCR反应体系的敏感性测定:用超微量分光光度计测量癣菌菌液的DNA浓度,再将癣菌菌液的DNA进行10倍连续稀释,获得不同浓度的癣菌菌液,随后进行PCR扩增,记录特定大小的阳性条带出现时的最小癣菌DNA浓度值,PCR重复做3次。结果所有癣菌菌株经PCR扩增后均出现366 bp的阳性条带,非癣菌菌株及人DNA经PCR扩增后均未出现任何条带。将浓度为600 ng/μl的癣菌DNA浓度经10倍连续稀释后,得到的7个浓度菌液,其中600 ng^6 pg经PCR扩增后出现366 bp的阳性条带,第7个600 fg浓度经PCR扩增后未出现任何条带。结论皮肤癣菌通用引物CHS1具有较高的特异性,可用作检测癣菌的特异性引物,本实验PCR体系的敏感度较高,为6 pg。 Objective To explore the clinical application of One-step Method to rapid identification of dermatophytes. Methods One-step Method was applied to the treatment of all the strains (including dermatophytes and non-dermatophytes) for DNA extrac- tion. For the specificity of the universal primers, DNA from the dermatophytes and the non-dermatophytes, as well as 2 human NDA, were amplified by the universal primers, and then observed whether there was a specific positive band. For the sensitivity of PCR, genomic DNA from the dermatophytes was serially diluted by 10 times, and the diluted DNA of different concentration was subjected to amplification, and then recorded the minimal concentration of DNA when the specific positive band presented. PCR procedure was repeated three times. Results All the dermatophyte strains yielded the same positive band (366 bp). In contrast, no band was seen in the non-dermatophyte strains and the human ones. The specific 366 bp band was observed in diluted dermato- phyte DNA samples ranging from 600 ng to 6 pg except 600 fg. Conclusion The universal primer CHS1 possesses high specificity, and the sensitivity of our PCR is 6 pg.
出处 《临床军医杂志》 CAS 2014年第8期831-833,839,共4页 Clinical Journal of Medical Officers
基金 军队十二五重点项目(BMS11C032)
关键词 皮肤癣菌 检测 引物CHS1 聚合酶链反应 dermatophyte detection primer CHS1 polymerase chain reaction
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