一种针对缺失基因的利什曼原虫物种鉴别的快速高分辨率熔解方法。
A rapid high-resolution melting method for differentiation of Leishmania species targeting lack gene.
作者信息
Kuang Ziwei, Zhang Chunying, Pang Huasheng, Ma Ying
机构信息
Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, China.
Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, China.
出版信息
Acta Trop. 2018 Feb;178:103-106. doi: 10.1016/j.actatropica.2017.10.016. Epub 2017 Oct 28.
OBJECTIVES
The aim of this research is to verify that if lack gene can be used for differentiation of Leishmania under HRM assay.
METHODS
Two specific primers were designed targeting polymorphic sites on the lack gene sequence. DNA from promastigotes of six species of Leishmania based on reference strains were tested following a HRM protocol. We also tested ten Chinese isolates in blind to validate our method.
RESULTS
Combined with amplicon of the two primers, the six reference strains can be easily discriminated without the effect of initial concentration of DNA templates. Ten Chinese isolates detected by our HRM method resulted in full accord with the standard identification results in previous study.
CONCLUSION
HRM is a rapid and reproducible method to discriminate different Leishmania species and lack gene is a potential novel biological characteristic for easy differentiation of Leishmania isolates in China.
目的
本研究旨在验证在高分辨率熔解曲线分析（HRM 分析）下，缺失基因是否可用于利什曼原虫的鉴别。
方法
针对缺失基因序列上的多态性位点设计了两条特异性引物。按照 HRM 分析方案，对基于参考菌株的六种利什曼原虫前鞭毛体的 DNA 进行了检测。我们还对十个中国分离株进行了盲测以验证我们的方法。
结果
结合两条引物的扩增子，六种参考菌株能够很容易地被区分开，不受 DNA 模板初始浓度的影响。我们的 HRM 方法检测的十个中国分离株的结果与先前研究中的标准鉴定结果完全一致。
结论
HRM 分析是一种快速且可重复的鉴别不同利什曼原虫种类的方法，缺失基因是中国利什曼原虫分离株易于鉴别的潜在新生物学特征。
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