Zianni Michael R, Nikbakhtzadeh Mahmood R, Jackson Bryan T, Panescu Jenny, Foster Woodbridge A
Plant-Microbe Genomics Facility, The Ohio State University, Columbus, OH 43210, USA.
J Biomol Tech. 2013 Apr;24(1):1-7. doi: 10.7171/jbt.13-2401-001.
There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software.
需要有更具成本效益的方法，以便更准确地区分冈比亚按蚊复合体的成员，特别是冈比亚按蚊和阿拉伯按蚊。这些物种在成虫阶段形态上无法区分，分布重叠，但行为和生态不同，然而它们都是赤道非洲疟疾的有效传播媒介。这里描述的方法，即高分辨率熔解（HRM）分析，利用了DNA熔解特性的微小差异，这取决于基于X染色体的核糖体DNA基因间隔区内不一致单核苷酸多态性的数量。所讨论的这两个物种平均有13个单核苷酸多态性差异，从而产生了差异很大的熔解曲线。使用实时PCR系统Bio-Rad CFX96与dsDNA特异性染料EvaGreen相结合，来检测和测量从选定蚊子腿部提取的DNA产生的扩增子的熔解特性。对来自已知物种纯菌落的7个个体以及通过DNA测序明确鉴定的10个野外捕获个体的检测结果表明，该方法具有很高的准确性。该方法用于通过将86只野外蚊子高度确定地分配到两个常见聚类中来进行鉴定。每个聚类由来自纯菌落的个体定义。HRM分析比大多数其他方法更易于使用，并且在这两个近缘物种之间提供了相当或更准确的区分，但需要专门的熔解分析仪器和软件。
