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首页> 外文期刊>The American Journal of Tropical Medicine and Hygiene >Identification of all members of the anopheles culicifacies complex using allele-specific polymerase chain reaction assays.
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Identification of all members of the anopheles culicifacies complex using allele-specific polymerase chain reaction assays.

机译：使用等位基因特异性聚合酶链反应测定法鉴定按蚊复合体的所有成员。

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摘要

Anopheles culicifacies, a complex of five isomorphic sibling species, is a major vector of malaria in India and neighboring countries. The five species are provisionally designated as species A, B, C, D, and E. Polytene chromosome examination has been the only method available that differentiates four members of this complex in areas where species E is not prevalent. However, this technique requires the mosquitoes to be in the half-gravid stage and thus limits its application to only about one fourth to one third of the total adult collection and excludes immature stages completely. For species E, both polytene chromosome examination and mitotic chromosome examination of F1 males are required. A polymerase chain reaction (PCR) assay based on the D3 domain (D3-PCR) of 28S rDNA and a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay involving ITS2 of rDNA are available for the discrimination of the members of the An. culicifacies complex. However, both these can only differentiate species A and D from species B, C, and E. We report here two allele-specific PCR assays (AD-PCR and BCE-PCR) using sequence differences in the mitochondrial cytochrome oxidase II (CO II) subunit. The AD-PCR assay distinguishes species A and D, whereas the BCE-PCR assay distinguishes species B, C, and E. Thus, with a combination of two PCR assays, namely the D3-PCR/ITS2-RsaI assay, followed by either the AD-PCR or the BCE-PCR assay, it is possible to identify individual specimens of any of the species of this complex. This assay system is the first, and the best available at present to distinguish all sibling species and especially to discriminate non-vector, species B from all the vector species, A, C, D, and E, of the An. culicifacies complex. Until another DNA-based method involving fewer steps is developed, this assay system can be used in all malaria epidemiologic studies where An. culicifacies is prevalent.
机译：库蚊按蚊是五个同形同胞物种的复合体，是印度及邻国疟疾的主要传播媒介。这五个物种被临时指定为物种A，B，C，D和E。多聚体染色体检查是唯一可用于在物种E不普遍的地区区分该复合体的四个成员的方法。但是，该技术要求蚊子处于半稠液阶段，因此将其应用限制在成虫总数的大约四分之一到三分之一之间，并且完全排除了未成熟阶段。对于物种E，需要对F1雄性进行多染色体染色体检查和有丝分裂染色体检查。可以使用基于28S rDNA的D3域（D3-PCR）的聚合酶链反应（PCR）分析和涉及ITS2的rDNA的聚合酶链反应-限制性片段长度多态性（PCR-RFLP）分析来区分安复杂。但是，这两种方法只能将A和D物种与B，C和E物种区分开来。我们在此报告了两种等位基因特异性PCR检测法（AD-PCR和BCE-PCR），利用线粒体细胞色素氧化酶II（CO II）中的序列差异） 亚基。 AD-PCR测定法可区分物种A和D，而BCE-PCR测定法可区分物种B，C和E。因此，结合两种PCR测定法，即D3-PCR / ITS2-RsaI测定法，然后进行任一种通过AD-PCR或BCE-PCR分析，可以鉴定该复合物中任何物种的单个标本。该分析系统是目前第一个也是最好的一种可用于区分所有同胞物种，尤其是将非载体物种B与An的所有载体物种A，C，D和E区别开的系统。复杂。在开发出另一种涉及较少步骤的基于DNA的方法之前，该分析系统可用于所有疟疾流行病学研究。平民化很普遍。

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