A multiplex-PCR assay based on mitochondrial cytochrome c oxidase subunit I (COI) sequences was developed for identification of five members of the Barbirostris Complex which occur in Thailand: Anopheles barbirostris s.s., An. dissidens, An. saeungae, An. wejchoochotei and An. barbirostris species A3. Anopheles campestris was not included in the assay due to the lack of unequivocal sequences. Allele-specific primers were designed for specific nucleotide segments of COI sequences of each species. Mismatch method and addition of long GC tail were applied for some primers. The assay provided products of 706 bp for An. barbirostris s.s., 238 bp for An. dissidens, 611 bp for An. saeungae, 502 bp for An. wejchoochotei and 365 bp for An. barbirostris A3. The assay was tested using 111 wild-caught female mosquitoes from Bhutan, Cambodia, Indonesia (Sulawesi) and Thailand. The results of the multiplex PCR were in complete agreement with COI sequencing; however, one of three specimens from Bhutan and all 11 specimens from Indonesia were not amplifiable by the assay due to their distinct COI sequences. This, together with the distinct rDNA sequences of these specimens, suggests the presence of at least two additional new species in the Barbirostris Complex.

中文翻译：

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基于线粒体COI序列的多重PCR用于鉴定泰国和该地区其他国家的按蚊复合体成员（双翅目：Cu科）。

开发了一种基于线粒体细胞色素C氧化酶亚基I（COI）序列的多重PCR检测方法，用于鉴定在泰国发生的Barbirostris Complex的五个成员：Anopheles barbirostris ss，An。dissidens，一种。saeungae，一种。wejchoochotei和安。巴比氏菌种A3。由于缺少明确的序列，因此没有将樟脑按蚊（Antroeles campestris）包括在内。针对每种物种的COI序列的特定核苷酸片段设计等位基因特异性引物。某些引物使用错配方法和增加长GC尾巴。该测定提供了706 bp的产物用于一个。barbirostris ss，An的238 bp 弃权，为An 611 bp 。saeungae，An的502 bp 。wejchoochotei和An的365 bp 。Barbirostris A3。使用不丹，柬埔寨，印度尼西亚（苏拉威西）和泰国的111只野外捕获的雌性蚊子测试了该检测方法。多重PCR的结果与COI测序完全一致。然而，由于不丹的三份标本的COI序列不同，因此无法对其进行扩增，这是不丹的三份标本之一和印尼的全部11份标本。这与这些标本的独特rDNA序列一起，表明在Barbirostris Complex中至少存在另外两个新物种。