Wilai Parinya, Namgay Rinzin, Made Ali Rusdiyah Sudirman, Saingamsook Jassada, Saeung Atiporn, Junkum Anuluck, Walton Catherine, Harbach Ralph E, Somboon Pradya
Center of Insect Vector Study, Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.
Vector-Borne Disease Control Programme, Ministry of Health, Gelephu 31101, Bhutan.
Insects. 2020 Jul 2;11(7):409. doi: 10.3390/insects11070409.
A multiplex-PCR assay based on mitochondrial cytochrome c oxidase subunit I () sequences was developed for identification of five members of the Barbirostris Complex which occur in Thailand: s.s., , , and species A3. was not included in the assay due to the lack of unequivocal sequences. Allele-specific primers were designed for specific nucleotide segments of 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 s.s., 238 bp for , 611 bp for , 502 bp for and 365 bp for 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 sequencing; however, one of three specimens from Bhutan and all 11 specimens from Indonesia were not amplifiable by the assay due to their distinct 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.
开发了一种基于线粒体细胞色素c氧化酶亚基I（）序列的多重PCR检测方法，用于鉴定在泰国出现的Barbirostris复合体的五个成员：指名亚种、、、和物种A3。由于缺乏明确的序列，未将其纳入该检测方法。针对每个物种的序列的特定核苷酸片段设计了等位基因特异性引物。一些引物采用了错配法和添加长GC尾的方法。该检测方法对指名亚种产生706 bp的产物，对产生238 bp的产物，对产生611 bp的产物，对产生502 bp的产物，对物种A3产生365 bp的产物。使用从不丹、柬埔寨、印度尼西亚（苏拉威西岛）和泰国野生捕获的111只雌性蚊子对该检测方法进行了测试。多重PCR的结果与测序完全一致；然而，由于其独特的序列，来自不丹的三个样本中的一个以及来自印度尼西亚的所有11个样本均无法通过该检测方法扩增。这与这些样本独特的rDNA序列一起，表明Barbirostris复合体中至少存在另外两个新物种。
