Department of Pathogen Biology and Immunology, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an, 710061, Shaanxi, People's Republic of China.
Assisted Reproduction Center, Northwest Women's and Children's Hospital, Xi'an, 710003, Shaanxi, People's Republic of China.
Parasit Vectors. 2020 May 13;13(1):251. doi: 10.1186/s13071-020-04124-z.
The morphological and molecular identification of mites is challenging due to the large number of species, the microscopic size of the organisms, diverse phenotypes of the same species, similar morphology of different species and a shortage of molecular data.
Nine medically important mite species belonging to six families, i.e. Demodex folliculorum, D. brevis, D. canis, D. caprae, Sarcoptes scabiei canis, Psoroptes cuniculi, Dermatophagoides farinae, Cheyletus malaccensis and Ornithonyssus bacoti, were collected and subjected to DNA barcoding. Sequences of cox1, 16S and 12S mtDNA, as well as ITS, 18S and 28S rDNA from mites were retrieved from GenBank and used as candidate genes. Sequence alignment and analysis identified 28S rDNA as the suitable target gene. Subsequently, universal primers of divergent domains were designed for molecular identification of 125 mite samples. Finally, the universality of the divergent domains with high identification efficiency was evaluated in Acari to screen DNA barcodes for mites.
Domains D5 (67.65%), D6 (62.71%) and D8 (77.59%) of the 28S rRNA gene had a significantly higher sequencing success rate, compared to domains D2 (19.20%), D3 (20.00%) and D7 (15.12%). The successful divergent domains all matched the closely-related species in GenBank with an identity of 74-100% and a coverage rate of 92-100%. Phylogenetic analysis also supported this result. Moreover, the three divergent domains had their own advantages. D5 had the lowest intraspecies divergence (0-1.26%), D6 had the maximum barcoding gap (10.54%) and the shortest sequence length (192-241 bp), and D8 had the longest indels (241 bp). Further universality analysis showed that the primers of the three divergent domains were suitable for identification across 225 species of 40 families in Acari.
This study confirmed that domains D5, D6 and D8 of 28S rDNA are universal DNA barcodes for molecular classification and identification of mites. 28S rDNA, as a powerful supplement for cox1 mtDNA 5'-end 648-bp fragment, recommended by the International Barcode of Life (IBOL), will provide great potential in molecular identification of mites in future studies because of its universality.
由于物种数量众多、生物体微观尺寸、同一物种不同表型、不同物种形态相似以及分子数据短缺等原因，螨虫的形态和分子鉴定具有挑战性。
收集并提取属于六个科的九种医学上重要的螨虫物种的 cox1、16S 和 12S mtDNA 以及 ITS、18S 和 28S rDNA 序列，这些序列来自 GenBank 并被用作候选基因。序列比对和分析确定 28S rDNA 是合适的目标基因。随后，针对 125 个螨虫样本设计了用于分子鉴定的通用分歧域引物。最后，在节肢动物中评估了具有高鉴定效率的分歧域的通用性，以筛选螨虫的 DNA 条码。
与 D2（19.20%）、D3（20.00%）和 D7（15.12%）相比，28S rRNA 基因的 D5（67.65%）、D6（62.71%）和 D8（77.59%）区域具有更高的测序成功率。成功的分歧域与 GenBank 中密切相关的物种完全匹配，相似度为 74-100%，覆盖率为 92-100%。系统发育分析也支持了这一结果。此外，这三个分歧域各有优势。D5 具有最低的种内差异（0-1.26%），D6 具有最大的条形码间隙（10.54%）和最短的序列长度（192-241 bp），D8 具有最长的插入缺失（241 bp）。进一步的通用性分析表明，这三个分歧域的引物适用于鉴定节肢动物 40 个科 225 种物种。
本研究证实，28S rDNA 的 D5、D6 和 D8 区域是螨虫分子分类和鉴定的通用 DNA 条码。28S rDNA 作为国际生命条形码倡议（IBOL）推荐的 cox1 mtDNA 5'-端 648-bp 片段的有力补充，由于其通用性，将在未来的螨虫分子鉴定研究中具有巨大潜力。
