One Approach to Insect Identification: Using DNA Characters to Identify Mixed Dipterans Simultaneously

Molecular characters have recently been used to identify insect species. In this paper, I introduce an approach aimed directly at mixed samples, in which each species introduced into the sample can be identified simultaneously. If two species are mixed in a DNA template, it will generate two forms of DNA products of the two species after PCR amplification. The DNA products were cloned into a plasmid after PCR amplification and each DNA copy of the DNA products could be cloned and then analyzed to identify the species' origin. DNA extracted from different species of the dipteran was artificially mixed as a DNA template for PCR amplification. A primer set was designed from the conserved fragments of mitochondrial 16S rDNA for this study. Three different couplings of dipteran DNA mixtures were produced for this analysis. Results confirmed that mixed PCR products from mixed DNA templates can be differentiated by cloning. In addition, PCR-RFLP was employed to distinguish the mixed DNA product. PCR products from a mixture of two species were digested by specific restriction enzymes. Digestion maps of mixed products revealed the specific patterns of the species. Thus, based on an established data bank of nucleic acids, identification of the unknown insects could be made.

關鍵字

molecular identification ； PCR-RFLP ； dipterans ； 16S rDNA

並列摘要

近年來常應用分子特徵於昆蟲的鑑定。本報告引介一種方法，可以將混合在一起的昆蟲物種分別鑑定出來。混有兩個物種的DNA模板，於PCR複製時會同時增殖出該2物種的產物，將產物送入質體後，每個質體僅可攜帶一條產物。不同質體選殖到不同物種的DNA產物。因此，分析這些質體的DNA特徵，即確知那些昆蟲物種存在。將不同蠅類的DNA混在一起，用一對從粒線體16S rDNA保守區域所設計之引子對，複製出16S rDNA片段。從三組混合兩種蠅類DNA的實驗顯示，透過選殖的步驟，的確可以將混合的DNA產物區分。另外，運用限制酵素分解PCR產物，也可以鑑定出混合樣品內的種類，限制圖譜顯示出不同的種類的圖譜。因此透過已建立的核酸資料庫，可以鑑定出混合標本內未知名的物種。

並列關鍵字

分子鑑定； PCR-RFLP ；雙翅目； 16S rDNA

國際替代計量

One Approach to Insect Identification: Using DNA Characters to Identify Mixed Dipterans Simultaneously

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