利用PCR-RFLP技術鑑定三種白輪盾介殼蟲（Aulacaspis spp.）（同翅目：盾蚧科）及其在檢疫上之應用

Identification of Three Aulacaspis Species (Homoptera: Diaspididae) by PCR-RFLP Analysis for Quarantine Application

邱一中(Yi- Chung Chiu) ； 吳文哲(Wen- Jer Wu) ； 石正人(Cheng - Jen Shih)

《台灣昆蟲》 21卷4期 (2001/12) Pp. vii+365-375

https://doi.org/10.6661/TESFE.2001030

摘要

介殼蟲蟲體微小且外形相似，幼體時期特徵不明顯，在鑑定上不易確定，造成在檢疫工作上困難重重。本研究主要利用基因組核酸標誌（genomic DNA markers）建立分子診斷技術，以鑑別三種白輪盾蚧屬（Aulacaspis spp.）之介殼蟲，分別為蘇鐵白輪盾蚧（A. yasumatsui Takagi）、月橘白輪盾蚧（A. murrayae Takahashi）和樟白輪盾蚧（A. yabunikkei Kuwana）。利用聚合酶連鎖反應－限制酶片段長度多態型（polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP）技術，探討核糖體形成基因（ribosomal DNA, rDNA），作為鑑別三種介殼蟲的可行性。我們使用單隻標本分別萃取DNA後，利用PCR引子對（Amur18S-3和ITS6）增幅rDNA之部分的18S、ITS1及部分的5.8S區域，蘇鐵白輪盾蚧和月橘白輪盾蚧均產生約500 bp的DNA片段；樟白輪盾蚧則產生約450 bp的DNA片段。利用多種核酸內切酶進行DNA片段切割，結果以TaqI、MspI、CfoI和HaeIII核酸內切酶，所得圖譜的種類特異性較高，可容易地區別三種白輪盾蚧。

關鍵字

白輪盾蚧 ； 快速鑑定 ； 聚合酶連鎖反應（PCR） ； 限制性片段長度多態型（RELP） ； 轉錄區內空白段1（ITS1）

並列摘要

Most scale insects are small and similar in appearance. It is difficult to identify these species especially in their immature stages, and this causes problems for quarantine officers. This study attempted to develop a molecular diagnostic technique using genomic DNA markers for identifying three species of the genus Aulacaspis, A. murrayae, A. yabunikkei, and A. yasumatsui. Amur 18S-3 and ITS6 primer sets were used to amplify ITS1 and its flanking regions of rDNA from genomic DNA as a template extracted from a single specimen. DNA extracted from these three species yielded a single fragment after PCR amplification. The DNA fragment sizes of the PCR products were about 450 bp (for A. yabunikkei) and 500 bp (for A. murrayae and A. yasumatsui). These PCR products were then cut with various restriction endonucleases in order to compare the results of the restriction fragment length polymorphism (RFLP). Our results also reveal that it is possible to discriminate these three Aulacaspis species based on the species-specific patterns acquired from digesting the PCR products with the endonucleases, TaqI, MspI, CfoI, and HaeIII.

並列關鍵字

Aulacaspis ； rapid diagnostic techniques ； polymerase chain reaction (PCR) ； restriction fragment length polymorphism (RFLP) ； internal transcribed spacer I (ITS1)