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研究生:胡可欣
研究生(外文):Ke-Shin Hu
論文名稱:運用多重聚合酶連鎖反應建立5種粉介殼蟲快速鑑定技術
論文名稱(外文):A multiplex PCR assay for the rapid identification of five mealybug pests (Hemiptera: Pseudococcidae)
指導教授:蔡志偉蔡志偉引用關係謝佳宏謝佳宏引用關係
指導教授(外文):Chi-Wei TsaiChia-Hung Hsieh
口試日期:2017-07-25
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:昆蟲學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:43
中文關鍵詞:分子標記檢疫粒線體基因核糖體基因溴化甲烷
外文關鍵詞:molecular markerquarantinemitochondrial generibosomal genemethyl bromide
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粉介殼蟲(半翅目:粉介殼蟲科)會造成嚴重農業危害,為世界重要經濟害蟲之一。進口新鮮水果輸入台灣經檢疫時,常常檢出粉介殼蟲,罹染粉介殼蟲之新鮮水果,必須採取退運、銷燬或是溴化甲烷 (methyl bromide) 燻蒸處理滅除該蟲方能輸入。粉介殼蟲主要依據雌成蟲形態特徵鑑定種類,但粉介殼蟲形態相似且早期發育期缺乏分類特徵,其種類鑑定困難與耗時。水果進口業者亦不願花時間等待粉介殼蟲鑑定結果,越來越多業者傾向不等待種類鑑定,而直接採取溴化甲烷燻蒸處理。本研究目的是建立一簡單、快速、靈敏、準確之分子診斷技術鑑別粉介殼蟲。應用粒線體基因及核糖體基因等分子標記,針對輸入鮮果時常檢出4種非檢疫有害生物康氏粉介殼蟲 (Pseudococcus comstocki (Kuwana))、長尾粉介殼蟲 (Ps. longispinus (Targioni Tozzetti))、桔臀紋粉介殼蟲 (Planococcus citri (Risso))、太平洋臀紋粉介殼蟲 (Pl. minor (Maskell)) 及檢疫有害生物木薯綿粉介殼蟲 (Phenacoccus manihoti Matile-Ferrero),開發多重聚合酶連鎖反應 (multiplex PCR) 快速鑑定技術。透過此技術可短時間同時鑑別5種粉介殼蟲種類。研究成果將有助於防止檢疫有害粉介殼蟲入侵,並減少溴化甲烷的使用量。
Mealybug (Hemiptera: Pseudococcidae), one of economically important insect pests causing serious damages on crops worldwide, is commonly found on the imported fresh fruits during the quarantine inspection in Taiwan. Importation of fresh fruits shall be subjected to re-shipment, destruction, or methyl bromide fumigation for mitigating mealybug pests if exotic species are inspected. Only adult female mealybugs are usually used for species identification by morphological characteristics. However, the taxonomic identification of mealybugs is difficult and time-consuming due to their morphological similarity and the absence of taxonomic morphological characteristics in their early life stages. Recently, more importers prefer methyl bromide fumigation rather than waiting for taxonomic identification when mealybugs are inspected on the imported fresh fruits. The objective of this study was to develop a simple, rapid, sensitive and accurate molecular diagnostic technique for the identification of mealybug pest species. The multiplex polymerase chain reaction (PCR) assay based on the molecular markers of mitochondrial and ribosomal genes was developed for the rapid identification of a quarantine mealybug species (Phenacoccus manihoti Matile-Ferrero) and four common non-quarantine mealybug species (Pseudococcus comstocki (Kuwana), Ps. longispinus (Targioni Tozzetti), Planococcus citri (Risso), and Pl. minor (Maskell)) in Taiwan. Multiplexing technique can accurately identify five species of mealybugs in a short time. The results of this study will be helpful for the ‎border control of exotic mealybug pests and reduction of the use of methyl bromide fumigation.
口試委員會審定書 i
誌謝 ii
中文摘要 iii
ABSTRACT iv
CONTENTS v
LIST OF TABLES vi
LIST OF FIGURES vii
1 Introduction 1
2 Materials and Methods 5
2.1 Sample collections 5
2.2 DNA extraction, amplification and sequencing 5
2.3 Basic Local Alignment Search Tool (BLAST) 6
2.4 Phylogenetic analyses 6
2.5 Design of species-specific primers 6
2.6 Multiplex PCR 7
3 Results 8
3.1 DNA sequence data 8
3.2 Species-specific primers 9
3.3 Multiplex PCR assay 10
4 Discussion 11
REFERENCES 16
APPENDIX 41
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