Development of a biochip for rapid discrimination of four tephritid fruit flies (Diptera: Tephritidae)

傳統上，成蟲的外部形態特徵是做為鑑別檢疫害蟲之主要依據；然而，在進出口水果中所常常發現的是果實蠅之幼蟲或卵，單要由這些時期之外部形態做鑑定是較為困難的。為了此問題，本研究選用四種重要果實蠅害蟲，包括東方果實蠅（Bactrocera dorsalis）、地中海果實蠅（Ceratitis capitata）、加勒比海果實蠅（Anastrepha suspensa）和蘋果果實蠅（Rhagoletis pomonella）之粒線體細胞色素氧化Traditionally, morphological characters have been adopted to discrimination insect pests that are intercepted in quarantine inspection. However, it is difficult to identify, based only on their morphology, most tephritid fruit flies which often occur in imported or exported fruits as eggs and/or larvae. Thus, an attempt was made to develop a biochip for efficient species identification. Cytochrome oxidase I (COI) gene of four tephritid fruit flies, i.e. Bactrocera dorsalis, Ceratitis capitata, Anastrepha suspensa and Rhagoletis pomonella, were selected for this study. We first obtained COI fragments from the genomic DNA of each species with PCR amplification using a pair of universal primer. After DNA sequencing and alignment, several potential species-specific oligonucleotide probes, i.e. 20~30 nt from the most variable regions of each species, were synthesized. After a series of screening and tests, a standard operative condition has been set up: hybridization temperature is 60℃, probe concentration is 40 µM/10 µl, and PCR cycle numbers for amplification is 25,with 100~10 ng of template DNA or 20 cycles, with 100 ng of template DNA. This biochip possesses the criteria of high sensitivity and species specificity for rapid discrimination of at least these four tephritid fruit flies.中文摘要----------------------------------------------- 1 英文摘要------------------------------------------------2 前言----------------------------------------------------3 前人研究------------------------------------------------5 材料與方法---------------------------------------------13 結果-------------------------------------------------- 23 討論---------------------------------------------------31 結論---------------------------------------------------37 參考文獻-----------------------------------------------39 圖-----------------------------------------------------49 表-----------------------------------------------------7

OLIGO-NUCLEOTIDE PROBES OF LEPIDOPTERAN PESTS IDENTIFICATION, BIOCHIP, AND IDENTIFYING METHOD THEREOF

鑑定鱗翅目害蟲之蘋果蠹蛾、東方果蛾、桃蛀果蛾、栗小卷蛾及淡棕色蘋果蠹蛾的寡核酸探針、生物晶片及其鑑定套組,包含以針對粒線體COI基因區段序列所設計之具有特異性的寡核酸探針,以及上述具有特異性的寡核酸探針所製成之生物晶片,可用以準確鑑定上述蛀食性害蟲

OLIGO-NUCLEOTIDE PROBES OF TEPHRITID FRUIT FLIES IDENTIFICATION, BIOCHIP, AND IDENTIFYING METHOD THEREOF

一種鑑定果實蠅之寡核酸探針、生物晶片及其鑑定套組,包含具有果實蠅種間特異性的寡核酸探針來鑑定不同種的果實蠅,以及上述具有種間特異性的寡核酸探針所製成之生物晶片

KIT FOR IDENTIFICATION OF CYDIA POMONELLA

一種鑑定蘋果蠹蛾之套組,提供可進行聚合連鎖反應之蘋果蠹蛾專一性引子對,藉增幅出具代表性之核酸片段作為蘋果蠹蛾之分子標記,為具有準確性及快速性之鑑定套組

OLIGO-NUCLEOTIDE PROBES OF LEPIDOPTERAN PESTS IDENTIFICATION, BIOCHIP, AND IDENTIFYING METHOD THEREOF

一種鑑定鱗翅目昆蟲之寡核酸探針、生物晶片及其鑑定方法,包含以鱗翅目昆蟲之細胞色素I(COI)基因序列所設計成之具有種間特異性的寡核酸探針來鑑定不同種的鱗翅目昆蟲,以及上述具有種間特異性的寡核酸探針所製成之生物晶片

OLIGO-NUCLEOTIDE PROBES OF LEPIDOPTERAN PESTS IDENTIFICATION, BIOCHIP, AND IDENTIFYING METHOD THEREOF

一種鑑定鱗翅目昆蟲之寡核苷酸探針、生物晶片及其鑑定方法，包含以鱗翅目昆蟲之細胞色素I(COI)基因序列所設計成之具有種間特異性的寡核苷酸探針來鑑定不同種的鱗翅目昆蟲，以及上述具有種間特異性的寡核苷酸探針所製成之生物晶片

OLIGO-NUCLEOTIDE PROBES OF TEPHRITID FRUIT FLIES IDENTIFICATION, BIOCHIP, AND IDENTIFYING METHOD THEREOF

一種鑑定果實蠅之寡核酸探針、生物晶片及其鑑定方法,包含以果實蠅之細胞色素I(COI)基因序列所設計成之具有屬間特異性的寡核酸探針來鑑定不同屬的果實蠅,以及上述具有屬間特異性的寡核酸探針所製成之生物晶片