200923365 九、發明說明： 【發明所屬之技術領域】 本發明是有關於一種鑑定蚜蟲類昆蟲的方法，且特別 是有關於一種鑑定蚜蟲類昆蟲的募核酸探針、生物晶片及 其鑑定方法。 【先前技術】BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for identifying aphid-like insects, and more particularly to a nucleic acid-promoting probe, a biochip, and an identification method thereof for identifying aphid-like insects. [Prior Art]
蚜蟲為常見的昆蟲’通常成群棲息在植物的莖、葉、 莢部上，以刺吸式Π器吸食植物體的汁液，被視為農業害 蟲0 財蟲身又群集於菜心部、花及種笑或葉背吸取營養 t並分泌蜜露誘發煤煙病，造成植株外觀不良，影響花 卉品質’危害嚴重的葉片常捲縮或萎〉周。此外，除直接危 害外，蚜蟲更可傳佈植物性病毒。 目刖蚜蟲類昆蟲的的鑑定—般以形態特徵鑑定為 主。然而，呀蟲體長約（U〜〇·5公分，由於蟲體微小再加 上蚜蟲具有多態型，個體間外部形態變異程度大，因此造 ^騎蟲種類較時的困難，需要由有經驗的鑑識人員依賴 =驗及專門能力加以判^ ’且傳統形態鑑^常因鑑定者主 觀的認知造成鑑定結果的不同，而難以客觀地判定。 【發明内容】 苷酸 因此本發明就是在提供一 探針（probe)及其使用方法 種鑑定蚜蟲類昆蟲之寡核 解決傳統型蚜蟲態鑑定正 200923365 確性受人為判斷影響的問題。 本發明的另一目的是在提供一種鑑定蚜蟲類昆蟲之 生物晶片及其使用方法，用以快速鑑定不同屬之蚜蟲類昆 蟲。 根據本發明，提出一種鑑定蚜蟲類昆蟲之寡核苷酸探 針及其使用方法，鑑定蚜蟲類昆蟲之寡核苷酸探針包含 SEQ ID NO ·· 1、SEQ ID NO : 2、SEQ ID NO : 3、SEQ ID NO : 4、SEQ ID NO ·· 5、SEQ ID NO : 6、SEQ ID NO ·· 7、 SEQ ID NO : 8、SEQ ID NO : 9、SEQ ID NO : 10、SEQ ID NO: 11、SEQ ID NO: 12所示之核苷酸序列、其互補股之 核苷酸序列、簡併序列及其衍生序列。 應用上述寡核苷酸探針鑑定蚜蟲類昆蟲之方法包 含：以待測蟲體之粒線體細胞色素氧化酶I基因 (cytochrome I，COI)與上述寡核苷酸探針進行雜合反應，並 由雜合反應之結果鑑定财蟲類昆蟲之屬（genus)。 根據本發明，提出一種快速鑑定蚜蟲類昆蟲之生物晶 片及其使用方法，鑑定蚜蟲類昆蟲之生物晶片包含一基 材，基材上可固著選自於SEQ ID NO : 1、SEQ ID NO : 2、 SEQ ID NO : 3、SEQ ID NO : 4、SEQ ID NO : 5、SEQ ID NO : 6、SEQ ID NO : 7、SEQ ID NO : 8、SEQ ID NO : 9、 SEQ ID NO : 10、SEQ ID NO : 11、SEQ ID NO : 12 所示 之核苷酸序列、其互補股之核苷酸序列、簡併序列、其衍 生序列及上述序列之任意組合所組成之族群。 應用上述生物晶片鑑定蚜蟲類昆蟲之方法包含：以待 200923365 測蟲體之粒線體細胞色素氧化酶I基因（COI)與上述生物 晶片上的寡核苷酸探針進行雜合反應（hybridization)，並由 雜合反應之結果鑑定財蟲類昆蟲之屬（genus)。 【實施方式】 本發明係利用棉財gossypii)、馬鈴薯财 (Aulacorthum ίο/β/·»·)、偽菜財、小桔財 (Toxoptera aurantii) ' 窥 1 场（Acythosiphon /7/5·ww)及才兆財 (Μ少ZM·? perhcae)等不同屬的坊蟲類昆蟲之細胞色素I基 因（COI)序列設計寡核苷酸探針，並進行探針之專一性測 試，除豌豆蚜及桃蚜外，均得到具有屬間特異性之寡核苷 酸探針，其序列分別編號為SEQ ID NO : 1、SEQ ID NO : 2、SEQ ID NO : 3、SEQ ID NO : 4、SEQ ID NO : 5、SEQ ID NO : 6、SEQ ID NO : 7、SEQ ID NO : 8、SEQ ID NO : 9、SEQ ID NO : 10、SEQ ID NO : 11、SEQ ID NO : 12。 所有使用之蟲體樣本均事先經鑑定確認種類。 其中編號 SEQ ID NO:卜 SEQ ID NO: 2、SEQ ID NO:3 及SEQ ID NO:4之寡核苷酸探針係利用棉蚜之細胞色素I 基因（COI)序列設計，可用以鑑定棉蚜。棉蚜之細胞色素I 基因序列參見序列識別號碼SEQ ID NO: 13。 SEQ ID NO: 5、SEQ ID NO: 6之募核苷酸探針係利用 馬鈴薯蚜之細胞色素I基因（COI)序列設計，可用以鑑定 馬鈴薯蚜。馬鈴薯蚜之細胞色素I基因序列參見序列識別 號碼 SEQ ID NO: 14。 200923365 SEQ ID NO: 7、SEQ ID NO: 8係利用偽菜蚜之細胞色 素I基因（COI)序列設計，可用以鑑定偽菜蚜，偽菜辑之 細胞色素I基因序列參見序列識別號碼SEQ ID NO: 15。 SEQ ID NO: 9、SEQ ID NO·· 10、SEQ ID ΝΟ:11 及 SEq ID NO: 12係利用小桔蚜之細胞色素i基因（c〇I)序列設 計，可用以鑑定小桔蚜，小桔蚜之細胞色素j基因序列參 見序列識別號碼SEQ ID NO: 16。 將上述之具有屬間特異性之寡核苷酸探針製成生物 晶片，可快速鑑定蚜蟲類昆蟲。用以鑑定蚜蟲之生物晶片 包含基材及固著於基材上之寡核苷酸探針，其中基材之材 質可包含尼龍膜、高分子材料、石夕片或玻璃。 生物晶片之製作方法包含將一空白晶片在45。〇下烘烤 約5分鐘，分別將40微莫耳濃度之（μΜ)合成的寡核苷酸探 針加入探針溶液混合，再用點片機點在設定的位置，置於 4rC烘烤1 -2分鐘，最後再以紫外線交聯器（UV Cr〇Sslinker) 於0.8焦爾’6分鐘的條件下將寡核#酸探針固定在晶片上。 喷參照第1圖，為本發明之生物晶片的寡核苷酸探針 施佈不意圖。每一點上所標示之數字代表序列識別編號之 數子英文予母’’C’’則代表控制组，用以確認雜合反應無 誤其中’數字1〜4代表序列為SEq ID N〇 : 1〜4之寡核 普酸探針，可與含有棉財之C〇I基因序列的去氧核醣核酸 ()樣1^產生專一性雜合，而在晶片相對應之位置呈 色*標不數子5〜6之點代表序列為SEQ ID N〇 : 5〜6之寡 核普酸探針’可與含有馬鈐薯呀之COI基因序列的DNA樣 200923365 品產生專一性雜合。標示數字為7〜8之點代表序列為SEQ ID NO: 7〜8之寡核苷酸探針，可與含有偽菜蚜之c〇I基因 序列的DNA樣品產生專一性雜合。標示數字為9〜12之點 代表序列為SEQ ID NO : 9〜12之募核苷酸探針，可與含有 小桔蚜之COI基因序列的DNA樣品產生專一性雜合。 請參照第2圖，為利用本發明之生物晶片鑑定蚜蟲類 昆蟲的方法流程圖’包含（a)萃取蟲體之基因體去氧核醣核 酸（DNA) ; (b)增幅待測蟲體之粒線體細胞色素氧化酶工基 因（COI)片段序列；（c)使用本發明之生物晶片與增幅之粒 線體細胞色素氧化酶I基因（COI)片段進行雜合反應；以及 (d)鑑定步驟（c)之雜合反應結果，並由雜合反應之結果鑑定 蚜蟲類昆蟲》 依照本發明之實施例，萃取蟲體之基因體DNA是使用 Wizard® Genomic DNA Purification Kit(Promega Corporation，Madison，USA)的方法，其步驟如下：將蟲體 置入内含緩衝液（0.5M EDTA，Nuclei Lysis Solution > 20 mg/ml Proteinase K)之離心管中研磨均勻，將研磨液置於 个亙溫培養箱中，於5 5 °C加熱反應隔夜，加入適量之核酷核 酸酶（RNase)溶液，反轉離心管數次，並於37°C培養15~30 分鐘。之後置於室溫5分鐘，再加入蛋白沈澱液（Protein Precipitation Solution)高速震盪20秒，置於冰上5分鐘。之 後以13,000xg之轉速離心4分鐘，取上清液至新的離心管内 並混入600微升（ul)異丙醇（Isopropanol)。輕輕翻轉直至白 色懸浮物產生。再離心去除上清液《加入70%之乙醇，輕 200923365Aphids are common insects 'usually inhabiting the stems, leaves, and pods of plants. They suck the sap of the plant with a sucking scorpion. They are regarded as agricultural pests. The worms are clustered in the heart of the cabbage and flowers. And the kind of laughter or leaf back absorbs nutrient t and secretes honeydew to induce soot disease, resulting in poor appearance of the plant, affecting the quality of the flower 'the leaf is often curled or wilted>. In addition, in addition to direct hazards, aphids can spread plant viruses. The identification of the insects of the eyeworms is generally characterized by morphological characteristics. However, the worm body length is about (U ~ 〇 · 5 cm, because the worm body is small and the mites are polymorphic, the external morphological variation between individuals is large, so it is difficult to make the raccoon species. The forensic personnel of the experience rely on the test and the special ability to judge ^' and the traditional form is often difficult to judge objectively because of the subjective cognition of the appraiser, and it is difficult to judge objectively. [Invention] The present invention is provided by the present invention. A probe and its method of use to identify oligocores of aphid insects to solve the problem of traditional locust state identification 200923365 Authenticity is affected by human judgment. Another object of the present invention is to provide an organism for identifying mites A wafer and a method of using the same for rapidly identifying aphid insects of different genus. According to the present invention, an oligonucleotide probe for identifying aphid insects and a method for using the same are provided, and an oligonucleotide probe for identifying aphid insects is provided. Included in SEQ ID NO.., SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO.. 5, SEQ ID NO: 6, SEQ ID NO.. 7, SEQ ID NO: 8 SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, the nucleotide sequence of the complementary strand, the degenerate sequence, and the sequence derived therefrom. The method for identifying an aphid insect by an oligonucleotide probe comprises: heterozygous reaction of the cytochrome oxidase I gene (COI) of the worm body to be tested with the above oligonucleotide probe, and The result of the heterozygous reaction identifies the genus of the insect insect (genus). According to the present invention, a biochip for rapidly identifying aphid insects and a method for using the same are provided, and the biochip for identifying the aphid insect comprises a substrate on the substrate. Can be fixedly selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO : 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, the nucleotide sequence, the nucleotide sequence of the complementary strand, the degenerate sequence, and the derivative thereof A population consisting of a sequence and any combination of the above sequences. The method for identifying aphid insects using the above biochip comprises: The mitochondrial cytochrome oxidase I gene (COI) of the 200923365 test organism was hybridized with the oligonucleotide probe on the above biochip, and the insect insects were identified by the result of the heterozygous reaction. Genus (genus). [Embodiment] The present invention utilizes cotton money gossypii), potato money (Aulacorthum ίο/β/·»·), fake vegetable money, and small orange money (Toxoptera aurantii) 'A field (Acythosiphon / 7/5·ww) Oligonucleotide probes were designed for the cytochrome I gene (COI) sequence of different genus insects such as Zengcai (ZM·? perhcae), and probe specificity tests were performed, except for peas and In addition to the peach aphid, oligonucleotide probes with inter-specificity are obtained, the sequences of which are numbered SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID, respectively. NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12. All samples of the insects used were identified and confirmed in advance. The oligonucleotide probes of the SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 are designed using the cytochrome I gene (COI) sequence of cotton aphid and can be used to identify cotton. aphid. The sequence of the cytochrome I gene of the cotton aphid is shown in SEQ ID NO: 13. The nucleotide probes of SEQ ID NO: 5 and SEQ ID NO: 6 were designed using the cytochrome I gene (COI) sequence of potato mash to identify potato mash. The sequence of the cytochrome I gene of the potato mash is shown in the sequence identification number SEQ ID NO: 14. 200923365 SEQ ID NO: 7. SEQ ID NO: 8 is designed using the cytochrome I gene (COI) sequence of Pseudosciaena chinensis. It can be used to identify the pseudocystis, and the cytochrome I gene sequence of the pseudo-species series. See sequence identification number SEQ ID NO: 15. SEQ ID NO: 9, SEQ ID NO.. 10, SEQ ID ΝΟ: 11 and SEq ID NO: 12 lines are designed using the cytochrome i gene (c〇I) sequence of citrus scutellariae, which can be used to identify small citrus, small The sequence of the cytochrome j gene of the orange bud is shown in SEQ ID NO: 16. The aphid-like insects can be quickly identified by making the above-described inter-specific-specific oligonucleotide probe into a biochip. The biochip for identifying aphids comprises a substrate and an oligonucleotide probe immobilized on the substrate, wherein the material of the substrate may comprise a nylon film, a polymer material, a stone tablet or a glass. The method of fabricating a biochip includes placing a blank wafer at 45. The underarm is baked for about 5 minutes, and the oligonucleotide probe of 40 μmol concentration (μΜ) is added to the probe solution for mixing, and then placed at a set position by a spotting machine, and placed in a 4rC baking 1 - 2 minutes, and finally the oligonuclear acid probe was immobilized on the wafer with a UV cross-linker (UV Cr〇Sslinker) at 0.8 Jol for 6 minutes. Referring to Fig. 1, the oligonucleotide probe of the biochip of the present invention is not intended to be applied. The number indicated on each point represents the number of the sequence identification number. The English to mother ''C'' represents the control group to confirm that the hybrid reaction is correct. The number 1~4 represents the sequence as SEq ID N〇: 1~ The oligonucleotide probe of 4 can be specifically hybridized with the DNA of the C〇I gene sequence containing the cotton, and the color is marked at the corresponding position of the wafer. The point 5 to 6 represents that the oligonucleotide of the sequence of SEQ ID N: 5 to 6 can be specifically hybridized with DNA-like 200923365 containing the sequence of the COI gene of the horse. The point indicated by the number 7 to 8 represents an oligonucleotide probe of the sequence of SEQ ID NOS: 7 to 8, which can be specifically hybridized with a DNA sample containing the sequence of the c〇I gene of the pseudophyta. The point indicated by the number 9 to 12 represents a nucleotide probe of the sequence of SEQ ID NO: 9 to 12, which can be specifically hybridized with a DNA sample containing the COI gene sequence of the citrus. Referring to FIG. 2, a flow chart for identifying a locust insect using the biochip of the present invention includes (a) extracting the genomic DNA of the worm (DNA); (b) increasing the size of the worm to be tested. a line cell cytochrome oxidase gene (COI) fragment sequence; (c) a hybridization reaction using the biochip of the invention with an amplified mitochondrial cytochrome oxidase I gene (COI) fragment; and (d) an identification step (c) Results of the heterozygous reaction and identification of aphid insects by the result of the heterozygous reaction. According to an embodiment of the present invention, the genomic DNA of the extracted worm is obtained using the Wizard® Genomic DNA Purification Kit (Promega Corporation, Madison, USA). The method is as follows: the worm is placed in a centrifuge tube containing a buffer (0.5 M EDTA, Nuclei Lysis Solution > 20 mg/ml Proteinase K), and the slurry is uniformly cultured. In the box, heat the reaction at 55 ° C overnight, add an appropriate amount of nuclear nuclease (RNase) solution, reverse the tube several times, and incubate at 37 ° C for 15 to 30 minutes. After being placed at room temperature for 5 minutes, it was further shaken for 20 seconds by adding a Protein Precipitation Solution and placed on ice for 5 minutes. Thereafter, it was centrifuged at 13,000 x g for 4 minutes, and the supernatant was taken to a new centrifuge tube and mixed with 600 μl of isopropyl alcohol (Isopropanol). Gently flip until the white suspension is produced. The supernatant was removed by centrifugation. Add 70% ethanol, light 200923365
反轉離心管數次，離心去除上清液再進行烘乾。烘乾後的 DNA以無菌水於65°C下回溶，此即為供試蟲體之模板DNA 來源。 利用萃取之蟲體模板DNA，並以COI基因之專一性引 子對進行聚合酶連鎖反應（PCR)增幅COI基因片段。其中， 使用的引子對之序列請參照SEQ ID NO:17 (正向序列）與 SEQ ID NO: 18 (反向序列）。 聚合酶連鎖反應係以迴溫反應器（Perkin-Elmer; GeneAmp® PCR System 2700)進行增幅。取得生物體COI 序列之反應：以微量離心管盛裝欲增幅的反應物，每一反 應之總體積為25 μΐ，内含50 ng模板DNA、250 μΜ dNTPs、lXPCR 緩衝液、0·2μΜ 引子，以及0.5UTaqPluse 耐熱聚合酶（Bio Basic Inc.，Canada)。COI之聚合酶連鎖 反應條件為94°C升溫2分鐘，之後以94°C 40秒、44°C 75 秒、72°C 40秒，進行30個循環，接著以72°C 10分鐘完成 反應，反應後之產物可進行產物片段之分離回收與純化。 將聚合酶連鎖反應增幅產物以洋菜瓊脂電泳分離，並 以溴化乙錠染色後，在紫外燈下將預定大小的DNA片段 自電泳膠片上切下，秤重後可以商用配方例如Micro-Elute DNA Clean/Extraction Kit ( Hopegen Biotechnology Development Enterprise, Taichung, Taiwan)進行回收，即 為COI基因片段。 取此COI基因片段利用商用配方例如pGEM®-T Easy Vector Systems(Promega Corporation, Madison, . USA)與 200923365Reverse the centrifuge tube several times, centrifuge to remove the supernatant and then dry. The dried DNA is reconstituted in sterile water at 65 ° C, which is the source of the template DNA for the test insects. The extracted plasmid DNA was extracted and the COI gene fragment was amplified by polymerase chain reaction (PCR) using a specific primer pair of the COI gene. For the sequence of primer pairs used, please refer to SEQ ID NO: 17 (forward sequence) and SEQ ID NO: 18 (reverse sequence). The polymerase chain reaction was amplified in a temperature-recovery reactor (Perkin-Elmer; GeneAmp® PCR System 2700). To obtain the reaction of the organism's COI sequence: the microfluidic tube contains the reactants to be increased, and the total volume of each reaction is 25 μΐ, containing 50 ng of template DNA, 250 μΜ dNTPs, lXPCR buffer, 0·2 μΜ primer, and 0.5 UTaq Pluse Thermostable Polymerase (Bio Basic Inc., Canada). The polymerase chain reaction conditions of COI were elevated at 94 ° C for 2 minutes, followed by 30 cycles of 94 ° C for 40 seconds, 44 ° C for 75 seconds, and 72 ° C for 40 seconds, followed by completion of the reaction at 72 ° C for 10 minutes. The product after the reaction can be subjected to separation, recovery and purification of the product fragments. The polymerase chain reaction amplification product is separated by electrophoresis on agar extract and stained with ethidium bromide, and the DNA fragment of a predetermined size is cut out from the electrophoresis film under ultraviolet light, and the product can be commercialized, for example, Micro-Elute. The DNA Clean/Extraction Kit ( Hopegen Biotechnology Development Enterprise, Taichung, Taiwan) is a COI gene fragment. Take this COI gene fragment using commercial formulas such as pGEM®-T Easy Vector Systems (Promega Corporation, Madison, . USA) and 200923365
Fast-Trans™ Comptent E. co/iXL-l-Blue cells(Prothec Technology Enterprise Co.，Ltd, Taiwan)的系統進行基因選 殖（Cloning) ’得到单一 COI基因序列的質體（plasmid)。 依照本發明之一實施例，利用含COI基因序列的質體 與具有生物素（Biotin)標定的引子進行聚合酶連鎖反應，其 每一個反應液總體積為25 μΐ，内含2 ng模板DNA、250 μΜ dNTPs、lXPCR 缓衝液、0·2/ιΜ 引子，以及 〇_5UTaqPluse 耐熱聚合酶（Bio Basic Inc.，Canada)。反應條件為94°C升 溫2分鐘，之後以94°C 40秒、44°C 75秒、72°C 40秒， 進行20個循環，接著以72°C 10分鐘完成反應，而得到具 有生物素標定的COI基因片段，即為雜合反應之標的 (target) ° 將生物素標定的target DNA片段與本發明之生物晶片 上之寡核苷酸探針進行雜合。依照本發明之實施例，進行 雜合反應之方法包含在晶片每一反應槽先加入雜合反應 液，再加入適量生物素標定的target DNA片段與之混合， 置於60°C環境下1小時，以進行雜合反應。之後洗去未雜合 的DNA片段，再加入顯色劑（4 μΐ NBT/BCIP + 196 μΐ Detection buffer)置於暗室中反應10分鐘，反應後以清水清 洗，並在45°C下烘乾，以出現色斑的有無判定蚜蟲種類。 請參照第3圖，為利用棉蚜之COI基因片段測試本 發明之生物晶片之探針的專一性結果。對照第1圖之募核 苷酸探針施佈示意圖，以棉蚜之COI基因片段與本發明 之生物晶片上之寡核苷酸探針雜合的結果顯示，試驗組只 11 200923365 有第1〜4號的點有呈色，且控制組亦有呈色故可確認雜 合反應無誤。其中，1〜4點代表序列為SEQIDN〇: i〜4 之募核苷酸探針，因此SEQIDN〇:丨〜4之寡核苷酸探針 可與含有棉蚜之coi基因序列的DNA樣品產生專一性雜 合，可用以準確鑑定棉蚜。 清參照第4圖，為利用馬鈴薯蚜之c〇I基因片段測 δ式本發明之生物晶片之探針的專一性結果。對照第1圖之 募核苷酸探針施佈示意圖，以馬鈴薯蚜之c〇I基因片段 與本發明之生物晶片上之寡核苷酸探針雜合的結果顯 示，試驗組第5、6號的點有呈色，且控制組亦有呈色故 可確認雜合反應無誤。其中，第5、6點代表序列為SEQ出 NO : 5、6之寡核苷酸探針，因此SEQ m N〇 : 5、6之寡 核苷酸探針可與含有馬鈴薯蚜之c〇I基因序列的DNA樣 品產生專一性雜合，可用以準確鑑定馬鈴薯蚜。此外以豌 豆蚜之COI基因序列設計之寡核苷酸探針由於不具有專一 性，因此亦與馬鈴薯蚜之c〇I基因片段雜合，出現於第4 圖中。 請參照第5圖，為利用偽菜蚜之c〇I基因片段測試 本發明之生物晶片之探針的專一性結果。對照第丨圖之寡 核苷酸探針施佈示意圖，以偽菜蚜之c〇I基因片段與本 發明之生物晶片上之寡核苷酸探針雜合的結果顯示，試驗 組只有弟7、8號的點有呈色，且控制組亦有呈色故可確 認雜合反應無誤。其中，第7、8點代表序列為SEQ ID NO : 7、8之寡核苷酸探針，因此SEq id NO : 7、8之寡核普 12 200923365 _針可與含有偽菜騎之c〇I基因序列的舰樣品產生 專一性雜合，可用以準確鑑定偽菜蚜。 叫參照第6圖，為利用小桔蚜之c〇I基因片段測試 本發明之生物晶片之探針的專一性結果。對照第丄圖之寡 核普酸探針施饰示意圖，以小桔騎之⑽基因片段與本 發明之生物晶片上之寡核苷酸探針雜合的結果顯示，試驗 組第9〜12號的點有呈色，且控制組亦有呈色故可確認雜 Γ: 合反應無誤。其中，第9〜12點代表序列為SEQidn〇: 9〜12之寡核苷酸探針，因此SEQidn〇: 7、8之寡核苷 酸探針可與含有小桔虫牙之c〇I基因序列的膽人樣品產生 專性雜5，可用以準確鑑定小桔坊。此外，專一性差的 豌豆辑募核#酸探針亦與小桔敎⑽基因片段雜合，於 第6圖中產生一微弱訊號。 依照本發明之實施例，本發明之生物晶片可包含上述 SEQ ID NQ:卜12所示之㈣酸序列、其互補股之核芽 D _列、簡併序列及其衍生序列。此處所指之衍生序列係 於序列SEQ ID NO: 1〜12之3|端或5·端修鋅核苦酸序列， 使其仍和原序列具有70%以上相似性之寡核苷酸序列。 雖然本發明已以數實施例揭露如上，然其並非用以限 定本發明，任何熟習此技藝者，在不脫離本發明之精神和 範圍内，當可作各種之更動與潤飾，因此本發明之保護範 圍當視後附之申請專利範圍所界定者為準。 【圖式簡單說明】 13 200923365 為讓本發明之上述和其他目的、特徵、優點與實施例 能更明顯易懂，所附圖式之詳細說明如下： 圖 第1圖為本發明之生物晶片的寡核苷酸探針施佈示意 第2圖係繪示依照本發明實施例的一種以生物晶片鑑 疋財蟲類昆蟲的方法流程圖。 第3圖為利用棉財之c〇I基因片段測試本發明之生 物晶片之探針的專一性結果。 第4圖為利用馬鈐薯財之C(3I基因片段測試本發明 生物晶片之探針的專-性結果。 生物料菜敎⑽基因片段測試本發明之 生物ΘΒ片之探針的專-性結果。 第6圖為利用小桔 生物晶片之探針的專_性結果。基因片段測試本發明之 主 要元件符號說明 無 14The system of Fast-TransTM Comptent E. co/iXL-l-Blue cells (Prothec Technology Enterprise Co., Ltd, Taiwan) performs gene selection (Cloning) to obtain a plasmid of a single COI gene sequence. According to an embodiment of the present invention, a polymerase chain reaction is carried out using a plastid containing a COI gene sequence and a biotin-labeled primer, each of which has a total volume of 25 μΐ and contains 2 ng of template DNA. 250 μΜ dNTPs, lXPCR buffer, 0·2/ιΜ primer, and 〇_5UTaqPluse thermostable polymerase (Bio Basic Inc., Canada). The reaction conditions were a temperature increase of 94 ° C for 2 minutes, followed by a cycle of 94 ° C for 40 seconds, 44 ° C for 75 seconds, and 72 ° C for 40 seconds, followed by completion of the reaction at 72 ° C for 10 minutes to obtain biotin. The calibrated COI gene fragment, which is the target of the hybrid reaction, is hybridized with the biotin-labeled target DNA fragment and the oligonucleotide probe on the biochip of the present invention. According to an embodiment of the present invention, the method for performing the hybrid reaction comprises first adding a hybrid reaction solution to each reaction tank of the wafer, and then adding an appropriate amount of the target DNA fragment labeled with biotin, and mixing it at 60 ° C for 1 hour. To carry out the heterozygous reaction. After that, the unhybridized DNA fragment was washed away, and then a color developing agent (4 μΐ NBT/BCIP + 196 μΐ Detection buffer) was added to the dark room for 10 minutes. After the reaction, it was washed with water and dried at 45 ° C. Determine the species of aphids by the presence or absence of stains. Referring to Figure 3, the specificity results of the probe of the biochip of the present invention were tested using the COI gene fragment of cotton aphid. According to the schematic diagram of the nucleotide probe of Figure 1, the hybridization of the COI gene fragment of cotton aphid with the oligonucleotide probe on the biochip of the present invention shows that the test group only has 11 200923365. The dots of ~4 have a coloration, and the control group also has a coloration to confirm that the hybrid reaction is correct. Wherein, 1 to 4 points represent a nucleotide probe having the sequence of SEQ IDN: i~4, and thus the oligonucleotide probe of SEQ IDN: 丨~4 can be produced with a DNA sample containing the coi gene sequence of cotton aphid. Specificity and heterogeneity can be used to accurately identify cotton aphid. Referring to Fig. 4, the specificity result of the probe of the biochip of the present invention is measured by using the c〇I gene fragment of potato mash. According to the schematic diagram of the nucleotide probe of Figure 1, the hybridization of the potato 蚜c〇I gene fragment with the oligonucleotide probe on the biochip of the present invention shows that the experimental group 5, 6 The point of the number has a color, and the control group also has a coloration to confirm that the hybrid reaction is correct. Wherein, points 5 and 6 represent oligonucleotide probes of the sequence SEQ out of NO: 5, 6, and thus the oligonucleotide probe of SEQ m N〇: 5, 6 can be combined with c〇I containing potato mash DNA samples of the gene sequence produce a specific heterozygosity that can be used to accurately identify potato mash. In addition, the oligonucleotide probe designed with the COI gene sequence of peas is also heterozygous and therefore hybridized with the potato 〇c〇I gene fragment, which appears in Fig. 4. Referring to Fig. 5, the specificity results of the probe of the biochip of the present invention were tested for the c〇I gene fragment of the pseudocaulis. According to the schematic diagram of the oligonucleotide probe of the digest map, the result of hybridization of the c〇I gene fragment of the pseudocaulis with the oligonucleotide probe on the biochip of the present invention shows that the experimental group only has the brother 7 The 8th point has a coloration, and the control group also has a coloration to confirm that the hybrid reaction is correct. Wherein, the 7th and 8th points represent the oligonucleotide probes of the sequence of SEQ ID NO: 7, 8 , so the SEq id NO : 7 and 8 of the oligonuclear 12 200923365 _ needle can be combined with the pseudo-dancing c〇 The ship samples of the I gene sequence produce a specific heterozygosity that can be used to accurately identify pseudo-vegetables. Referring to Fig. 6, a specific result of testing the probe of the biochip of the present invention was tested using the c〇I gene fragment of the citrus. According to the schematic diagram of the oligonucleotide probe of the digraph, the heterozygous (10) gene fragment of the Xiaoyanqi and the oligonucleotide probe on the biochip of the present invention showed that the test group No. 9 to No. 12 The dots are colored, and the control group is also colored to confirm the miscellaneous: the reaction is correct. Wherein, the 9th to 12th points represent the oligonucleotide probes of the sequence SEQidn〇: 9~12, and thus the oligonucleotide probes of SEQidn〇: 7, 8 can be combined with the c〇I gene sequence containing the small orange worm teeth. The bile sample produces a specialized impurity 5 that can be used to accurately identify the small orange square. In addition, the poorly specific pea-collecting nuclear #acid probe is also heterozygous with the small orange (10) gene fragment, which produces a weak signal in Figure 6. According to an embodiment of the present invention, the biochip of the present invention may comprise the (tetra) acid sequence shown in SEQ ID NO: 12 above, the nucleus D _ column of the complementary strand, the degenerate sequence and the derivative sequence thereof. The derivative sequence referred to herein is an oligonucleotide sequence which has a 3'-end or a 5'-end of the SEQ ID NO: 1 to 12, and which has a similarity to the original sequence of 70% or more. The present invention has been disclosed in the above embodiments, and is not intended to limit the present invention, and it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application attached. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; Oligonucleotide Probe Application Diagram 2 is a flow chart showing a method for identifying insects and insects on a biological wafer according to an embodiment of the present invention. Fig. 3 is a view showing the specificity of the probe for testing the biochip of the present invention using the c〇I gene fragment of M. chinensis. Figure 4 is a photograph showing the specificity of the probe of the biochip of the present invention using the C (3I gene fragment) of the horse yam. The biological material 敎 (10) gene fragment is tested for the specificity of the probe of the biochip of the present invention. Results. Figure 6 is a specific result of the probe using the small orange biochip. Gene fragment test The main component symbol description of the present invention is 14