1338135 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鱗翅目昆蟲的方法，且特別 是有關於一種鑑定鱗翅目昆蟲的寡核酸探針、生物晶片及 其鑑定方法。 【先前技術】 鱗翅目為世界性分布之昆蟲，許多重要的經濟害蟲亦 屬之’於幼蟲期危害或處卵期而藏於農作物之中，增加了 鑑定上的困擾。例如蘋果蠹蛾，其幼蟲於蘋果、梨、桃及 杏等鮮果内鑽蛀啃食，檢疫時發現者大多為幼蟲，由於幼 **之外部形態鑑疋較為困難，是故建立起一套快速、簡便 之鑑定方法已成為當務之急。 目前的鱗翅目昆蟲鑑定一般以形態特徵鑑定為主。然 而，由於昆蟲生活史存在變態的現象，分別有卵、幼蟲、 蛹及成蟲等各時期，而各時期的形態迥異，因此在形態鑑 疋上需要由有經驗的鑑識人員依賴經驗及專門能力加以 判定。此外，由於存在於農產品中的害蟲，被發現時常僅 剩下某些殘肢碎片，缺乏足夠之外部特徵而使得鑑定困 難，而蟲體完整者往往處於幼蟲'蛹甚至是卵的生長階 段，多需將其飼育至羽化方得以鑑定，如此便喪失了檢疫 的時效，造成貿易的損失。因此，目前在港口與機場的檢 疫工作上，要能夠快速且準確的鑑定害蟲仍具有許多實務 上的限制。 5 鈐果：：：蟲的形態鑑定常因鑑定者主觀的認知造成鑑定 :进续而難以客觀地狀，且形態鑑^亦無法應用 #間的分類’昆蟲幼蟲的特徵更難以辨 形態鑑定㈣果亦可㈣好議。 【發明内容】 4 A因此本發明就是在提供一種鑑定鱗翅目昆蟲之寡核 苦1¼針（prc)be)及其使用方法，解決傳統鱗翅目昆蟲形 態鑑定正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鱗翅目昆蟲之 生物晶片及其使用方法，用以快速鑑定不同種之鱗翅目昆 蟲。 根據本發明，提出一種鑑定鱗翅目昆蟲之募核苷酸探 針及其使用方法，鑑定鱗翅目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鱗翅目昆蟲之方法包 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鱗翅目昆蟲之種（species)。 根據本發明，提出一種快速鑑定鱗翅目昆蟲之生物晶 片及其使用方法，鑑定鱗翅目昆蟲之生物晶片包含一基 1338135 材，基材上可固著選自於SEQ ID N〇 :】〜9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鱗翅目昆蟲之方法包含：以待 測蟲體之粒線體細胞色素氧化酶I基因（C〇i )與上述生 物ββ片上的券核苷酸探針進行雜合反應（hybridizati〇n )， 並由雜合反應之結果進行鑑定。1338135 IX. Description of the Invention: [Technical Field] The present invention relates to a method for identifying lepidopteran insects, and more particularly to an oligonucleic acid probe, a biochip, and an identification method thereof for identifying lepidopteran insects. [Prior Art] Lepidoptera is a worldwidely distributed insect, and many important economic pests are also hidden in crops during larval stage damage or ovulation, which increases the identification problem. For example, the cockroach moth, whose larvae are foraged in fresh fruits such as apples, pears, peaches, and apricots, are mostly larvae found during quarantine. Because the external form of the young cockroaches is difficult, it is a quick set up. Simple identification methods have become a top priority. The current identification of lepidopteran insects is generally characterized by morphological characteristics. However, due to the metamorphosis of insect life history, there are different periods of eggs, larvae, pupa and adults, and the patterns of each period are different. Therefore, it is necessary to rely on experience and expertise by experienced forensic personnel in the form of identification. determination. In addition, due to the pests present in agricultural products, it is often found that only some fragments of residual limbs remain, lacking sufficient external features to make identification difficult, and those with intact worms are often in the growth stage of larvae's cockroaches and even eggs. It needs to be reared to the feathers to be identified, thus losing the quarantine time and causing trade losses. Therefore, there are still many practical limitations in the rapid and accurate identification of pests in port and airport quarantine work. 5 Capsule::: The morphological identification of the worm is often identified by the subjective cognition of the appraiser: it is difficult to objectively shape, and the morphological identification can not be applied. The classification of the larvae is more difficult to distinguish the morphological identification (4) It can also be (4) good discussion. SUMMARY OF THE INVENTION 4 A Therefore, the present invention provides an oligonuclear pectorus (prc) which is used to identify lepidopteran insects and a method for using the same, and solves the problem that the correctness of the traditional lepidopteran shape identification is influenced by human judgment. Another object of the present invention is to provide a biochip for identifying lepidopteran insects and methods of use thereof for rapidly identifying different species of Lepidoptera. According to the present invention, a nucleotide probe for identifying lepidopteran insects and a method for using the same are provided. The oligonucleotide probe for identifying lepidopteran insects comprises SEQ ID NO: 1, SEQ ID NO: 2, and 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 nucleotide sequence, nucleus of its complementary strand A nucleotide sequence, a degenerate sequence, and a derivative thereof. The method for identifying lepidopteran insects by using the above oligonucleotide probe comprises: heterozygous reaction of the cytochrome oxidase I gene (COI) of the worm body to be tested with the above oligonucleotide probe, Species of Lepidoptera are identified from the results of the heterozygous reaction. According to the present invention, a biochip for rapidly identifying lepidopteran insects and a method for using the same are provided. The biochip for identifying lepidopteran insects comprises a base 1338135 material, and the substrate can be fixed and selected from the group consisting of SEQ ID N::~9 A population consisting of the nucleotide sequence shown, the nucleotide sequence of its complementary strand, the degenerate sequence, the derivative sequence thereof, and any combination of the above sequences. The method for identifying a Lepidoptera insect using the above biochip comprises: heterozygous reaction of a mitochondrial cytochrome oxidase I gene (C〇i) of the worm to be tested with a nucleus probe on the above-mentioned biological ββ sheet (hybridizati) 〇n ) and identified by the results of the heterozygous reaction.
【實施方式】 本發明係利用蘋果蠢蛾（)、梨小食心 蟲（C少、桃虹果蛾（Carpos/wa said//)等不 同種的鱗翅目昆蟲之細胞色素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，得到具有種間特 異性之寡核苷酸探針’其序列分別編號為SEQ ID NO : 1 〜9。所有使用之蟲體樣本均事先經鑑定確認種類。[Embodiment] The present invention utilizes the cytochrome I gene (COI) sequence design of different species of lepidopteran insects such as apple stupid moth (), P. sinensis (C less, Carpos/wa said//). Oligonucleotide probes, and probe specificity tests were performed to obtain oligonucleotide probes with interspecies specificity. The sequences were numbered as SEQ ID NOs: 1 to 9. The samples of all used insects were The type is confirmed by identification in advance.
其中編號SEQ ID NO: 1〜3之寡核苷酸探針係利用蘋 果蠹蛾之細胞色素I基因（COI )序列設計，可用以鑑定 韻果蠹蛾。蘋果蠢蛾之細胞色素I基因序列參見序列識別 號碼 SEQ ID NO: 10。 SEQ ID NO: 4〜6之寡核苷酸探針係利用梨小食心蟲 之細胞色素I基因（COI )序列設計，可用以梨小食心蟲。 梨小食心蟲之細胞色素I基因序列參見序列識別號碼SEQ ID NO: 11。 SEQ ID NO: 7〜9之寡核苷酸探針係係利用桃蛀果蛾 7 之細胞色素I基因（COI)序列設計，可用以鑑定桃蛀果 蛾，桃蛀果蛾之細胞色素丨基因序列參見序列識別號碼 SEQ ID NO:12。 將上述之具有屬間特異性之寡核苷酸探針製成生物 晶片，可快速鑑定鱗翅目昆蟲。用以鑑定鱗翅目昆蟲之生 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（μΜ)之合成的寡核苷酸 探針加入探針溶液混合，再用點片機點在設定的位置置 於45°C烘烤1-2分鐘，最後再以紫外線交聯器（uv cr〇ss linker)於0.8焦爾，6分鐘的條件下將寡核苷酸探針固定在 晶片上。 請參照第1圖，為本發明之生物晶片的寡核苷酸探針 施佈示意圖。每一點上所標示之數字代表序列識別編號之 數字，英文字母’’C’’則代表控制組，用以確認雜合反應無 誤。其中’數字1〜3代表序列為SEQ ID NO : 1〜3之寡核 苷酸探針，可與含有蘋果蠹蛾之c〇I基因序列的去氧核醣 核酸樣品產生專一性雜合，而在晶片相對應之位置呈色。 標示數字4〜6之點代表序列為SEq ID N〇 : 4〜6之寡核苷 酸探針，可與含有梨小食心蟲之(：〇1基因序列的去氧核醣 核酸樣品產生專一性雜合。標示數字為7〜9之點代表序列 分別為SEQ ID NO : 7〜9之寡核苷酸探針，可與含有桃蛀 果蛾之COI基因序列的去氧核醣核酸樣品產生專—性雜 1338135 合0 '•月參照第2圖，為利用本發明之生物晶片鑑定鱗翅目 昆蟲的方法流程圖，包含（a)萃取蟲體之基因體去氧核醣 核自文，（b )增幅待測蟲體之粒線體細胞色素氧化酶I基因 (CO〇片段序列；（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丨基因（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 之結果鑑定鱗翅目昆蟲。 依照本發明之實施例，萃取蟲體之基因體去氧核醣核 酸的方法包含將蟲體置入内含緩衝液（100 mM NaCl，10 mM Tris-Cl, 25 mM EDTA, 0.5% SDS, 0.1 mg/ml protinase k)之離心管中研磨均勻，將研磨液置於恆溫培養箱中， 於65 C加熱反應30分鐘，期間每隔3_5分鐘，將樣品搖晃混 合均勻一次；之後加入1/7體積之8 Μ醋酸氨（ammonium acetate)，緩慢搖勻1〇分鐘，置於冰上45分鐘。之後以 14,5000 X g之轉速離心15分鐘，取上清液加入等體積95% 酒精，並置於-8(TC下10-15分鐘，使DNA沉搬，再離心去 除上清液。加入70%之乙醇，輕反轉離心管數次，離心去 除上清液再進行烘乾。烘乾後的去氧核醣核酸以無菌水於 65°C下回溶一段時間，此即為供試蟲體之模板1)]^八來源。 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c〇I基因片段。其 中，使用的引子對之序列請參照SEQ ID NO: 13 (正向序列） 與SEQ ID NO:14 (反向序列）。 9 聚合酶連鎖反應係以迴溫反應器（Perkin-Elmer; GeneAmp® PCR System 2700 )進行增幅。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、IX PCR緩衝液、0.5 μΜ 引子， 以及0_5U rTaq财熱聚合酶（Takara，Japan )。COI之聚合酶 連鎖反應條件為94°C升溫2分鐘，之後以94°C 45秒、44°C 75 秒、72°C 45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雜合反應。依照本發明之一實施 例，利用具有生物素（Biotin )標定的引子進行聚合酶連 鎖反應，可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片段與之混 合，置於60t環境下1小時，以進行雜合反應。之後洗去未 雜合的target片段，再加入顯色劑（4plNBT/BCIP+ 196 μΐ Detection buffer)置於暗室中反應30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鱗翅目昆 蟲種類。 請參照第3圖，為利用蘋果蠢蛾之C0I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圖之寡 核苷酸探針施佈示意圖，以蘋果蠹蛾之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 組只有第1〜3號的點之呈色較明顯，控制組亦有呈色故 1338135 可確認雜合反應無誤。其中，卜3點代表序列為叫⑴ NO : 1〜3之寡核苷酸探針，因此SEq ID N〇 : 1〜3 核苷酸探針可與含有蘋果蠹蛾之C〇I基因序列的去氧核^ 核酸樣品產生專一性雜合，可用以準確鑑定蘋果蠹蛾。 請參照第4圖，為利用梨小食心蟲之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1圖之 寡核苷酸探針施佈示意圖，以梨小食心蟲之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= 示，試驗組只有第4〜6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4〜6點代表序列為 SEQ ID NO : 4〜6之寡核苷酸探針，因此SEQ m N〇 :‘4 〜6之寡核苷酸探針可與含有梨小食心蟲之c〇I基因序列 的去氧核醣核酸樣品產生專一性雜合，可用以準確鑑定梨 小食心蟲。 請參照第5圖，為利用桃蛀果蛾之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1圖之寡 核苷酸探針施佈示意圖，以桃蛀果蛾之COl基因片段與本 發明之生物晶片上之募核苷酸探針雜合的結果顯示，試驗 組只有第7〜9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7〜9點代表序列為SEQ m NO: 7〜9之寡核苷酸探針，因此SEQIdn〇: 7〜9之募 核苷酸探針可與含有桃蛀果蛾之c 〇丨基因序列的去氧核醣 核酸樣品產生專一性雜合，可用以準確鑑定桃蛀果蛾。 依照本發明之實施例，本發明之生物晶片可包含上述 11 1338135 SEQIDNO:卜9所示之核苦酸序列、其互補股之核苦酸 序列、簡併序列及其衍生序列。此處所指之衍生序列係於 序列SEQIDNO:卜9之3,端或5ι端修飾核苦酸序列，使 其仍和原序列具有70%以上相似性之寡核苦酸序列。 —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’ t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】 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： 第1圖為本發明之生物晶片的寡核苷酸探針施佈示意 圖。 第2圖係繪示依照本發明實施例的一種以生物晶片鑑 定鱗翅目昆蟲的方法流程圖。 第3圖為利用蘋果蠹蛾之COI基因片段測試本發明之 生物晶片之探針的專一性結果。 第4圖為利用梨小食心蟲之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5圖為利用桃蛀果蛾之COI基因片段測試本發明之 生物晶片之探針的專一性結果。 12 1338135 【主要元件符號說明】The oligonucleotide probes numbered SEQ ID NOS: 1 to 3 were designed using the cytochrome I gene (COI) sequence of the scorpion moth, and can be used to identify the snail. The sequence of the cytochrome I gene of the apple moth is shown in the sequence identification number SEQ ID NO: 10. The oligonucleotide probes of SEQ ID NOS: 4 to 6 were designed using the cytochrome I gene (COI) sequence of P. grisea, and can be used for P. grisea. The cytochrome I gene sequence of P. grisea is shown in SEQ ID NO: 11. The oligodeoxynucleotide probe sequences of SEQ ID NOS: 7 to 9 were designed using the cytochrome I gene (COI) sequence of Myzus persicae 7 to identify the cytochrome 丨 gene of the peach locust moth and the peach moth. See Sequence Identification Number SEQ ID NO: 12 for the sequence. The above-described oligonucleotide probe having an intergeneric specificity is made into a biochip, and the lepidopteran insect can be quickly identified. The biochip for identifying lepidopteran insects comprises a substrate and an oligonucleotide probe immobilized on the substrate, wherein the material of the substrate may comprise a nylon film 'polymer material, stone tablet or glass. The method of fabricating a biochip includes placing a blank wafer at 45. The underarm was baked for about 5 minutes, and the synthetic oligonucleotide probe of 40 micromolar concentration (μΜ) was added to the probe solution for mixing, and then baked at a set position at 45 ° C using a spotting machine. For 1-2 minutes, the oligonucleotide probe was finally immobilized on the wafer with a UV crosslinker (vr 〇ss linker) at 0.8 J, for 6 minutes. Please refer to Fig. 1 for a schematic diagram of the oligonucleotide probe of the biochip of the present invention. The number indicated at each point represents the number of the serial identification number, and the English letter ''C'' represents the control group to confirm that the hybrid reaction is correct. Wherein 'numbers 1 to 3 represent an oligonucleotide probe having the sequence of SEQ ID NO: 1 to 3, which can be specifically hybridized with a DNA sample containing the c〇I gene sequence of the codling moth, and The position corresponding to the wafer is colored. The point labeled with the numbers 4 to 6 represents an oligonucleotide probe of the sequence SEq ID N〇: 4 to 6, which can be specifically hybridized with a DNA sample containing the P. sinensis (: 〇1 gene sequence). The dot numbered 7 to 9 represents an oligonucleotide probe having the sequence of SEQ ID NO: 7 to 9, respectively, which can produce a specific heterozygous DNA sample with a COI gene sequence of the peach hazel. 1338135 and 0'• month refer to Fig. 2, which is a flow chart of a method for identifying lepidopteran insects using the biochip of the present invention, comprising (a) extracting the genus deoxyribose core of the worm, and (b) increasing the amplitude to be tested The mitochondrial cytochrome oxidase I gene of the worm (CO 〇 fragment sequence; (C) hybridization reaction using the biochip of the invention with the amplified mitochondrial cytochrome oxidase 丨 gene (COI) fragment; d) identifying the results of the hybridization reaction of step (C) and identifying the lepidopteran insects as a result of the hybridization reaction. According to an embodiment of the invention, the method of extracting the genetic DNA of the worm body comprises placing the worm body Buffer (100 mM NaCl, 10 mM Tris-Cl, 25) Grind evenly in the centrifuge tube of mM EDTA, 0.5% SDS, 0.1 mg/ml protinase k), place the slurry in a constant temperature incubator, heat the reaction at 65 C for 30 minutes, and mix the sample evenly every 3 to 5 minutes. Once; add 1/7 volume of 8 amammonium acetate, shake slowly for 1 , minutes, put on ice for 45 minutes, then centrifuge at 14,5000 x g for 15 minutes, add the supernatant to join Equal volume of 95% alcohol, and placed at -8 (TC for 10-15 minutes, so that the DNA is heavier, then centrifuge to remove the supernatant. Add 70% ethanol, gently reverse the tube several times, centrifuge to remove the supernatant and then Drying. The dried deoxyribonucleic acid is reconstituted in sterile water at 65 ° C for a period of time, which is the template for the test insect body 1)] VIII source. Using the extracted worm template DNA, and The polymerase chain reaction (PCR) is used to increase the c〇I gene fragment by the specific primer pair of the COI gene. For the sequence of the primer pair used, please refer to SEQ ID NO: 13 (forward sequence) and SEQ ID NO: 14 ( Reverse sequence) 9 Polymerase chain reaction with a regenerative reactor (Perkin-Elmer; GeneAmp® PCR System 2700) is augmented with a microcentrifuge tube containing the desired volume of the reaction, with a total volume of 25 μM per reaction containing 50 ng of template DNA, 250 μΜ dNTPs, IX PCR buffer, 0.5 μΜ primer, and 0_5U rTaq Treasury polymerase (Takara, Japan). The polymerase chain reaction conditions of COI were elevated at 94 ° C for 2 minutes, followed by 30 cycles of 94 ° C for 45 seconds, 44 ° C for 75 seconds, and 72 ° C for 45 seconds, followed by completion of the reaction at 72 ° C for 10 minutes. The product after the reaction can be subjected to a heterozygous reaction. According to an embodiment of the present invention, a polymerase-linked reaction using a biotin-labeled primer can obtain a biotin-labeled COI gene fragment, which is a target of a hybrid reaction. The biotin-labeled target fragment is hybridized to 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, adding an appropriate amount of the biotin-labeled target fragment, mixing it, and placing it in a 60 t environment for 1 hour. Hybrid reaction. Then wash away the unhybrid target fragment, then add the color developer (4plNBT/BCIP+ 196 μΐ Detection buffer) and let it react in the dark room for 30 minutes. After the reaction, wash it with water and dry at 45 °C to appear color. The presence or absence of a spot determines the species of Lepidoptera. Referring to Fig. 3, the specificity results of the probe of the biochip of the present invention were tested using the C0I gene fragment of the apple stupid moth. According to the schematic diagram of the oligonucleotide probe of Fig. 1, the result of hybridization of the COI gene fragment of the codling moth with the oligonucleotide probe on the biochip of the present invention shows that the test group only has the first to third The color of the dots is more obvious, and the control group also has a coloration. Therefore, 1338135 can confirm that the hybrid reaction is correct. Wherein, the 3-point representative sequence is an oligonucleotide probe called (1) NO: 1~3, so the SEq ID N〇: 1~3 nucleotide probe can be combined with the C〇I gene sequence containing the codling moth The deoxygenated nuclear nucleic acid sample produces a specific heterozygosity that can be used to accurately identify the codling moth. Referring to Fig. 4, the specificity results of the probe of the biochip of the present invention were tested using the c〇I gene fragment of P. grisea. Referring to the schematic diagram of the oligonucleotide probe of Figure 1, the result of hybridization of the c〇I gene fragment of P. grisea with the oligonucleotide probe on the biochip of the present invention indicates that the test group has only the first The dots from 4 to 6 have a coloration, and the control group also has a coloration to confirm that the hybrid reaction is correct. Wherein, the 4th to 6th points represent the oligonucleotide probes of the sequence of SEQ ID NO: 4 to 6, and thus the oligonucleotide probe of SEQ m N〇: '4 to 6 can be combined with the c. Deoxyribonucleic acid samples of the 〇I gene sequence produce a specific heterozygosity that can be used to accurately identify P. grisea. Referring to Fig. 5, the specificity results of the probe of the biochip of the present invention were tested using the c〇I gene fragment of the peach fruit moth. According to the schematic diagram of the oligonucleotide probe of FIG. 1 , the result of hybridization of the CO1 gene fragment of the peach moth and the nucleotide probe on the biochip of the present invention shows that the test group only has the 7th~ The spot on the 9th has a coloration, and the control group also has a coloration to confirm that the hybrid reaction is correct. Wherein, the 7th to 9th points represent the oligonucleotide probes of the sequence of SEQ m NO: 7 to 9, and thus the nucleotide probe of SEQIdn〇: 7 to 9 can be combined with the c含有 containing the peach hazelnut moth. Deoxyribonucleic acid samples of the gene sequence produce a specific hybrid that can be used to accurately identify the peach fruit moth. According to an embodiment of the present invention, the biochip of the present invention may comprise the nucleotide sequence shown in the above-mentioned 11 1338135 SEQ ID NO: 2, the nucleotide sequence of the complementary strand thereof, the degenerate sequence and the derivative sequence thereof. The derivative sequence referred to herein is a sequence of SEQ ID NO: </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; The present invention has been disclosed in the above several embodiments, but it is not intended to limit the invention, and the present invention may be modified and modified 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. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; Schematic diagram of acid probe application. Figure 2 is a flow chart showing a method for identifying lepidopteran insects using a biochip in accordance with an embodiment of the present invention. Fig. 3 is a view showing the specificity of the probe of the biochip of the present invention using the COI gene fragment of the codling moth. Fig. 4 is a view showing the specificity of the probe of the biochip of the present invention using the c〇I gene fragment of P. grisea. Fig. 5 is a view showing the specificity of the probe of the biochip of the present invention using the COI gene fragment of the peach moth. 12 1338135 [Key component symbol description]