TWI449787B - Oligo-nucleotide probes of thrips identification, biochip, and identifying method thereof - Google Patents
Oligo-nucleotide probes of thrips identification, biochip, and identifying method thereof Download PDFInfo
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本發明是有關於一種鑑別薊馬的方法,且特別是有關於一種鑑別薊馬的寡核酸探針、生物晶片及其鑑別方法。The present invention relates to a method for identifying thrips, and more particularly to an oligonucleic acid probe for identifying thrips, a biochip, and a method for identifying same.
薊馬(Thrips)是一種靠植物汁液維生的昆蟲,其屬於昆蟲綱纓翅目。薊馬的個體小且具有隱匿性,多生活在植物花中取食花粉和花蜜,或以植物的嫩梢、葉片及果實為生,而造成葉子與花朵的損傷,成為農作物、花卉及林果的一害。Thrips are insects that depend on plant juices and belong to the order Insectidae. The individual of Hummer is small and occult. It lives in plant flowers to feed on pollen and nectar, or to feed on the young shoots, leaves and fruits of plants, causing damage to leaves and flowers and becoming crops, flowers and fruit trees. One harm.
薊馬在農作物上之危害,在最近數年來愈來愈趨於嚴重,舉凡花卉、蔬菜、果樹等均有不同種類的薊馬存在,除直接刺吸危害外,且傳播病毒病,造成農業生產上重大的損失。The hazards of thrips on crops have become more and more serious in recent years. There are different kinds of thrips in flowers, vegetables, fruit trees, etc., in addition to direct sucking hazards, and spread viral diseases, resulting in agricultural production. A major loss.
薊馬之鑑別方式可藉由傳統生物學特徵的鑑別方法或應用分子生物學的技術進行鑑別。然而,利用傳統生物學特徵鑑別方法進行鑑別,所需時間長。The identification method of the thrips can be identified by the identification method of traditional biological features or the technique of applying molecular biology. However, the use of traditional biological feature identification methods for identification takes a long time.
目前應用分子生物學的技術,以專一性引子進行聚合酶連鎖反應,配合洋菜瓊脂電泳雖能一次鑑別數種薊馬的種類,然而由於開發出來的專一性引子較少,且使用膠體電泳方式分析仍無法一次達到快速且大量的鑑別需求。At present, the technology of molecular biology is applied, and the polymerase chain reaction is carried out with specific primers. Although it can identify several species of thrips at the same time with agar extract, the development of specific primers is less, and colloidal electrophoresis is used. Analysis still does not achieve fast and large-scale identification needs at once.
因此本發明之一態樣就是在提供一種鑑別薊馬之寡核苷酸探針(probe)及其使用方法,解決傳統薊馬形態鑑別正確性受人為 判斷影響的問題。Therefore, one aspect of the present invention is to provide an oligonucleotide probe for identifying a thrips and a method for using the same, which solves the problem that the correct identification of the traditional thrips is artificial. Determine the impact of the problem.
本發明的另一態樣是在提供一種鑑別薊馬之生物晶片及其使用方法,用以快速同時鑑別不同種之薊馬。Another aspect of the present invention is to provide a biochip for identifying a thrips and a method of using the same for quickly and simultaneously identifying different species of thrips.
根據本發明,提出一種鑑別薊馬之寡核苷酸探針及其使用方法,鑑別薊馬之寡核苷酸探針包含SEQ ID NO:1至SEQ ID NO:59所示之核苷酸序列。According to the present invention, there is provided an oligonucleotide probe for identifying a thrips and a method for using the same, wherein the oligonucleotide probe for identifying a thrips comprises the nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 59 .
應用上述寡核苷酸探針鑑別薊馬之方法包含:以待測蟲體之間隔2區(intergenic spacer 2;ITS2)cDNA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別薊馬之種(species)。The method for identifying a thrips using the above oligonucleotide probe comprises: heterozygous reaction of the intergenic spacer 2 (ITS2) cDNA with the above oligonucleotide probe, and heterozygous reaction The result identifies the species of the thrips.
根據本發明,提出一種快速鑑別薊馬之生物晶片及其使用方法,鑑別薊馬之生物晶片包含一基材,基材上可固著SEQ ID NO:1至SEQ ID NO:6之至少其中之一、SEQ ID NO:7至SEQ ID NO:12之至少其中之一、SEQ ID NO:13至SEQ ID NO:18之至少其中之一、SEQ ID NO:19至SEQ ID NO:24之至少其中之一、SEQ ID NO:25至SEQ ID NO:30之至少其中之一、SEQ ID NO:31至SEQ ID NO:36之至少其中之一、SEQ ID NO:37至SEQ ID NO:42之至少其中之一、SEQ ID NO:43至SEQ ID NO:48之至少其中之一、SEQ ID NO:49至SEQ ID NO:54之至少其中之一、SEQ ID NO:55至SEQ ID NO:59之至少其中之一所示核苷酸序列之寡核苷酸探針。According to the present invention, a biochip for rapidly identifying a thrips and a method of using the same are provided. The biochip for identifying a thrips comprises a substrate on which at least one of SEQ ID NO: 1 to SEQ ID NO: 6 can be immobilized. 1. At least one of SEQ ID NO: 7 to SEQ ID NO: 12, at least one of SEQ ID NO: 13 to SEQ ID NO: 18, at least one of SEQ ID NO: 19 to SEQ ID NO: One of at least one of SEQ ID NO: 25 to SEQ ID NO: 30, at least one of SEQ ID NO: 31 to SEQ ID NO: 36, and at least one of SEQ ID NO: 37 to SEQ ID NO: One of them, at least one of SEQ ID NO: 43 to SEQ ID NO: 48, at least one of SEQ ID NO: 49 to SEQ ID NO: 54, and SEQ ID NO: 55 to SEQ ID NO: 59 An oligonucleotide probe of at least one of the nucleotide sequences shown.
應用上述生物晶片鑑別薊馬之方法包含:以待測蟲體之ITS2之DNA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別薊馬之種。The method for identifying a thrips using the above biochip comprises: performing hybridization of the DNA of the ITS2 to be tested with the oligonucleotide probe on the above biochip, and identifying the thrips by the result of the heterozygous reaction. Kind of.
本發明實施例係利用西方花薊馬(Frankliniella occidentalis )、蔥薊馬(Thrips tabaci )、台灣花薊馬(Frankliniella intonsa )、菊花薊馬(Mircocephalothrips abdominalis )、豆花薊馬(Megalurothrips usitatus )、尖角薊馬(Frankliniella cephalica )、澳洲疫薊馬(Thrips imaginis )、花薊馬(Thrips hawaiiensis )、南黃薊馬(Thrips palmi )、小黃薊馬(Scirtothrips dorsalis )等不同種的薊馬之ITS2 DNA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至SEQ ID NO:59。所有使用之蟲體樣本均事先經鑑別確認種類。In the embodiment of the present invention, Frankliniella occidentalis , Thrips tabaci , Frankliniella intonsa , Mircocephalothrips abdominalis , Megalurothrips usitatus , sharp angles are used. ITS2 DNA of different species of thrips such as Frankliniella cephalica , Thrips imaginis , Thrips hawaiiensis , Thrips palmi , and Scirtothrips dorsalis Oligonucleotide probes were designed in sequence and probe specificity assays were performed to obtain oligonucleotide probes with interspecies specificity, the sequences of which are numbered SEQ ID NO: 1 to SEQ ID NO: 59, respectively. All samples of the insects used were identified and identified in advance.
其中編號SEQ ID NO:1至SEQ ID NO:6之寡核苷酸探針係利用西方花薊馬之ITS2 DNA序列設計,可用以鑑別西方花薊馬。The oligonucleotide probes numbered SEQ ID NO: 1 to SEQ ID NO: 6 were designed using the ITS2 DNA sequence of Western flower scorpion and can be used to identify western flower thrips.
編號SEQ ID NO:7至SEQ ID NO:12之寡核苷酸探針係利用蔥薊馬之ITS2 DNA序列設計,可用以鑑別蔥薊馬。Oligonucleotide probes, numbered SEQ ID NO: 7 to SEQ ID NO: 12, were designed using the ITS2 DNA sequence of Onion scorpion and can be used to identify onion thrips.
編號SEQ ID NO:13至SEQ ID NO:18之寡核苷酸探針係利用台灣花薊馬之ITS2 DNA序列設計,可用以鑑別台灣花薊馬。The oligonucleotide probes numbered SEQ ID NO: 13 to SEQ ID NO: 18 were designed using the ITS2 DNA sequence of T. chinensis, and can be used to identify T. chinensis.
編號SEQ ID NO:19至SEQ ID NO:24之寡核苷酸探針係利用菊花薊馬之ITS2 DNA序列設計,可用以鑑別菊花薊馬。The oligonucleotide probes numbered SEQ ID NO: 19 to SEQ ID NO: 24 were designed using the ITS2 DNA sequence of Chrysanthemum, which can be used to identify chrysanthemum thrips.
編號SEQ ID NO:25至SEQ ID NO:30之寡核苷酸探針係利用豆花薊馬之ITS2 DNA序列設計,可用以鑑別豆花薊馬。Oligonucleotide probes, numbered SEQ ID NO: 25 to SEQ ID NO: 30, were designed using the ITS2 DNA sequence of the Beans Thrips and can be used to identify the Beans Thrips.
編號SEQ ID NO:31至SEQ ID NO:36之寡核苷酸探針係利用尖角薊馬之ITS2 DNA序列設計,可用以鑑別尖角薊馬。Oligonucleotide probes, numbered SEQ ID NO: 31 to SEQ ID NO: 36, were designed using the ITS2 DNA sequence of Sharp-horned Thrips to identify sharp-horned thrips.
編號SEQ ID NO:37至SEQ ID NO:42之寡核苷酸探針係利 用澳洲疫薊馬之ITS2 DNA序列設計,可用以鑑別澳洲疫薊馬。Oligonucleotide probes numbered SEQ ID NO: 37 to SEQ ID NO: 42 Designed with the Australian ITS2 DNA sequence, it can be used to identify Australian plagues.
編號SEQ ID NO:43至SEQ ID NO:48之寡核苷酸探針係利用花薊馬之ITS2 DNA序列設計,可用以鑑別花薊馬。Oligonucleotide probes, numbered SEQ ID NO: 43 to SEQ ID NO: 48, were designed using the ITS2 DNA sequence of Phyllostachys pubescens and can be used to identify Thrips tabaci.
編號SEQ ID NO:49至SEQ ID NO:54之寡核苷酸探針係利用南黃薊馬之ITS2 DNA序列設計,可用以鑑別南黃薊馬。The oligonucleotide probes numbered SEQ ID NO: 49 to SEQ ID NO: 54 were designed using the ITS2 DNA sequence of the Southern Yellow Horse, which can be used to identify Southern Yellow Horse.
編號SEQ ID NO:55至SEQ ID NO:59之寡核苷酸探針係利用小黃薊馬之ITS2 DNA序列設計,可用以鑑別小黃薊馬。Oligonucleotide probes, numbered SEQ ID NO: 55 to SEQ ID NO: 59, were designed using the ITS2 DNA sequence of P. striata and can be used to identify P. striata.
將上述之具有種間特異性之寡核苷酸探針製成生物晶片,可快速鑑別薊馬。用以鑑別薊馬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、矽片或玻璃。The above-described oligonucleotide probe having interspecies specificity is made into a biochip, and the thrips can be quickly identified. The biochip for identifying the thrips comprises a substrate and an oligonucleotide probe fixed on the substrate, wherein the material of the substrate may comprise a nylon film, a polymer material, a cymbal or a glass.
生物晶片之製作方法包含將一空白晶片在45℃下烘烤約5分鐘,分別將20微莫耳(μM)濃度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℃烘烤12分鐘,最後再以紫外線交聯器(UV crosslinker)於0.8至1.0焦耳將寡核苷酸探針固定在晶片上。The method for manufacturing a biochip comprises baking a blank wafer at 45 ° C for about 5 minutes, separately adding a synthetic probe of 20 micromolar (μM) concentration to the probe solution, and then using a spotting machine. The spot was placed in the set position, baked at 45 ° C for 12 minutes, and finally the oligonucleotide probe was immobilized on the wafer at 0.8 to 1.0 Joule with a UV crosslinker.
請參照第1圖,為利用本發明之生物晶片鑑別薊馬的方法流程圖,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ITS2 DNA片段序列;(c)使用本發明之生物晶片與增幅之ITS2 DNA片段進行雜合反應;以及(d)鑑別步驟(c)之雜合反應結果,並由雜合反應之結果鑑別薊馬。Referring to Fig. 1, a flow chart of a method for identifying a thrips using the biochip of the present invention comprises (a) extracting the genetic DNA of the worm (DNA); (b) increasing the ITS2 DNA of the worm to be tested. a sequence of fragments; (c) using the biochip of the present invention to hybridize with the amplified ITS2 DNA fragment; and (d) identifying the result of the hybridization of step (c), and identifying the thrips as a result of the hybridization reaction.
依照本發明之實施例,萃取蟲體之基因體DNA是使用Protec公司所生產的純化試劑EPICENTRE® Biotechnologies Kit的溶液60μl加入放置蟲體的離心管中,震盪20秒,試管放入乾熱器內溫 度調至65℃,300rpm 反應15~20分鐘。再將試管取出震盪20秒,此次乾熱器溫度調至98℃,300rpm ,反應2分鐘,之後保存於20℃冰箱中備用,此即為供試蟲體之模板DNA來源。In accordance with embodiments of the present invention, genome DNA was extracted using a solution of parasites 60μl Protec Company produced purified EPICENTRE ® Biotechnologies Kit reagent tubes are placed in the addition of the parasite and shaken for 20 seconds into the test tube on dry heat The temperature was adjusted to 65 ° C and reacted at 300 rpm for 15 to 20 minutes. Then, the test tube was taken out and shaken for 20 seconds. The temperature of the dry heat was adjusted to 98 ° C, 300 rpm , and reacted for 2 minutes, and then stored in a refrigerator at 20 ° C for use. This is the source of template DNA for the test insect body.
利用萃取之蟲體模板DNA,並以適用於各種類薊馬的ITS2之引子對進行聚合酶連鎖反應(PCR)增幅ITS2 DNA片段。其中,使用的引子對之序列請參照SEQ IDNO:60(正向序列)與SEQ IDNO:61(反向序列)。The ITS2 DNA fragment was amplified by polymerase chain reaction (PCR) using the extracted worm template DNA and the primer pair of ITS2 suitable for various types of thrips. For the sequence of primer pairs used, please refer to SEQ ID NO: 60 (forward sequence) and SEQ ID NO: 61 (reverse sequence).
聚合酶連鎖反應係以迴溫反應器進行增幅。取得生物體ITS2 DNA之反應為:以微量離心管盛裝欲增幅的反應物,每一反應之總體積為50μl,內含4μl模板DNA、25mM dNTP混合液0.4μl、10X Taq緩衝液(含20mM MgCl2 )5μl、10μM之引子各1μl,以及1μl Taq DNA聚合酶(2U/μl)。聚合酶連鎖反應條件為95℃升溫2分鐘,之後以95℃ 40秒、45℃ 50秒、72℃ 40秒,進行35個循環,接著以72℃ 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,即為ITS2 DNA片段。回收與純化得到的ITS2 DNA片段經由核酸定序獲得各物種之ITS2 DNA序列。The polymerase chain reaction was increased in a temperature-recovery reactor. The reaction of obtaining the ITS2 DNA of the organism is as follows: the microparticles are filled with the reactants to be increased, and the total volume of each reaction is 50 μl, which contains 4 μl of template DNA, 25 μm of dNTP mixture, 0.4 μl, and 10× Taq buffer (containing 20 mM MgCl). 2 ) 1 μl of 5 μl, 10 μM primers, and 1 μl of Taq DNA polymerase (2 U/μl). The polymerase chain reaction conditions were elevated at 95 ° C for 2 minutes, followed by 35 cycles of 95 ° C for 40 seconds, 45 ° C for 50 seconds, and 72 ° C for 40 seconds, followed by completion of the reaction at 72 ° C for 10 minutes. The separation and purification of the fragment is the ITS2 DNA fragment. The ITS2 DNA fragment obtained by the recovery and purification was subjected to nucleic acid sequencing to obtain the ITS2 DNA sequence of each species.
各物種之專一性引子利用BioEdit(1999)軟體將各個分類單元的ITS2 DNA序列放在一起比對分析,並搜尋多個物種序列差異的地方設計。The specificity primers of each species use BioEdit (1999) software to put the ITS2 DNA sequences of each taxon together for alignment analysis and to search for local differences in sequence differences of multiple species.
依照本發明之一實施例,生物晶片之製備係利用含ITS2序列的DNA樣品與具有生物素(Biotin)標定的引子進行聚合酶連鎖反應,每一反應之總體積為25μl,內含2μl模板DNA、25mM dNTP混合液0.2μl、10X Taq緩衝液(含20mM MgCl2 )2.5μl、 10μM之引子各0.5μl,以及0.5μl Taq DNA聚合酶(2U/μl)。聚合酶連鎖反應條件為95℃升溫2分鐘,之後以95℃ 40秒、45℃ 50秒、72℃ 40秒,進行35個循環,接著以72℃ 10分鐘完成反應,而得到具有生物素標定的ITS2 DNA片段,即為雜合反應之標的(target)。According to an embodiment of the present invention, a biochip is prepared by a polymerase chain reaction using a DNA sample containing an ITS2 sequence and a biotin-labeled primer, each of which has a total volume of 25 μl and contains 2 μl of template DNA. 0.2 μl of a 25 mM dNTP mixture, 2.5 μl of 10X Taq buffer (containing 20 mM MgCl 2 ), 0.5 μl of each primer of 10 μM, and 0.5 μl of Taq DNA polymerase (2 U/μl). The polymerase chain reaction conditions were elevated at 95 ° C for 2 minutes, followed by 35 cycles of 95 ° C for 40 seconds, 45 ° C for 50 seconds, and 72 ° C for 40 seconds, followed by completion of the reaction at 72 ° C for 10 minutes to obtain a biotin-labeled The ITS2 DNA fragment is the target of the hybrid reaction.
將生物素標定的target DNA片段與上述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含200μl DR.HybTM Buffer之雜合反應液,再加入10μl生物素標定的target DNA片段與之混合,置於45℃環境下1小時進行雜合反應。之後倒掉雜合反應液,以200μl Wash Buffer洗去未雜合的DNA片段。以200μl Blocking Reagent混合0.2μl Strep-AP,移入晶片反應30分鐘。再倒掉反應液,以200μl Wash Buffer洗2次,以紙巾吸乾殘留液體。再加入顯色劑(4μl NBT/BCIP+196μl Detection buffer),置於暗處進行3-5分鐘呈色反應。最後用去離子水清洗晶片。以出現色斑的位置判定薊馬種類。The biotin-labeled target DNA fragment is hybridized to the oligonucleotide probe on the above-described biochip. In accordance with embodiments of the present invention, a hybrid method comprising the reaction of the wafer in each reaction vessel is added to 200μl DR.Hyb TM Buffer containing the hybrid reaction solution was added 10μl of biotin target DNA fragment is mixed with the calibration, set The hybrid reaction was carried out at 45 ° C for 1 hour. Thereafter, the hybrid reaction solution was discarded, and the unhybridized DNA fragment was washed away with 200 μl of Wash Buffer. 0.2 μl of Strep-AP was mixed with 200 μl of Blocking Reagent and transferred to the wafer for 30 minutes. The reaction solution was again poured off, washed twice with 200 μl of Wash Buffer, and the residual liquid was blotted with a paper towel. Further, a color developer (4 μl of NBT/BCIP + 196 μl Detection buffer) was added, and the reaction was carried out in the dark for 3-5 minutes. Finally, the wafer is washed with deionized water. The type of thrips is determined by the position where the stain appears.
請參照第2圖,為分別利用不同種類薊馬之ITS2 DNA片段測試本發明之生物晶片之探針的專一性結果。Referring to Figure 2, the specificity results of the probes of the biochip of the present invention were tested using ITS2 DNA fragments of different types of thrips, respectively.
第2圖(A)部分係繪示本實施例之生物晶片的寡核苷酸探針施佈示意圖。其中,每一點上所標示之數字代表序列識別編號之數字,右下方五個點為正控制組,用以確認雜合反應無誤。所示數字1至6代表序列為SEQ ID NO:1至SEQ ID NO:6之寡核苷酸探針,可與含有西方花薊馬之ITS2 DNA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。數字7至12代表序列 為SEQ ID NO:7至SEQ ID NO:12之寡核苷酸探針,可與含有蔥薊馬之ITS2 DNA序列的去氧核醣核酸樣品產生專一性雜合。數字13至18代表序列為SEQ ID NO:13至SEQ ID NO:18之寡核苷酸探針,可與含有台灣花薊馬之ITS2 DNA序列的去氧核醣核酸樣品產生專一性雜合。數字19至24代表序列為SEQ ID NO:19至SEQ ID NO:24之寡核苷酸探針,可與含有菊花薊馬之ITS2 DNA序列的去氧核醣核酸樣品產生專一性雜合。數字25至30代表序列為SEQ ID NO:25至SEQ ID NO:30之寡核苷酸探針,可與含有豆花薊馬之ITS2 DNA序列的去氧核醣核酸樣品產生專一性雜合。Fig. 2(A) is a schematic view showing the implantation of an oligonucleotide probe of the biochip of the present embodiment. Among them, the number indicated on each point represents the number of the serial identification number, and the five points on the lower right side are the positive control group to confirm that the hybrid reaction is correct. The numbers 1 to 6 shown represent oligonucleotide probes of the sequence SEQ ID NO: 1 to SEQ ID NO: 6, which can be specifically hybridized with a DNA sample containing the ITS2 DNA sequence of the western flower scorpion. And color at the position corresponding to the wafer. Numbers 7 through 12 represent sequences The oligonucleotide probes of SEQ ID NO: 7 to SEQ ID NO: 12 can be specifically hybridized with a DNA sample containing the ITS2 DNA sequence of Onion. Numerals 13 to 18 represent oligonucleotide probes of the sequence of SEQ ID NO: 13 to SEQ ID NO: 18, which are specifically hybridized to a DNA sample containing the ITS2 DNA sequence of T. chinensis. Numerals 19 to 24 represent oligonucleotide probes of the sequence of SEQ ID NO: 19 to SEQ ID NO: 24, which are specifically hybridized to a DNA sample containing the ITS2 DNA sequence of Chrysanthemum. Numerals 25 to 30 represent oligonucleotide probes of the sequence of SEQ ID NO: 25 to SEQ ID NO: 30, which can be specifically hybridized with a DNA sample containing the ITS2 DNA sequence of the bean scorpion.
第2圖(B)、(C)、(D)、(E)及(F)部分,為本實施例之探針施佈方式鑑別五種薊馬種類的生物晶片分析結果。其中(B)部分為利用西方花薊馬之ITS2 DNA片段為target DNA;(C)部分為利用蔥薊馬之ITS2 DNA片段為target DNA;(D)部分為利用台灣花薊馬之ITS2 DNA片段為target DNA;(E)部分為利用菊花薊馬之ITS2 DNA片段為target DNA;(F)部分為利用豆花薊馬之ITS2 DNA片段為target DNA。Fig. 2 (B), (C), (D), (E) and (F), the results of the biowafer analysis of the five species of thrips were identified by the probe application method of the present embodiment. Part (B) is the target DNA using the ITS2 DNA fragment of Western flower thrips; (C) is the target DNA using the ITS2 DNA fragment of the onion horse; (D) is the ITS2 DNA fragment using the Taiwan flower For the target DNA; (E) is the target DNA using the ITS2 DNA fragment of the chrysanthemum thrips; (F) is the target DNA of the ITS2 DNA fragment using the bean flower thrips.
從第2圖(B)部分的結果,對照第2圖(A)部分所示之寡核苷酸探針施佈示意圖,試驗組只有代表序列為SEQ ID NO:1至6之寡核苷酸探針之第1至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。因此SEQ ID NO:1至6之寡核苷酸探針可與含有西方花薊馬之ITS2序列的DNA樣品產生專一性雜合,可用以準確鑑別西方花薊馬。From the results of the section (B) of Fig. 2, the oligonucleotide probes shown in the section (A) of Fig. 2 were subjected to the schematic diagram, and only the oligonucleotides representing the sequences of SEQ ID NOS: 1 to 6 were detected in the test group. The spots from the first to the sixth of the probe are colored, and the control group is also colored to confirm that the hybrid reaction is correct. Thus, the oligonucleotide probes of SEQ ID NOS: 1 to 6 can be specifically heterozygous for DNA samples containing the ITS2 sequence of the western flower scorpion, and can be used to accurately identify western flower hummers.
第2圖(C)部分的結果顯示,試驗組只有代表序列為SEQ ID NO:7至12之寡核苷酸探針之第7至12號的點有呈色。因此SEQ ID NO:7至12之寡核苷酸探針可與含有蔥薊馬之ITS2序列的DNA樣品產生專一性雜合,可用以準確鑑別蔥薊馬。The results in part (C) of Fig. 2 show that the test group has only the dots of the 7th to 12th numbers representing the oligonucleotide probes of the sequences of SEQ ID NOS: 7 to 12. Therefore SEQ The ID NO: 7 to 12 oligonucleotide probe can be specifically hybridized with a DNA sample containing the ITS2 sequence of Onion scorpion, and can be used to accurately identify onion thrips.
第2圖(D)部分的結果顯示,試驗組只有代表序列為SEQ ID NO:13至18之寡核苷酸探針之第13至18號的點有呈色。因此SEQ ID NO:13至18之寡核苷酸探針可與含有台灣花薊馬之ITS2序列的DNA樣品產生專一性雜合,可用以準確鑑別台灣花薊馬。The results of the part (D) of Fig. 2 show that the test group has only the dots of the 13th to the 18th of the oligonucleotide probes of the sequences of SEQ ID NOS: 13 to 18. Therefore, the oligonucleotide probes of SEQ ID NOS: 13 to 18 can be specifically hybridized with a DNA sample containing the ITS2 sequence of T. chinensis, and can be used to accurately identify T. chinensis.
第2圖(E)部分的結果顯示,試驗組只有代表序列為SEQ ID NO:19至24之寡核苷酸探針之第19至24號的點有呈色。因此SEQ ID NO:19至24之寡核苷酸探針可與含有菊花薊馬之ITS2序列的DNA樣品產生專一性雜合,可用以準確鑑別菊花薊馬。The results in part (E) of Fig. 2 show that only the dots of the experimental group representing the 19th to 24th oligonucleotide probes of the sequences of SEQ ID NOS: 19 to 24 were colored. Thus, the oligonucleotide probes of SEQ ID NOS: 19 to 24 can be specifically hybridized to a DNA sample containing the ITS2 sequence of Chrysanthemum chinense, which can be used to accurately identify chrysanthemum thrips.
第2圖(F)部分的結果顯示,試驗組只有代表序列為SEQ ID NO:25至30之寡核苷酸探針之第25至30號的點有呈色。因此SEQ ID NO:25至30之寡核苷酸探針可與含有豆花薊馬之ITS2序列的DNA樣品產生專一性雜合,可用以準確鑑別豆花薊馬。The results in part (F) of Fig. 2 show that the test group has only the color of the dots 25 to 30 representing the oligonucleotide probes of the sequences of SEQ ID NOS: 25 to 30. Therefore, the oligonucleotide probes of SEQ ID NOS: 25 to 30 can be specifically hybridized with a DNA sample containing the ITS2 sequence of the Beans, and can be used to accurately identify the humus thrips.
第2圖(G)部分係繪示本實施例之生物晶片的寡核苷酸探針施佈示意圖。其中,每一點上所標示之數字代表序列識別編號之數字,右下方五個點為正控制組,用以確認雜合反應無誤。所示數字31至36代表序列為SEQ ID NO:31至SEQ ID NO:36之寡核苷酸探針,可與含有尖角薊馬之ITS2 DNA序列的去氧核醣核酸樣品產生專一性雜合。數字37至42代表序列為SEQ ID NO:37至SEQ ID NO:42之寡核苷酸探針,可與含有澳洲疫薊馬之ITS2 DNA序列的去氧核醣核酸樣品產生專一性雜合。數字43至48代表序列為SEQ ID NO:43至SEQ ID NO:48之寡核苷酸探針,可與含有花薊馬之ITS2 DNA序列的去氧核醣核酸樣品產生專一性 雜合。數字49至54代表序列為SEQ ID NO:49至SEQ ID NO:54之寡核苷酸探針,可與含有南黃薊馬之ITS2 DNA序列的去氧核醣核酸樣品產生專一性雜合。數字55至59代表序列為SEQ ID NO:54至SEQ ID NO:59之寡核苷酸探針,可與含有小黃薊馬之ITS2 DNA序列的去氧核醣核酸樣品產生專一性雜合。Fig. 2(G) is a schematic view showing the application of the oligonucleotide probe of the biochip of the present embodiment. Among them, the number indicated on each point represents the number of the serial identification number, and the five points on the lower right side are the positive control group to confirm that the hybrid reaction is correct. The numbers 31 to 36 represent the oligonucleotide probes of SEQ ID NO: 31 to SEQ ID NO: 36, which can be specifically hybridized with a DNA sample containing the ITS2 DNA sequence of the scorpion scorpion. . Numerals 37 to 42 represent oligonucleotide probes of SEQ ID NO: 37 to SEQ ID NO: 42 which are specifically hybridized to a DNA sample containing the ITS2 DNA sequence of the Australian plague. Numerals 43 to 48 represent oligonucleotide probes of the sequence of SEQ ID NO: 43 to SEQ ID NO: 48, which are specific for DNA samples containing the ITS2 DNA sequence of the flower bud horse. mixed. Numerals 49 to 54 represent oligonucleotide probes of the sequence of SEQ ID NO: 49 to SEQ ID NO: 54 which are specifically hybridized to a DNA sample containing the ITS2 DNA sequence of the Southern Scutellaria. Numerals 55 to 59 represent oligonucleotide probes of the sequence of SEQ ID NO: 54 to SEQ ID NO: 59, which can be specifically hybridized with a DNA sample containing the ITS2 DNA sequence of the small yellow horse.
第2圖(H)、(I)、(J)、(K)及(L)部分,為本實施例之探針施佈方式鑑別五種薊馬種類的生物晶片分析結果。其中(H)部分為利用尖角薊馬之ITS2 DNA片段為target DNA;(I)部分為利用澳洲疫薊馬之ITS2 DNA片段為target DNA;(J)部分為利用花薊馬之ITS2 DNA片段為target DNA;(K)部分為利用南黃薊馬之ITS2 DNA片段為target DNA;(L)部分為利用小黃薊馬之ITS2 DNA片段為target DNA。Fig. 2 (H), (I), (J), (K) and (L), the results of biofilm analysis of the five species of thrips were identified by the probe application method of the present embodiment. The (H) part is the target DNA using the ITS2 DNA fragment of the sharp-pointed thrips; (I) is the target DNA using the ITS2 DNA fragment of the Australian plague horse; (J) is the ITS2 DNA fragment using the flower scorpion horse. For the target DNA; (K) is the target DNA using the ITS2 DNA fragment of the southern yellow pheasant; (L) is the target DNA using the ITS2 DNA fragment of the small yellow pheasant.
從第2圖(H)部分的結果顯示,試驗組只有代表序列為SEQ ID NO:31至36之寡核苷酸探針之第31至36號的點有呈色。因此SEQ ID NO:31至36之寡核苷酸探針可與含有尖角薊馬之ITS2序列的DNA樣品產生專一性雜合,可用以準確鑑別尖角薊馬。From the results of the section (H) of Fig. 2, it was revealed that only the dots 31 to 36 representing the oligonucleotide probes of the sequences of SEQ ID NOS: 31 to 36 were colored. Thus, the oligonucleotide probes of SEQ ID NOS: 31 to 36 can be specifically hybridized to a DNA sample containing the ITS2 sequence of the scorpion scorpion, which can be used to accurately identify sharp-horned hummers.
第2圖(I)部分的結果顯示,試驗組只有代表序列為SEQ ID NO:37至42之寡核苷酸探針之第37至42號的點有呈色。因此SEQ ID NO:37至42之寡核苷酸探針可與含有澳洲疫薊馬之ITS2序列的DNA樣品產生專一性雜合,可用以準確鑑別澳洲疫薊馬。The results of the section (I) of Fig. 2 show that the test group has only the dots of the 37th to 42nd points representing the oligonucleotide probes of the sequences of SEQ ID NOS: 37 to 42. Thus, the oligonucleotide probes of SEQ ID NOS: 37 to 42 can be specifically heterozygous for DNA samples containing the ITS2 sequence of the Australian plague, and can be used to accurately identify the Australian plague.
第2圖(J)部分的結果顯示,試驗組只有代表序列為SEQ ID NO:43至48之寡核苷酸探針之第43至48號的點有呈色。因此SEQ ID NO:43至48之寡核苷酸探針可與含有花薊馬之ITS2序列的DNA樣品產生專一性雜合,可用以準確鑑別花薊馬。The results of the section (J) of Fig. 2 show that the test group has only the color of the dots 43 to 48 representing the oligonucleotide probes of the sequences of SEQ ID NOS: 43 to 48. Thus, the oligonucleotide probes of SEQ ID NOS: 43 to 48 can be specifically hybridized to a DNA sample containing the ITS2 sequence of the hibiscus, which can be used to accurately identify the scorpion horse.
第2圖(K)部分的結果顯示,試驗組只有代表序列為SEQ ID NO:49至54之寡核苷酸探針之第49至54號的點有呈色。因此SEQ ID NO:49至54之寡核苷酸探針可與含有南黃薊馬之ITS2序列的DNA樣品產生專一性雜合,可用以準確鑑別南黃薊馬。The results of the section (K) of Fig. 2 show that only the dots of the experimental group representing the oligonucleotide probes of the sequences of SEQ ID NOS: 49 to 54 are colored. Therefore, the oligonucleotide probes of SEQ ID NOS: 49 to 54 can be specifically hybridized with a DNA sample containing the ITS2 sequence of the southern scorpion horse, and can be used to accurately identify the southern scorpion horse.
第2圖(L)部分的結果顯示,試驗組只有代表序列為SEQ ID NO:55至59之寡核苷酸探針之第55至59號的點有呈色。因此SEQ ID NO:55至59之寡核苷酸探針可與含有小黃薊馬之ITS2序列的DNA樣品產生專一性雜合,可用以準確鑑別小黃薊馬。The results in part (L) of Fig. 2 show that the test group has only the color of the dots representing the 55th to 59th of the oligonucleotide probes of the sequences of SEQ ID NOS: 55 to 59. Therefore, the oligonucleotide probes of SEQ ID NOS: 55 to 59 can be specifically hybridized with a DNA sample containing the ITS2 sequence of P. striata, and can be used to accurately identify P. striata.
根據第2圖的結果,可知本發明實施例所揭露方法及具有SEQ ID NO:1至SEQ ID NO:59所示序列之寡核甘酸探針,能使用具SEQ IDNO:60與SEQ IDNO:61序列之引子對待測蟲體之模板DNA進行聚合酶連鎖反應增幅出ITS2的DNA片段,在同一晶片上同時鑑別西方花薊馬、蔥薊馬、台灣花薊馬、菊花薊馬、豆花薊馬、尖角薊馬、澳洲疫薊馬、南黃薊馬及小黃薊馬等10種薊馬。因此,本發明實施例之寡核甘酸探針具有高專一性的特點,製成生物晶片能快速且正確的鑑別多種薊馬。According to the results of FIG. 2, the method disclosed in the examples of the present invention and the oligonucleotide probe having the sequence of SEQ ID NO: 1 to SEQ ID NO: 59 can be used to enable SEQ ID NO: 60 and SEQ ID NO: 61. The primer of the sequence is subjected to polymerase chain reaction of the template DNA of the test body to increase the DNA fragment of ITS2, and simultaneously identify the western flower thrip horse, the onion thrip horse, the Taiwan flower thrip horse, the chrysanthemum thrip horse, the bean flower thrip horse on the same wafer. There are 10 kinds of thrips such as sharp-horned horses, Australian plague horses, southern yellow horses and small yellow horses. Therefore, the oligonucleotide probe of the embodiment of the invention has the characteristics of high specificity, and the biochip can quickly and correctly identify a variety of thrips.
雖然本發明已以實施方式揭露如上,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內,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。Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention, and the present invention can be modified and modified without departing from the spirit and scope of the present invention. The scope is subject to the definition of the scope of the patent application attached.
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,所附圖式之說明如下:The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.
第1圖為利用本發明之生物晶片鑑別薊馬的方法流程圖。Figure 1 is a flow chart of a method for identifying a thrips using the biochip of the present invention.
第2圖為分別利用不同種類薊馬之ITS2 DNA片段測試本發明之生物晶片之探針的專一性結果。Figure 2 is a graph showing the specificity of the probes of the biochip of the present invention using the ITS2 DNA fragments of different species of thrips, respectively.
<110> 國立中興大學<110> National Chung Hsing University
<120> 鑑別薊馬之寡核苷酸探針、生物晶片及其鑑別方法<120> Identification of oligonucleotide probes, biochips and identification methods thereof
<160> 61<160> 61
<210> SEQ ID NO:1<210> SEQ ID NO: 1
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別西方花薊馬(Frankliniella occidentalis )之寡核苷酸探針<223> Identification of thrips (Frankliniella occidentalis) of an oligonucleotide probe
<400> 1 <400> 1
<210> SEQ ID NO:2<210> SEQ ID NO: 2
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別西方花薊馬(Frankliniella occidentalis )之寡核苷酸探針<223> Identification of oligonucleotide probes of Frankliniella occidentalis
<400> 2 <400> 2
<210> SEQ ID NO:3<210> SEQ ID NO: 3
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別西方花薊馬(Frankliniella occidentalis )之寡核苷酸探針<223> Identification of oligonucleotide probes of Frankliniella occidentalis
<400> 3 <400> 3
<210> SEQ ID NO:4<210> SEQ ID NO: 4
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別西方花薊馬(Frankliniella occidentalis )之寡核苷酸探針<223> Identification of oligonucleotide probes of Frankliniella occidentalis
<400> 4 <400> 4
<210> SEQ ID NO:5<210> SEQ ID NO: 5
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別西方花薊馬(Frankliniella occidentalis )之寡核苷酸探針<223> Identification of oligonucleotide probes of Frankliniella occidentalis
<400> 5 <400> 5
<210> SEQ ID NO:6<210> SEQ ID NO: 6
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別西方花薊馬(Frankliniella occidentalis )之寡核苷酸探針<223> Identification of oligonucleotide probes of Frankliniella occidentalis
<400> 6 <400> 6
<210> SEQ ID NO:7<210> SEQ ID NO: 7
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別蔥薊馬(Thrips tabaci )之寡核苷酸探針<223> Identification of oligonucleotide probes for Thrips tabaci
<400> 7 <400> 7
<210> SEQ ID NO:8<210> SEQ ID NO: 8
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別蔥薊馬(Thrips tabaci )之寡核苷酸探針<223> Identification of oligonucleotide probes for Thrips tabaci
<400> 8 <400> 8
<210> SEQ ID NO:9<210> SEQ ID NO: 9
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別蔥薊馬(Thrips tabaci )之寡核苷酸探針<223> Identification of oligonucleotide probes for Thrips tabaci
<400> 9 <400> 9
<210> SEQ ID NO:10<210> SEQ ID NO: 10
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別蔥薊馬(Thrips tabaci )之寡核苷酸探針<223> Identification of oligonucleotide probes for Thrips tabaci
<400> 10 <400> 10
<210> SEQ ID NO:11<210> SEQ ID NO: 11
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別蔥薊馬(Thrips tabaci )之寡核苷酸探針<223> Identification of oligonucleotide probes for Thrips tabaci
<400> 11 <400> 11
<210> SEQ ID NO:12<210> SEQ ID NO: 12
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別蔥薊馬(Thrips tabaci )之寡核苷酸探針<223> Identification of oligonucleotide probes for Thrips tabaci
<400> 12 <400> 12
<210> SEQ ID NO:13<210> SEQ ID NO: 13
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別台灣花薊馬(Frankliniella intonsa )之寡核苷酸探針<223> Identification of oligonucleotide probes from Frankliniella intonsa
<400> 13 <400> 13
<210> SEQ ID NO:14<210> SEQ ID NO: 14
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別台灣花薊馬(Frankliniella intonsa )之寡核苷酸探針<223> Identification of oligonucleotide probes from Frankliniella intonsa
<400> 14 <400> 14
<210> SEQ ID NO:15<210> SEQ ID NO: 15
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別台灣花薊馬(Frankliniella intonsa )之寡核苷酸探針<223> Identification of oligonucleotide probes from Frankliniella intonsa
<400> 15 <400> 15
<210> SEQ ID NO:16<210> SEQ ID NO: 16
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別台灣花薊馬(Frankliniella intonsa )之寡核苷酸探針<223> Identification of oligonucleotide probes from Frankliniella intonsa
<400> 16 <400> 16
<210> SEQ ID NO:17<210> SEQ ID NO: 17
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別台灣花薊馬(Frankliniella intonsa )之寡核苷酸探針<223> Identification of oligonucleotide probes from Frankliniella intonsa
<400> 17 <400> 17
<210> SEQ ID NO:18<210> SEQ ID NO: 18
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別台灣花薊馬(Frankliniella intonsa )之寡核苷酸探針<223> Identification of oligonucleotide probes from Frankliniella intonsa
<400> 18 <400> 18
<210> SEQ ID NO:19<210> SEQ ID NO: 19
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別菊花薊馬(Mircocephalothrips abdominalis )之寡核苷酸探針<223> Identification of oligonucleotide probes for Mircocephalothrips abdominalis
<400> 19 <400> 19
<210> SEQ ID NO:20<210> SEQ ID NO: 20
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別菊花薊馬(Mircocephalothrips abdominalis )之寡核苷酸探針<223> Identification of oligonucleotide probes for Mircocephalothrips abdominalis
<400> 20 <400> 20
<210> SEQ ID NO:21<210> SEQ ID NO: 21
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別菊花薊馬(Mircocephalothrips abdominalis )之寡核苷酸探針<223> Identification of oligonucleotide probes for Mircocephalothrips abdominalis
<400> 21 <400> 21
<210> SEQ ID NO:22<210> SEQ ID NO: 22
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別菊花薊馬(Mircocephalothrips abdominalis )之寡核苷酸探針<223> Identification of oligonucleotide probes for Mircocephalothrips abdominalis
<400> 22 <400> 22
<210> SEQ ID NO:23<210> SEQ ID NO: 23
<211> 22<211> 22
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別菊花薊馬(Mircocephalothrips abdominalis )之寡核苷酸探針<223> Identification of oligonucleotide probes for Mircocephalothrips abdominalis
<400> 23 <400> 23
<210> SEQ ID NO:24<210> SEQ ID NO: 24
<211> 22<211> 22
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別菊花薊馬(Mircocephalothrips abdominalis )之寡核苷酸探針<223> Chrysanthemum Thrips discrimination (Mircocephalothrips abdominalis) of an oligonucleotide probe
<400> 24 <400> 24
<210> SEQ ID NO:25<210> SEQ ID NO: 25
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別豆花薊馬(Magalurothrips usitatus )之寡核苷酸探針<223> Identification of oligonucleotide probes for Magalurothrips usitatus
<400> 25 <400> 25
<210> SEQ ID NO:26<210> SEQ ID NO:26
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別豆花薊馬(Magalurothrips usitatus )之寡核苷酸探針<223> Identification of oligonucleotide probes for Magalurothrips usitatus
<400> 26 <400> 26
<210> SEQ ID NO:27<210> SEQ ID NO:27
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別豆花薊馬(Magalurothrips usitatus )之寡核苷酸探針<223> Identification of oligonucleotide probes for Magalurothrips usitatus
<400> 27 <400> 27
<210> SEQ ID NO:28<210> SEQ ID NO: 28
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別豆花薊馬(Magalurothrips usitatus )之寡核苷酸探針<223> Identification of oligonucleotide probes for Magalurothrips usitatus
<400> 28 <400> 28
<210> SEQ ID NO:29<210> SEQ ID NO: 29
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別豆花薊馬(Magalurothrips usitatus )之寡核苷酸探針<223> Identification of oligonucleotide probes for Magalurothrips usitatus
<400> 29 <400> 29
<210> SEQ ID NO:30<210> SEQ ID NO: 30
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別豆花薊馬(Magalurothrips usitatu s)之寡核苷酸探針<223> Identification of oligonucleotide probes for Magalurothrips usitatu s
<400> 30 <400> 30
<210> SEQ ID NO:31<210> SEQ ID NO: 31
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別尖角薊馬(Frankliniella cephalica )之寡核苷酸探針<223> Identification of Oligonucleotide Probes for Frankliniella cephalica
<400> 31 <400> 31
<210> SEQ ID NO:32<210> SEQ ID NO: 32
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別尖角薊馬(Frankliniella cephalica )之寡核苷酸探針<223> Identification of Oligonucleotide Probes for Frankliniella cephalica
<400> 32 <400> 32
<210> SEQ ID NO:33<210> SEQ ID NO: 33
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別尖角薊馬(Frankliniella cephalica )之寡核苷酸探針<223> Identification of Oligonucleotide Probes for Frankliniella cephalica
<400> 33 <400> 33
<210> SEQ ID NO:34<210> SEQ ID NO: 34
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別尖角薊馬(Frankliniella cephalica )之寡核苷酸探針<223> Identification of Oligonucleotide Probes for Frankliniella cephalica
<400> 34 <400> 34
<210> SEQ ID NO:35<210> SEQ ID NO: 35
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別尖角薊馬(Frankliniella cephalica )之寡核苷酸探針<223> Identification of Oligonucleotide Probes for Frankliniella cephalica
<400> 35 <400> 35
<210> SEQ ID NO:36<210> SEQ ID NO: 36
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別尖角薊馬(Frankliniella cephalica )之寡核苷酸探針<223> Identification of Oligonucleotide Probes for Frankliniella cephalica
<400> 36 <400> 36
<210> SEQ ID NO:37<210> SEQ ID NO: 37
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別澳洲疫薊馬(Thrips imaginis )之寡核苷酸探針<223> Identification of oligonucleotide probes for the Australian Thrips imaginis
<400> 37 <400> 37
<210> SEQ ID NO:38<210> SEQ ID NO: 38
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別澳洲疫薊馬(Thrips imaginis )之寡核苷酸探針<223> Identification of oligonucleotide probes for the Australian Thrips imaginis
<400> 38 <400> 38
<210> SEQ ID NO:39<210> SEQ ID NO: 39
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別澳洲疫薊馬(Thrips imaginis )之寡核苷酸探針<223> Identification of Australian plague thrips (Thrips imaginis) of an oligonucleotide probe
<400> 39 <400> 39
<210> SEQ ID NO:40<210> SEQ ID NO: 40
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別澳洲疫薊馬(Thrips imaginis )之寡核苷酸探針<223> Identification of oligonucleotide probes for the Australian Thrips imaginis
<400> 40 <400> 40
<210> SEQ ID NO:41<210> SEQ ID NO: 41
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別澳洲疫薊馬(Thrips imaginis )之寡核苷酸探針<223> Identification of oligonucleotide probes for the Australian Thrips imaginis
<400> 41 <400> 41
<210> SEQ ID NO:42<210> SEQ ID NO: 42
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別澳洲疫薊馬(Thrips imaginis )之寡核苷酸探針<223> Identification of oligonucleotide probes for the Australian Thrips imaginis
<400> 42 <400> 42
<210> SEQ ID NO:43<210> SEQ ID NO: 43
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別花薊馬(Thrips hawaiiensis )之寡核苷酸探針<223> Identification of oligonucleotide probes of Thrips hawaiiensis
<400> 43 <400> 43
<210> SEQ ID NO:44<210> SEQ ID NO: 44
<211> 18<211> 18
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別花薊馬(Thrips hawaiiensis )之寡核苷酸探針<223> Identification of oligonucleotide probes of Thrips hawaiiensis
<400> 44 <400> 44
<210> SEQ ID NO:45<210> SEQ ID NO: 45
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別花薊馬(Thrips hawaiiensis )之寡核苷酸探針<223> Identification of oligonucleotide probes of Thrips hawaiiensis
<400> 45 <400> 45
<210> SEQ ID NO:46<210> SEQ ID NO: 46
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別花薊馬(Thrips hawaiiensis )之寡核苷酸探針<223> Identification of oligonucleotide probes of Thrips hawaiiensis
<400> 46 <400> 46
<210> SEQ ID NO:47<210> SEQ ID NO: 47
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別花薊馬(Thrips hawaiiensis )之寡核苷酸探針<223> Identification of oligonucleotide probes of Thrips hawaiiensis
<400> 47 <400> 47
<210> SEQ ID NO:48<210> SEQ ID NO: 48
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別花薊馬(Thrips hawaiiensis )之寡核苷酸探針<223> Identification flower thrips (Thrips hawaiiensis) of an oligonucleotide probe
<400> 48 <400> 48
<210> SEQ ID NO:49<210> SEQ ID NO: 49
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別南黃薊馬(Thrips palmi )之寡核苷酸探針<223> Identification of oligonucleotide probes for Thrips palmi
<400> 49 <400> 49
<210> SEQ ID NO:50<210> SEQ ID NO: 50
<211> 18<211> 18
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別南黃薊馬(Thrips palmi )之寡核苷酸探針<223> Identification of oligonucleotide probes for Thrips palmi
<400> 50 <400> 50
<210> SEQ ID NO:51<210> SEQ ID NO: 51
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別南黃薊馬(Thrips palmi )之寡核苷酸探針<223> Identification of oligonucleotide probes for Thrips palmi
<400> 50 <400> 50
<210> SEQ ID NO:51<210> SEQ ID NO: 51
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別南黃薊馬(Thrips palmi )之寡核苷酸探針<223> Identification of oligonucleotide probes for Thrips palmi
<400> 50 <400> 50
<210> SEQ ID NO:51<210> SEQ ID NO: 51
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別南黃薊馬(Thrips palmi )之寡核苷酸探針<223> Identification of oligonucleotide probes for Thrips palmi
<400> 51 <400> 51
<210> SEQ ID NO:52<210> SEQ ID NO: 52
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別南黃薊馬(Thrips palmi )之寡核苷酸探針<223> Identification of oligonucleotide probes for Thrips palmi
<400> 52 <400> 52
<210> SEQ ID NO:53<210> SEQ ID NO: 53
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別南黃薊馬(Thrips palmi )之寡核苷酸探針<223> Identification of oligonucleotide probes for Thrips palmi
<400> 53 <400> 53
<210> SEQ ID NO:54<210> SEQ ID NO: 54
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別南黃薊馬(Thrips palmi )之寡核苷酸探針<223> Identification of oligonucleotide probes for Thrips palmi
<400> 54 <400> 54
<210> SEQ ID NO:55<210> SEQ ID NO: 55
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別小黃薊馬(Scirtothrips dorsalis ;Sdor)之寡核苷酸探針<223> Identification of small yellow thrips (Scirtothrips dorsalis; Sdor) of an oligonucleotide probe
<400> 56 <400> 56
<210> SEQ ID NO:56<210> SEQ ID NO: 56
<211> 21<211> 21
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別小黃薊馬(Scirtothrips dorsalis ;Sdor)之寡核苷酸探針<223> Identification of oligonucleotide probes for Scirtothrips dorsalis ; Sdor
<400> 56 <400> 56
<210> SEQ ID NO:57<210> SEQ ID NO: 57
<211> 28<211> 28
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別小黃薊馬(Scirtothrips dorsalis ;Sdor)之寡核苷酸探針<223> Identification of oligonucleotide probes for Scirtothrips dorsalis ; Sdor
<400> 56 <400> 56
<210> SEQ ID NO:58<210> SEQ ID NO: 58
<211> 24<211> 24
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別小黃薊馬(Scirtothrips dorsalis ;Sdor)之寡核苷酸探針<223> Identification of oligonucleotide probes for Scirtothrips dorsalis ; Sdor
<400> 24 <400> 24
<210> SEQ ID NO:59<210> SEQ ID NO: 59
<211> 24<211> 24
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 鑑別小黃薊馬(Scirtothrips dorsalis ;Sdor)之寡核苷酸探針<223> Identification of oligonucleotide probes for Scirtothrips dorsalis ; Sdor
<400> 24 <400> 24
<210> SEQ ID NO:60<210> SEQ ID NO: 60
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220> Primer bind<220> Primer bind
<223> 用於增幅薊馬ITS2區域之專一性引子(正向序列)<223> Specificity primer (forward sequence) for increasing the ITS2 region of Hummer
<400> 60 <400> 60
<210> SEQ ID NO:61<210> SEQ ID NO: 61
<211> 17<211> 17
<212> DNA<212> DNA
<213> 人工序列<213> Artificial sequence
<220> Primer bind<220> Primer bind
<223> 用於增幅薊馬ITS2區域之專一性引子(反向序列)<223> A specific primer for the amplification of the ITS2 region of Hummer (reverse sequence)
<400> 61 <400> 61
Claims (7)
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TW101118715A TWI449787B (en) | 2012-05-25 | 2012-05-25 | Oligo-nucleotide probes of thrips identification, biochip, and identifying method thereof |
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
TWI335432B (en) * | 2007-11-28 | 2011-01-01 | Univ Nat Chunghsing | Oligo-nucleotide probes of aphids identification, biochip, and identifying method thereof |
TW201114909A (en) * | 2009-10-21 | 2011-05-01 | Univ Nat Chunghsing | Primers and kit of thrips identification and identification method for the same |
-
2012
- 2012-05-25 TW TW101118715A patent/TWI449787B/en not_active IP Right Cessation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
TWI335432B (en) * | 2007-11-28 | 2011-01-01 | Univ Nat Chunghsing | Oligo-nucleotide probes of aphids identification, biochip, and identifying method thereof |
TW201114909A (en) * | 2009-10-21 | 2011-05-01 | Univ Nat Chunghsing | Primers and kit of thrips identification and identification method for the same |
Non-Patent Citations (3)
Title |
---|
、K. S. Huang, S. E. Lee, Y. Yeh, G. S. Shen, E. Mei and C. M. Chang," Taqman real-time quantitative PCR for identification of western flower thrip ( Frankliniella occidentalis) for plant Taqman real-time quantitative PCR for identification of quarantine", Biol. Lett.,2010,page 1-3 * |
國際植物檢疫措施標準,"棕梠薊馬診斷規程草案",2007年5月 * |
農業生物技術國家型科技計畫辦公室,"農業生物技術國家型科技計畫 重要農業有害薊馬分子檢測系統之開發",2009,page 1-373 * |
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