TW201348443A - Oligo-nucleotide probes of thrips identification, biochip, and identifying method thereof - Google Patents

Abstract

Oligo-nucleotide probes of thrips identification, a biochip, and the identify method thereof are disclosed. The present invention includes a method of employing a plurality of species-specific probes to identify the thrip of different species and a biochip spotted with the species-specific probes.

Description

Oligonucleotide probe for identifying thrips, biochip and identification method thereof

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.

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.

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.

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.

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.

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.

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.

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 .

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.

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.

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.

In the embodiment of the present invention, Frankliniella occidentalis , Thrips tabaci , Frankliniella intonsa , Mircocephalothrips abdominalis , Megalurothrips usitatus , and combs are utilized . Iris2 of different species such as Framkliniella schultzei , Thrips imaginis , Thrips hawaiiensis , Thrips palmi , Scirtothrips dorsalis Oligonucleotide probes were designed for DNA sequences and probe specificity tests 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.

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.

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.

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.

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.

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.

Oligonucleotide probes, numbered SEQ ID NO: 31 to SEQ ID NO: 36, were designed using the ITS2 DNA sequence of Brassica chinensis, which can be used to identify comb-necked thrips.

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.

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.

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.

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.

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.

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.

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.

In accordance with embodiments of the present invention, the worm genome DNA was extracted using a solution of 60 μl Protec Company produced purified EPICENTRE ^® Biotechnologies Kit reagent tubes are placed in the addition of the parasite and shaken for 20 seconds, the tubes in the dry heat The internal 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.

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).

The polymerase chain reaction was increased in a temperature-recovery reactor. The reaction of obtaining the ITS2 DNA of the organism is as follows: a microcentrifuge tube is used to hold the reactants to be increased, and the total volume of each reaction is 50 μl, which contains 4 μl of template DNA, a mixture of 25 mM dNTPs, 0.4 μl, and 10X Taq buffer ( 1 μl each containing 20 μM MgCl ₂ ) 5 μl, 10 μM, and 1 μl 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.

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.

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 having a total volume of 25 μl and containing 2 μl. Template DNA, 0.2 μl of 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.

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 method of mixing the reaction hybrid of each wafer contained in the first reaction vessel was added 200 μl DR.Hyb ^TM Buffer containing hybrid of the reaction solution was added 10 μl of biotin calibration target DNA fragment with The mixture was subjected to a heterozygous reaction at room temperature of 45 ° C for 1 hour. After that, 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. Add a color developer (4 μl NBT/BCIP+196 μl Detection buffer) and place it 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.

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.

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.

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.

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.

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.

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.

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.

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.

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 shown represent oligonucleotide probes of the sequence of SEQ ID NO: 31 to SEQ ID NO: 36, which are capable of generating specificity with a DNA sample containing the ITS2 DNA sequence of the scutellaria Hehe. 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.

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 scorpion scorpion; (I) is the target DNA of the ITS2 DNA fragment of the Australian plague horse; (J) is the ITS2 DNA of the genus The fragment is target DNA; the (K) part is the target DNA using the ITS2 DNA fragment of the southern yellow horse; the (L) part is the target DNA using the ITS2 DNA fragment of the small yellow horse.

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. Therefore, the oligonucleotide probes of SEQ ID NOS: 31 to 36 can be specifically hybridized with a DNA sample containing the ITS2 sequence of the scutellaria, and can be used to accurately identify the scorpion scorpion.

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.

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. Therefore, the oligonucleotide probes of SEQ ID NOS: 41 to 47 can be combined with the ITS2 sequence containing the flower scorpion horse. The DNA samples of the column produce a specific heterozygosity that can be used to accurately identify the flower thrips.

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.

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.

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 species of hummers, such as the scorpion scorpion, the Australian scorpion scorpion horse, the southern yellow scorpion horse and the small yellow scorpion horse. 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.

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.

The above and other objects, features, advantages and embodiments of the present invention will become more apparent. It is obvious and easy to understand. The description of the drawings is as follows: Fig. 1 is a flow chart of a method for identifying a thrips using the biochip of the present invention.

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> National Chung Hsing University

<120> Identification of oligonucleotide probes, biochips and identification methods thereof

<160> 61

<210> SEQ ID NO: 1

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Frankliniella occidentalis

<400> 1 GCTTCACCGC TTCCCCCGTA A

<210> SEQ ID NO: 2

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Frankliniella occidentalis

<400> 2 TCAGGGTGGT CGCTTCACC

<210> SEQ ID NO: 3

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Frankliniella occidentalis

<400> 3 CAAAGAGCAA CCGCGCAGTT

<210> SEQ ID NO: 4

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Frankliniella occidentalis

<400> 4 TCGCTTCACC GCTTCCCCC

<210> SEQ ID NO: 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Frankliniella occidentalis

<400> 5 AGTGCGAGAA AATAATGCAA

<210> SEQ ID NO: 6

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Frankliniella occidentalis

<400> 6 CTTTCTCTTT ACGGGGGAAG C

<210> SEQ ID NO: 7

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Thrips tabaci

<400> 7 CGATTACCAG ACTGCCCAA

<210> SEQ ID NO: 8

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Thrips tabaci

<400> 8 CACCTCGTGT GTGTTGGAGC

<210> SEQ ID NO: 9

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Thrips tabaci

<400> 9 ACGATTACCA GACTGCCCAA

<210> SEQ ID NO: 10

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Thrips tabaci

<400> 10 CAAGCGACAG CACACACCT

<210> SEQ ID NO: 11

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Thrips tabaci

<400> 11 AGTAAGACTG AGAGAAGCC

<210> SEQ ID NO: 12

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Thrips tabaci

<400> 12 CAGCACAACA CATTCCACTC

<210> SEQ ID NO: 13

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes from Frankliniella intonsa

<400> 13 TTCCGAGAGA AATCTCTGG

<210> SEQ ID NO: 14

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes from Frankliniella intonsa

<400> 14 AGACTGTTCC GAGAGAAATC

<210> SEQ ID NO: 15

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes from Frankliniella intonsa

<400> 15 ACTGTTCCGA GAGAAATCTC

<210> SEQ ID NO: 16

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes from Frankliniella intonsa

<400> 16 CCTCGCTCCA GAGATTTCT

<210> SEQ ID NO: 17

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes from Frankliniella intonsa

<400> 17 TTTTGTTGCA CACTTTTCCG

<210> SEQ ID NO: 18

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes from Frankliniella intonsa

<400> 18 CGCTTTGTAC CGGCAGAGT

<210> SEQ ID NO: 19

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Mircocephalothrips abdominalis

<400> 19 TTGTGTGTTT GGCCTCGGCC

<210> SEQ ID NO: 20

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Mircocephalothrips abdominalis

<400> 20 TTTGGCCTCG GCCTGCACG

<210> SEQ ID NO: 21

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Mircocephalothrips abdominalis

<400> 21 TGCACGCTCG GCGAGCTTG

<210> SEQ ID NO: 22

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Mircocephalothrips abdominalis

<400> 22 CGCTCCTTCG GGAGGGGTCC T

<210> SEQ ID NO: 23

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Mircocephalothrips abdominalis

<400> 23 GGTCCTCTTA AATGCCTCAA AA

<210> SEQ ID NO: 24

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Mircocephalothrips abdominalis

<400> 24 ACGCAGCAAA CACTTATCAC

<210> SEQ ID NO: 25

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Magalurothrips usitatus

<400> 25 CAGAAGAGCT AGTGTCGCAT

<210> SEQ ID NO:26

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Magalurothrips usitatus

<400> 26 TCAGCGCGTT GGAGTTTTC

<210> SEQ ID NO:27

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Magalurothrips usitatus

<400> 27 GTGAGTCCCC TCCCTAGTAA

<210> SEQ ID NO: 28

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Magalurothrips usitatus

<400> 28 AGGGTGACAG ACAAACTTGA A

<210> SEQ ID NO: 29

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Magalurothrips usitatus

<400> 29 TGGGAACGTC GTGCGAATTC

<210> SEQ ID NO: 30

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Magalurothrips usitatus

<400> 30 TAAGACAACA GTGTGTATCG

<210> SEQ ID NO: 31

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of Oligonucleotide Probes of Frankliniella schultzei

<400> 31 GTCTTCGCGA CAGAGCGAG

<210> SEQ ID NO: 32

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of Oligonucleotide Probes of Frankliniella schultzei

<400> 32 GCACTCCGCA CTATTTGTG

<210> SEQ ID NO: 33

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of Oligonucleotide Probes of Frankliniella schultzei

<400> 33 GATTGCGAGA TATGCACGG

<210> SEQ ID NO: 34

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of Oligonucleotide Probes of Frankliniella schultzei

<400> 34 TGCACGGGTC CGCTTGCGGT

<210> SEQ ID NO: 35

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of Oligonucleotide Probes of Frankliniella schultzei

<400> 35 GTCTTCAAGA CGGGAAGCAT

<210> SEQ ID NO: 36

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of Oligonucleotide Probes of Frankliniella schultzei

<400> 36 TGAGGCGAAA CGCTGGATTG

<210> SEQ ID NO: 37

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for the Australian Thrips imaginis

<400> 37 TCGGTTGAGG GTTGGTCATG

<210> SEQ ID NO: 38

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for the Australian Thrips imaginis

<400> 38 TATCACCAAA CTGCGTGTCA T

<210> SEQ ID NO: 39

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for the Australian Thrips imaginis

<400> 39 GTTGGTCATG GAGATATCA

<210> SEQ ID NO: 40

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for the Australian Thrips imaginis

<400> 40 ACCAAACTGC GTGTCATGGA

<210> SEQ ID NO: 41

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for the Australian Thrips imaginis

<400> 41 TCACCAAACT GCGTGTCATG G

<210> SEQ ID NO: 42

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for the Australian Thrips imaginis

<400> 42 ATGGTCTTCG GCGCGCATTT

<210> SEQ ID NO: 43

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Thrips hawaiiensis

<400> 43 CTGGCGTCTT CGGACGACT

<210> SEQ ID NO: 44

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Thrips hawaiiensis

<400> 44 CTTCGGACGA CTGCGCGC

<210> SEQ ID NO: 45

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Thrips hawaiiensis

<400> 45 CCTCTGGCGT AGTCCTCTT

<210> SEQ ID NO: 46

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Thrips hawaiiensis

<400> 46 AAATCCATGT GGACCGTATT G

<210> SEQ ID NO: 47

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Thrips hawaiiensis

<400> 47 ACCGTATTGC CGTACGGTAG

<210> SEQ ID NO: 48

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Thrips hawaiiensis

<400> 48 TAGACAAAGT ACCCAACATC

<210> SEQ ID NO: 49

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Thrips palmi

<400> 49 TAACCAGACT GTGATGTACGT

<210> SEQ ID NO: 50

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Thrips palmi

<400> 50 TGGACTTGCT CTCACGGG

<210> SEQ ID NO: 51

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Thrips palmi

<400> 50 ACGGGCGAGT CCTCCCAGT

<210> SEQ ID NO: 51

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Thrips palmi

<400> 50 AAACGGGGAA AGGCCTTGCA

<210> SEQ ID NO: 51

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Thrips palmi

<400> 51 ACGGGCGAGT CCTCCCAGT

<210> SEQ ID NO: 52

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Thrips palmi

<400> 52 AAACGGGGAA AGGCCTTGCA

<210> SEQ ID NO: 53

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Thrips palmi

<400> 53 CGGAGCGAGG AGGCATTTA

<210> SEQ ID NO: 54

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Thrips palmi

<400> 54 CGGAGCGAGG AGGCATTTA

<210> SEQ ID NO: 55

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Scirtothrips dorsalis ; Sdor

<400> 56 ACGACCAGAC TGTCCGCTTT A

<210> SEQ ID NO: 56

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Scirtothrips dorsalis ; Sdor

<400> 56 CGTGGTACTC TTAAATAGAG T

<210> SEQ ID NO: 57

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Scirtothrips dorsalis ; Sdor

<400> 56 CGCCGCGTTT ATCGAAATCG AAAACGAG

<210> SEQ ID NO: 58

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Scirtothrips dorsalis ; Sdor

<400> 24 AAATCGAAAA CGAGCGCACG GGAC

<210> SEQ ID NO: 59

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Scirtothrips dorsalis ; Sdor

<400> 24 AAAACCCACT CGATTGCCTT CCGTGC

<210> SEQ ID NO: 60

<211> 19

<212> DNA

<213> Artificial sequence

<220> Primer bind

<223> Specificity primer (forward sequence) for increasing the ITS2 region of Hummer

<400> 60 GTGGATCCCT GGGCTTGTG

<210> SEQ ID NO: 61

<211> 17

<212> DNA

<213> Artificial sequence

<220> Primer bind

<223> Specificity primer (forward sequence) for increasing the ITS2 region of Hummer

<400> 61 GTTAGTTTCT TTTCCTC

Claims (7)

A type of identification of Frankliniella occidentalis , Thrips tabac , Frankliniella intonsa , Mircocephalothrips abdominalis , Magalurothrips usitatus , and comb- nose horses Frankliniella schultzei ), Thrips imaginis , Thrips hawaiiensis , Thrips palmi and Scirtothrips dorsalis , including: (a) extracting test insects (b) Polymerase chain reaction using SEQ ID NO: 60 and SEQ ID NO: 61 to increase the interval 2 region (ITS2) DNA fragment of the test worm; (c) use of oligo The nucleotide probe is hybridized with the product of step (b), wherein the oligonucleotide probe is the nucleotide sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 59; and (d) is identified The result of the heterozygous reaction of step (c). A type of identification of Frankliniella occidentalis , Thrips tabac , Frankliniella intonsa , Mircocephalothrips abdominalis , Magalurothrips usitatus , and comb- nose horses Frankliniella schultzei ), Thrips imaginis , Thrips hawaiiensis , Thrips palmi , and Scirtothrips dorsalis , including: (a) extracting test insects (b) Polymerase chain reaction using SEQ ID NO: 60 and SEQ ID NO: 61 to increase the interval 2 region (ITS2) DNA fragment of the test worm; (c) use of oligo The nucleotide probe is hybridized with the product of step (b), wherein the oligonucleotide probe comprises at least one of SEQ ID NO: 1 to SEQ ID NO: 6, SEQ ID NO: 7 to SEQ At least one of 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: 24, and SEQ ID NO: 25 to At least one of SEQ ID NO: 30, SEQ ID NO: 31 to SEQ ID NO At least one of: 36, at least one of SEQ ID NO: 37 to SEQ ID NO: 42, at least one of SEQ ID NO: 43 to SEQ ID NO: 487, SEQ ID NO: 49 to SEQ ID a nucleotide sequence of at least one of NO: 54, at least one of SEQ ID NO: 554 to SEQ ID NO: 59; and (d) the result of the hybridization of step (c). One is used to identify Frankliniella occidentalis , Thrips tabaci , Frankliniella intonsa , Mircocephalothrips abdominalis , Megalurothrips usitatus , and calyx A biochip of the genus Frankliniella schultzei , Thrips imaginis , Thrips hawaiiensis , Thrips palmi , and Scirtothrips dorsalis , comprising a substrate; An oligonucleotide probe immobilized on the substrate, the oligonucleotide probes being the nucleotide sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 59. As used in claim 3, it is used to identify Frankliniella occidentalis , Thrips tabaci , Frankliniella intonsa , Mircocephalothrips abdominalis , and Megalurothrips usitatus . , biofilms of Frankliniella schultzei , Thrips imaginis , Thrips hawaiiensis , Thrips palmi , and Scirtothrips dorsalis , of which The material of the substrate comprises a nylon film, a polymer material, a slab or a glass. One is used to identify Frankliniella occidentalis , Thrips tabaci , Frankliniella intonsa , Mircocephalothrips abdominalis , Megalurothrips usitatus , and calyx A biochip of the genus Frankliniella schultzei , Thrips imaginis , Thrips hawaiiensis , Thrips palmi , and Scirtothrips dorsalis , comprising a substrate; An oligonucleotide probe affixed to the substrate, the oligonucleotide probe comprising at least one of SEQ ID NO: 1 to SEQ ID NO: 6, SEQ ID NO: 7 to SEQ ID At least one of 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: 24, SEQ ID NO: 25 to SEQ At least one of ID NO: 30, at least one of SEQ ID NO: 31 to SEQ ID NO: 36, at least one of SEQ ID NO: 37 to SEQ ID NO: 42, and SEQ ID NO: 43 At least one of SEQ ID NO: 48, SEQ ID NO: 49 to SEQ ID NO: 5 A nucleotide sequence of at least one of 4, at least one of SEQ ID NO: 55 to SEQ ID NO: 59. A biochip identified in claim 3 or claim 5 identifies a Frankliniella occidentalis , a Thrips tabaci , a Frankliniella intonsa , a Mircocephalothrips abdominalis , and a bean flower. Megalurothrips usitatus , Frankliniella schultzei , Thrips imaginis , Thrips hawaiiensis , Thrips palmi , Scirtothrips dorsalis The method comprises: (a) extracting the deoxyribonucleic acid of the worm to be tested; (b) using the polymerase chain reaction to increase the interval 2 of the worm to be tested using SEQ ID NO: 60 and SEQ ID NO: 61 ( ITS2) a deoxyribonucleic acid fragment; (c) performing a hybrid reaction using the biochip of claim 3 or claim 5; and (d) identifying the result of the hybridization reaction of step (c). A type of identification of Frankliniella occidentalis , Thrips tabaci , Frankliniella intonsa , Mircocephalothrips abdominalis , Megalurothrips usitatus , and comb- nose horses Frankliniella schultzei ), Thrips imaginis , Thrips hawaiiensis , Thrips palmi , Scirtothrips dorsalis , containing at least ten oligonucleosides The acid probe, the ten oligonucleotide probes comprising at least one of SEQ ID NO: 1 to SEQ ID NO: 6, at least one of SEQ ID NO: 7 to SEQ ID NO: 12, SEQ ID NO At least one of: 13 to SEQ ID NO: 18, at least one of SEQ ID NO: 19 to SEQ ID NO: 24, at least one of SEQ ID NO: 25 to SEQ ID NO: 30, SEQ ID At least one of NO: 31 to SEQ ID NO: 36, at least one of SEQ ID NO: 37 to SEQ ID NO: 42, at least one of SEQ ID NO: 43 to SEQ ID NO: 48, SEQ ID NO: 49 to at least one of SEQ ID NO: 54, SEQ ID NO: 55 to a nucleotide sequence of at least one of SEQ ID NO: 59; and a specific primer pair having the sequences set forth in SEQ ID NO: 60 and SEQ ID NO: 61.