TWI473881B - Oligo-nucleotide probes of psyllids identification, biochip, and identifying method thereof - Google Patents

Description

Oligonucleotide probe for identifying hibiscus, biochip and identification method thereof

The present invention relates to a method for identifying hibiscus, and more particularly to an oligonucleic acid probe for identifying hibiscus, a biochip, and a method for identifying same.

The psyllids are insects of the Hemiptera Sternorrhyncha genus Psylloidea, which have been recorded in the world by about 3,000 species. All hibiscus are herbivorous and feed on woody plants. There are many economic crops such as fruit trees. There are quite a lot of damage records in temperate and subtropical regions, such as citrus, pear, apple, persimmon, fig, cotton, potato. Etc. There are also some harmful ornamental plants or street trees, such as eucalyptus, poplar, boxwood and eucalyptus.

The effect of hibiscus on plants is multi-faceted. They use the sucking mouthparts to feed the sap of the plant phloem, causing direct damage to the plants, which may cause the shoots or leaves to curl and wither; and many species emit a lot of honey. Exposure, causing soot disease, a large number of cover the surface of the leaves will block the photosynthesis of plants, causing the tree to weaken; especially, some species play the carrier of plant pathogens, leading to plant rickets and even death, the most famous is the spread of blight Citrus hibiscus and pear hibiscus that spread pears and weakened diseases.

Take the example of pear hibiscus in Taiwan. Since 1994, pear gardens in central Taiwan have been affected by new pests and diseases, causing serious losses in agriculture. After several years of research, it was confirmed that the newly discovered disease is pear decline (PD), which is caused by the infection of pear trees by plant mycelium, and this disease has caused serious harm to the pear industry in Europe and the United States for a long time. The diseased pear trees will wither, dwarf, and early defoliation, resulting in poor fruit quality, reduced yield symptoms, and eventually death, causing significant losses. This disease can be transmitted through grafting and hibiscus. The vector insects of PD in Europe and the United States have been identified as Cacopsylla pyricola Förster and C. pyri (Linnaeus). Taiwan has never discovered hibiscus in pear trees before the 1990s. After the debilitating disease, two species of hibiscus native to mainland China, namely Cacopsylla qianli and Chinese pear hibiscus ( C. Chinensis ), the latter has long been an important pest in agriculture in China, and it has been confirmed that both Alder and Chinese Pear are the vectors of Taiwan pear debilitation.

The identification method of hibiscus can be identified by the identification method of traditional biological features or the technique of applying molecular biology. However, the use of traditional biological characterization methods for identification takes a long time to match the timeliness required for quarantine. Therefore, it is important to develop a rapid and accurate identification method for hibiscus.

Therefore, one aspect of the present invention is to provide an oligonucleotide probe for identifying hibiscus and a method for using the same, which solves the problem that the correctness of the traditional hibiscus morphological identification is influenced by human judgment.

Another aspect of the present invention is to provide a biochip for identifying hibiscus and a method of using the same for quickly and simultaneously identifying different species of hibiscus.

According to the present invention, it is proposed to identify Neophacopteron euphoriae , Trioza outeiensis , T. magnicauda , Paurocephala sauteri , Microceropsylla nigr , and citrus. Diaphorina citri , Cacopsylla chinensis , Cacopsylla qianli , Cacopsylla pyri , Bactericera cockerell , Cacopsylla oluanpiensis and Cacopsylla tobirae An oligonucleotide probe comprising the nucleotide sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 49.

A method for identifying the above-mentioned species of hibiscus using an oligonucleotide probe, comprising: (a) extracting deoxyribonucleic acid of the worm to be tested; (b) using the specificity of SEQ ID NO: 48 and SEQ ID NO: 49 The primer pair is subjected to a polymerase chain reaction to increase the amount of the intergenic spacer 2 (ITS2; ITS2) deoxyribonucleic acid fragment; (c) using the oligonucleotide probe and the product of step (b) a hybridization reaction, wherein the oligonucleotide probe comprises at least one of SEQ ID NO: 1 to SEQ ID NO: 4, at least one of SEQ ID NO: 5 to SEQ ID NO: 8, SEQ ID NO : at least one of 9 to SEQ ID NO: 11, at least one of SEQ ID NO: 12 to SEQ ID NO: 15, at least one of SEQ ID NO: 16 to SEQ ID NO: 19, SEQ ID NO: 20 to at least one of SEQ ID NO: 23, at least one of SEQ ID NO: 24 to SEQ ID NO: 27, at least one of SEQ ID NO: 28 to SEQ ID NO: 31, SEQ At least one of ID NO: 32 to SEQ ID NO: 35, at least one of SEQ ID NO: 36 to SEQ ID NO: 39, and at least one of SEQ ID NO: 40 to SEQ ID NO: 43 A nucleotide sequence of at least one of SEQ ID NO: 44 to SEQ ID NO: 47; and (d) identifying the result of the hybridization reaction of step (c).

According to the present invention, there is provided a biochip for rapidly identifying a hibiscus, the biochip for identifying hibiscus comprising a substrate on which at least one of SEQ ID NO: 1 to SEQ ID NO: 4 can be immobilized, SEQ ID NO: 5 to at least one of SEQ ID NO: 8, at least one of SEQ ID NO: 9 to SEQ ID NO: 11, at least one of SEQ ID NO: 12 to SEQ ID NO: 15, SEQ At least one of ID NO: 16 to SEQ ID NO: 19, at least one of SEQ ID NO: 20 to SEQ ID NO: 23, at least one of SEQ ID NO: 24 to SEQ ID NO: 27, At least one of SEQ ID NO: 28 to SEQ ID NO: 31, at least one of SEQ ID NO: 32 to SEQ ID NO: 35, at least one of SEQ ID NO: 36 to SEQ ID NO: 39 An oligonucleotide probe of at least one of SEQ ID NO: 40 to SEQ ID NO: 43 and a nucleotide sequence of at least one of SEQ ID NO: 44 to SEQ ID NO: 47.

The method for identifying a hibiscus using the above biochip comprises heterozygous reaction of the DNA fragment DNA of the interval 2 (ITS2) of the worm to be tested with the oligonucleotide probe on the above biochip (hybridization) ), and the type of hibiscus is identified by the result of the heterozygous reaction.

According to the present invention, it is proposed to identify longan hibiscus, Brazilian rosewood hibiscus, ivory hibiscus, mulberry hibiscus, lemon hibiscus, citrus hibiscus, Chinese pear hibiscus, avocado hibiscus, pear hibiscus, Bactericera cockerell , Taiwan paulownia a kit of cockroaches, sea paulownia, hawthorn hibiscus, pear hibiscus and vegetable and fruit paulownia, comprising a first oligonucleotide probe combination comprising at least one of SEQ ID NO: 1 to SEQ ID NO: a second oligonucleotide probe combination comprising at least one of SEQ ID NO: 5 to SEQ ID NO: 8; a third oligonucleotide probe combination comprising SEQ ID NO: 9 to SEQ ID NO: At least one of 11; a fourth oligonucleotide probe combination comprising at least one of SEQ ID NO: 12 to SEQ ID NO: 15; a fifth oligonucleotide probe combination comprising SEQ ID NO: 16 to at least one of SEQ ID NO: 19; a sixth oligonucleotide probe combination comprising at least one of SEQ ID NO: 20 to SEQ ID NO: 23; a seventh oligonucleotide probe combination , comprising at least one of SEQ ID NO: 24 to SEQ ID NO: 27; an eighth oligonucleotide probe combination, package At least one of SEQ ID NO: 28 to SEQ ID NO: 31; a ninth oligonucleotide probe combination comprising at least one of SEQ ID NO: 32 to SEQ ID NO: 35; a tenth oligonucleoside An acid probe combination comprising at least one of SEQ ID NO: 36 to SEQ ID NO: 39, an eleventh oligonucleotide probe combination comprising at least one of SEQ ID NO: 40 to SEQ ID NO: And a twelfth oligonucleotide probe combination comprising at least one of SEQ ID NO: 45 to SEQ ID NO: 47.

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

Figure 2 is a graph showing the specificity of the probes of the biochip of the present invention using ITS2 DNA fragments of different species of hibiscus, respectively.

Embodiments of the present invention utilize Neophacopteron euphoriae , Trioza outeiensis , T. magnicauda , Paurocephala sauteri , Microceropsylla nigr , citrus Diaphorina citri , Cacopsylla chinensis , Cacopsylla qianli , Cacopsylla pyri , Bactericera cockerell , Cacopsylla oluanpiensis , Cacopsylla tobirae , etc. Oligonucleotide probes were designed for different types of ITS2 DNA sequences, and probe specificity tests were performed to obtain specific oligonucleotide probes whose sequences were numbered SEQ ID NO: 1 to SEQ, respectively. ID NO: 47.

The oligonucleotide probes numbered SEQ ID NO: 1 to SEQ ID NO: 4 are designed using the ITS2 DNA sequence of Longan hibiscus and can be used to identify Longan hibiscus.

The oligonucleotide probes numbered SEQ ID NO: 5 to SEQ ID NO: 8 were designed using the ITS2 DNA sequence of the Brazilian rosewood stalk to identify the Brazilian hibiscus.

The oligonucleotide probes numbered SEQ ID NO: 9 to SEQ ID NO: 11 were designed using the ITS2 DNA sequence of Ivory Hibiscus, which can be used to identify ivory wood rafts.

The oligonucleotide probes numbered SEQ ID NO: 12 to SEQ ID NO: 15 were designed using the ITS2 DNA sequence of Mulberry, and can be used to identify mulberry.

The oligonucleotide probes numbered SEQ ID NO: 16 to SEQ ID NO: 19 were designed using the ITS2 DNA sequence of citrus sylvestris and can be used to identify lemon hibiscus.

The oligonucleotide probes numbered SEQ ID NO: 20 to SEQ ID NO: 23 were designed using the ITS2 DNA sequence of citrus hibiscus and can be used to identify citrus hibiscus.

Oligonucleotides numbered SEQ ID NO: 24 to SEQ ID NO: 27 The probe system is designed by using the ITS2 DNA sequence of Chinese pear hibiscus, which can be used to identify Chinese pear hibiscus.

The oligonucleotide probes numbered SEQ ID NO: 28 to SEQ ID NO: 31 were designed using the ITS2 DNA sequence of A. sinensis to identify avocado hibiscus.

The oligonucleotide probes numbered SEQ ID NO: 32 to SEQ ID NO: 35 were designed using the ITS2 DNA sequence of P. sylvestris and can be used to identify P. sylvestris.

Oligonucleotide probes numbered SEQ ID NO: 36 to SEQ ID NO: 39 were designed using Bactericera cockerell 's ITS2 DNA sequence and can be used to identify Bactericera cockerell .

The oligonucleotide probes numbered SEQ ID NO: 40 to SEQ ID NO: 43 were designed using the ITS2 DNA sequence of P. taiwanensis, and can be used to identify P. taiwanensis.

The oligonucleotide probes numbered SEQ ID NO: 44 to SEQ ID NO: 47 were designed using the ITS2 DNA sequence of P. sylvestris and can be used to identify P. sylvestris.

The above-mentioned specific oligonucleotide probe is made into a biochip, and the raft can be quickly identified.

Referring to Fig. 1, a flow chart of a method for identifying hibiscus using the biochip of the present invention comprises (a) extracting the genomic DNA of the worm (DNA); (b) increasing the ITS2 DNA of the worm to be tested. a sequence of fragments; (c) performing a hybrid reaction with the amplified ITS2 DNA fragment using the biochip of the present invention; and (d) identifying the result of the hybridization reaction of the step (c), and identifying the hibiscus from the result of the heterozygous reaction.

Extraction of genomic DNA from worms according to an embodiment of the present invention This was carried out in accordance with the method of Lu et al. (2002). The whole worm was weighed and placed on the wiped paper, and then a single worm was placed in the buffer (100 mM NaCl, 10 mM Tris-Cl, 25 mM EDTA, 0.5% SDS, 0.1 mg/ml). Protinase k) in a glass grinder (1.2 ml buffer per 100 mg of tissue) is evenly ground; place the slurry in a water bath, heat the reaction at 65 ° C for 30 minutes, and mix the sample every 3-5 minutes. Uniformly; then add 1/7 volume of 8 M ammonium acetate, shake slowly for 10 minutes, place on ice for 45 minutes, centrifuge at 14500 g for 15 minutes, and take the supernatant to an equal volume of 95% alcohol. And placed at -80 ° C for 10-15 minutes, the DNA was precipitated, and then centrifuged at 14500 g for 15 minutes, the supernatant was removed, the precipitate was washed with 75% alcohol, and dried by a vacuum centrifugal dryer, followed by Appropriate amount of sterile water. The DNA was reconstituted, and the purified DNA was stored at -20 ° C, which was the source of the template DNA for testing the worms. The appropriate amount of purified genomic DNA was diluted 200 X with sterile water, and the concentration of DNA was estimated by a spectrophotometer (Gene Spec III, Naka Instriments, Japan) and recorded with an optical density (OD) of 260 mm. Between 1.6 and 2.0 is a DNA of higher purity.

The DNA rapid extraction method was carried out using an Easy DNA High-Speed extraction kit (Saturn Biotech Limited, Perth, Western Australia) and in accordance with the instructions. Place a single worm into a 1.5 ml centrifuge tube containing 32 μl of solution 1A and 8 μl of solution 1B, grind and place at 95 ° C for 30 minutes (cover with an explosion-proof cover), remove and let stand to cool down to room After warming, add 10 μl of solution 2, mix and centrifuge for about 10 seconds, then take the supernatant for PCR.

Polymerase chain reaction of the extracted worms with SEQ ID NO: 60 (forward sequence) and SEQ ID NO: 61 (reverse sequence) using degenerate primers (PCR) Amplification of the ITS2 DNA fragment.

The PCR amplification reaction was carried out in a GeneAmp ^® PCR System 2700 or 2400 Rewarm Reactor (Perkin-Elmer Inc., Singapore). In a 200 μ l Eppendorf tube containing the DNA to be increased, the total volume of each reaction was 25 μ l, containing 100 ~ 0.01 ng template DNA, 250 μ M dNTPs, 1X PCR buffer, 0.5 μ M primer, and 0.5U rTaq thermostable polymerase (TaKaRa, Japan), PCR reaction conditions, first 94 ° C for 5 minutes, then 94 ° C 30 seconds, 55 ° C 45 seconds, 72 ° C 60 seconds, 35 cycles, followed by 72 ° C 10 minutes reaction.

For the preparation of biochips, the same method is used to carry out a polymerase chain reaction with a biotin-labeled primer to obtain a biotin-labeled ITS2 DNA fragment, which is the target of the hybrid reaction.

The biochip comprises a substrate and an oligonucleotide probe affixed to the substrate, the sequences of which are numbered SEQ ID NO: 1 to SEQ ID NO: 48, respectively. The material of the substrate may include a nylon film, a polymer material, a cymbal sheet or a glass. In this embodiment, the method for manufacturing the bio-wafer includes placing a blank wafer at a specific position of the manual spotting machine, and preparing 10 μl . The good probe solution is separately added to the spot hole tray, and then the fine needle placed on the spotting machine is arrayed at the set position, and then the wafer finished with the dot is baked at 45 ° C for 1-2 minutes. The probe was then fixed on the wafer by UV irradiation with a UV linker (UVitec, EEC) at 1 J for 6 minutes. Then add 500 μl of sterile water to wash, let stand for 6 minutes, then pour off, repeat twice, and finally take 100 μl of 99.5% alcohol into each reaction tank and immediately pour off, then bake at 45 ° C for 10 minutes. To dry.

The hybridization reaction was carried out with biotin-labeled target DNA fragment and oligonucleotide probes SEQ ID NO: 1 to SEQ ID NO: 47. Use DR.Chip The DIYTM Kit (DR.Chip Biotechnology Incorporation, Taiwan) is a heterozygous reaction step as follows: PCR amplification: PCR amplification of the worm sample is performed first, because the color rendering result is used in the process of making the biochip, so PCR amplification must be performed using primers with biotin calibration. On the one hand, the target DNA to be tested can be amplified by PCR to increase the amount of DNA; in addition, the target can be biotin-calibrated via a PCR process. The resulting PCR product was first placed in a -20 ° C refrigerator for later use.

Prepared probe solution: Take 10 μ l at a concentration of 40 μ M oligonucleotide probe synthesized with 10 μ l 2X probe solution (probe solution) mixed into 0.6 ml microfuge tubes to the standby.

Spotting and fixing of the probe: first place the blank wafer on a specific position of the manual spotting machine, take 10 μl of the prepared probe solution into the spot hole plate, and then use the fine placed on the spotting machine. The needle dot is in the set position, and then the dot-finished wafer is baked at 45oC for 1-2 minutes, and then subjected to UV linker (UVitec, EEC) at 1 J for 6 minutes. UV irradiation immobilizes the probe on the wafer. Then add 500 μl of sterile water to wash, let stand for 6 minutes, then pour off, repeat twice, and finally take 100 μl of 99.5% alcohol into each wafer reaction tank and immediately pour off, then bake at 45oC for 10 minutes. dry.

Hybridization reaction: 200 μl of hybrid reaction solution was added to each reaction tank of the wafer, and 10 μl of the PCR product (with biotin calibration) heated and denatured at 95 ° C for 5 minutes was mixed with it and placed in a heterozygous reaction at 48 ° C. 1 hour. After that, the hybrid reaction solution was discarded, and the unhybridized DNA fragment was washed away with 200 μl of Wash Buffer. After the heterozygous reaction, wash each well with 200 μl washing buffer for 1 minute, and then block the buffer in one reaction tank, and block 0.2 μl of streptavidin conjugated alkaline phosphatase (Strep-AP) with 200 μl . The reagent mixture was shaken vigorously for 10 seconds, then 180 μl blocking buffer was added to each reaction tank for 30 minutes, and then washed 3 times with 200 μl washing buffer; then the developer was prepared (mixed with 4 μl NBT/BCIP and 196 μl detection buffer) And slowly shake it), add 190 μl to the wafer reaction tank, and let it react in the dark room for 5-10 minutes. After the reaction, it is washed repeatedly with 500 μl of reverse osmosis pure water for 3 times, dried at 45 ° C, and finally placed. Store at room temperature. The result is mainly based on the presence or absence of stains as a basis for discriminating results.

Referring to Figure 2, the specificity results of the probes of the biochip of the present invention were tested for the ITS2 DNA fragments of different species of hibiscus, respectively.

Fig. 2(A) is a schematic view showing the application of an oligonucleotide probe of a biochip of the present embodiment. Wherein, the number indicated on each point represents the number of the sequence identification number, the number C1 is the control group for confirming the vp1 gene with the correct PCR reaction, and the number C2 is the control group of the highly conserved region of the gene to confirm the correctness of the hybrid reaction. .

The numbers 1 to 4 shown represent oligonucleotide probes of the sequence of SEQ ID NO: 1 to SEQ ID NO: 4, which are specifically hybridized to a DNA sample containing the ITS2 DNA sequence of Longan hibiscus. It is colored at the position corresponding to the wafer.

The numbers 5 to 8 shown represent oligonucleotide probes of the sequence SEQ ID NO: 5 to SEQ ID NO: 8, which can be specifically hybridized to a DNA sample containing the ITS2 DNA sequence of the Brazilian rosewood. And color at the position corresponding to the wafer.

The numbers 9 to 11 shown represent oligonucleotide probes of the sequence SEQ ID NO: 9 to SEQ ID NO: 11 and can be associated with ITS2 DNA containing ivory wood hibiscus The sequence of DNA samples produces a specific heterozygote that is colored at the corresponding location of the wafer.

The numbers 12 to 15 shown represent oligonucleotide probes of the sequence SEQ ID NO: 12 to SEQ ID NO: 15 which can be specifically hybridized to a DNA sample containing the ITS2 DNA sequence of M. sylvestris. The color is colored at the position corresponding to the wafer.

The numbers 16 to 19 shown represent oligonucleotide probes of the sequence of SEQ ID NO: 16 to SEQ ID NO: 19, which are specifically hybridized to a DNA sample containing the ITS2 DNA sequence of citrus hibiscus. It is colored at the position corresponding to the wafer.

The numbers 20 to 23 represent the oligonucleotide probes of SEQ ID NO: 20 to SEQ ID NO: 23, which can be specifically hybridized with a DNA sample containing the ITS2 DNA sequence of citrus hibiscus. And color at the position corresponding to the wafer.

The numbers 24 to 27 represent the oligonucleotide probes of SEQ ID NO: 24 to SEQ ID NO: 27, which can be specifically hybridized with a DNA sample containing the ITS2 DNA sequence of P. chinensis. It is colored at the position corresponding to the wafer.

The numbers 28 to 31 represent the oligonucleotide probes of SEQ ID NO: 28 to SEQ ID NO: 31, which can be specifically hybridized with a DNA sample containing the ITS2 DNA sequence of A. sinensis. It is colored at the position corresponding to the wafer.

The numbers 32 to 35 represent the oligonucleotide probes of SEQ ID NO: 32 to SEQ ID NO: 35, which can be specifically hybridized with a DNA sample containing the ITS2 DNA sequence of P. sylvestris. The color is colored at the position corresponding to the wafer.

The numbers 36 to 39 shown represent oligonucleotide probes of the sequence SEQ ID NO: 36 to SEQ ID NO: 39, which are specifically hybridized to a DNA sample containing the ITS2 DNA sequence of Bactericera cockerell , and The color is colored at the position corresponding to the wafer.

The numbers 40 to 43 represent the oligonucleotide probes of SEQ ID NO: 40 to SEQ ID NO: 43 and can be specifically hybridized with a DNA sample containing the ITS2 DNA sequence of Taiwan paulownia. And color at the position corresponding to the wafer.

The numbers 44 to 47 are shown to represent oligonucleotide probes of SEQ ID NO: 44 to SEQ ID NO: 47, which can be specifically hybridized to a DNA sample containing the ITS2 DNA sequence of P. sylvestris. It is colored at the position corresponding to the wafer.

Figure 2 (B), (C), (D), (E), (F), (G), (H), (I), (J), (K), (L) and (M) In part, the biowafer analysis results of the raft species were identified by the probe application method of the present embodiment. Part (B) is the target DNA using the ITS2 DNA fragment of Longan hibiscus; (C) is the target DNA using the ITS2 DNA fragment of the Brazilian hibiscus; (D) is the ITS2 DNA fragment using the ivory wood hibiscus. Target DNA; (E) is the target DNA using the ITS2 DNA fragment of Mulberry; (F) is the target DNA using the ITS2 DNA fragment of the citrus sylvestris; (G) is the ITS2 DNA using the citrus hibiscus The fragment is target DNA; (H) is the target DNA using the ITS2 DNA fragment of P. sylvestris; (I) is the target DNA using ATS2 DNA fragment of A. chinensis; (J) is the ITS2 DNA using Pearwood The fragment is target DNA; the (K) part uses the ITS2 DNA fragment of Bactericera cockerell as the target DNA; the (L) part uses the ITS2 DNA fragment of Taiwan sea paulownia as the target DNA; and the (M) part uses the ITS2 of the sea paulownia. The DNA fragment is target DNA.

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 4 were detected in the test group. The spots of the probe are colored, and the control group also has a coloration to confirm that the hybrid reaction is correct. Therefore, the oligonucleotide probes of SEQ ID NOS: 1 to 4 can be specifically hybridized with a DNA sample containing the ITS2 sequence of longan hibiscus, and can be used to accurately identify longan hibiscus.

Part 2 (C) shows the results of the oligonucleotide probes shown in part (A) of Figure 2, and the experimental group only has the oligonucleotides representing the sequences of SEQ ID NOs: 5 to 8. The needle points are colored, and the control group also has a coloration to confirm that the hybrid reaction is correct. Thus, the oligonucleotide probes of SEQ ID NOS: 5 to 8 can be specifically hybridized to a DNA sample containing the ITS2 sequence of the Brazilian hibiscus, which can be used to accurately identify the Brazilian hibiscus.

Part (D) of the results in Figure 2 shows the oligonucleotide probes shown in the section (A) of Figure 2, and the oligonucleotides in the test group are only SEQ ID NOs: 9 to 11. The needle points are colored, and the control group also has a coloration to confirm that the hybrid reaction is correct. Thus, the oligonucleotide probes of SEQ ID NOS: 9 to 11 can be specifically heterozygous for DNA samples containing the ITS2 sequence of Ivory Hibiscus, and can be used to accurately identify ivory wood rafts.

Part (E) of Figure 2 shows the results of the oligonucleotide probes shown in section (A) of Figure 2, and the experimental group only has oligonucleotides representing the sequences of SEQ ID NOs: 12 to 15. The needle points are colored, and the control group also has a coloration to confirm that the hybrid reaction is correct. Thus, the oligonucleotide probes of SEQ ID NOS: 12 to 15 can be specifically hybridized to a DNA sample containing the ITS2 sequence of Mulberry, and can be used to accurately identify mulberry.

Part (F) of Figure 2 shows the results of the oligonucleotide probes shown in the section (A) of Figure 2, and only the oligonucleotides representing the sequences of SEQ ID NOS: 16 to 19 were tested. The needle points are colored, and the control group also has a coloration to confirm that the hybrid reaction is correct. Therefore, the oligonucleotide probes of SEQ ID NOS: 16 to 19 can be specifically hybridized with a DNA sample containing the ITS2 sequence of citrus hibiscus, and can be used to accurately identify lemon hibiscus.

The results of part (G) of Fig. 2 show that the oligonucleotide probes shown in the section (A) of Figure 2 are mapped, and only the oligonucleotides representing the sequences of SEQ ID NOS: 20 to 23 are tested. The needle points are colored, and the control group also has a coloration to confirm that the hybrid reaction is correct. Therefore, the oligonucleotide probes of SEQ ID NOS: 20 to 23 can be specifically hybridized with a DNA sample containing the ITS2 sequence of citrus hibiscus, and can be used to accurately identify citrus hibiscus.

Part (H) of Figure 2 shows the results of the oligonucleotide probes shown in the section (A) of Figure 2, and the experimental group only has the oligonucleotides representing the sequences of SEQ ID NOs: 24 to 27. The needle points are colored, and the control group also has a coloration to confirm that the hybrid reaction is correct. Therefore, the oligonucleotide probes of SEQ ID NOS: 24 to 27 can be specifically hybridized with a DNA sample containing the ITS2 sequence of P. chinensis, and can be used to accurately identify Chinese pear hibiscus.

Part (I) of Figure 2 shows the results of the oligonucleotide probes shown in section (A) of Figure 2, and the experimental group only has oligonucleotides representing the sequences of SEQ ID NOs: 28 to 31. The needle points are colored, and the control group also has a coloration to confirm that the hybrid reaction is correct. Therefore, the oligonucleotide probes of SEQ ID NOS: 28 to 31 can be specifically hybridized with a DNA sample containing the ITS2 sequence of A. sinensis, and can be used to accurately identify A. sinensis.

Part 2 (J) results show that the oligonucleotide probes shown in the second panel (A) are shown in the schematic diagram. The spots of the oligonucleotide probes of SEQ ID NOS: 32 to 35 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: 32 to 35 can be specifically heterozygous for DNA samples containing the ITS2 sequence of P. sylvestris, and can be used to accurately identify P. sylvestris.

Part (K) of the results in Figure 2 shows the oligonucleotide probes shown in the section (A) of Figure 2, and the oligonucleotides in the test group are only SEQ ID NO: 36 to 39. The needle points are colored, and the control group also has a coloration to confirm that the hybrid reaction is correct. Thus, the oligonucleotide probes of SEQ ID NOS: 36 to 39 can be specifically hybridized to a DNA sample containing the ITS2 sequence of Bactericera cockerell , which can be used to accurately identify Bactericera cockerell .

The results of part (L) of Fig. 2 show that the oligonucleotide probes shown in the section (A) of Fig. 2 are mapped, and only the oligonucleotides representing the sequences of SEQ ID NOS: 40 to 43 are tested. The needle points are colored, and the control group also has a coloration to confirm that the hybrid reaction is correct. Therefore, the oligonucleotide probes of SEQ ID NOS: 40 to 43 can be specifically hybridized with a DNA sample containing the ITS2 sequence of P. taiwanensis, and can be used to accurately identify P. sinensis.

The results of part (M) of Fig. 2 show that the oligonucleotide probes shown in the section (A) of Fig. 2 are mapped, and only the oligonucleotides representing the sequences of SEQ ID NOS: 44 to 47 are tested. The needle points are colored, and the control group also has a coloration to confirm that the hybrid reaction is correct. Therefore, the oligonucleotide probes of SEQ ID NOS: 44 to 47 can be specifically hybridized with a DNA sample containing the ITS2 sequence of P. sylvestris, and can be used to accurately identify P. sylvestris.

According to the results of Fig. 2, the method disclosed in the examples of the present invention and the oligonucleotide probe having the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 47 can be used to enable SEQ ID NO: 48 and SEQ ID NO: 49. 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 longan hibiscus, Brazilian rosewood hibiscus, ivory wood hibiscus, mulberry hibiscus, lemon fruit hibiscus, mandarin on the same wafer. Twelve species of hibiscus , such as orange hibiscus, Chinese pear hibiscus, avocado hibiscus , pear hibiscus, Bactericera cockerell , Taiwan sea paulownia and sea paulownia.

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 plurality of hibiscus.

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.

<110> National Chung Hsing University

<120> Oligonucleotide probe for identifying hibiscus, biochip and identification method thereof

<160> 61

<210> SEQ ID NO: 1

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<223> Identification of Oligonucleotide Probes for Neophacopteron euphoriae

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<223> Identification of Oligonucleotide Probes for Neophacopteron euphoriae

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<223> Identification of Oligonucleotide Probes for Neophacopteron euphoriae

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<223> Identification of Oligonucleotide Probes for Neophacopteron euphoriae

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<210> SEQ ID NO: 5

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<223> Identification of oligonucleotide probes from Trioza outeiensis

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<210> SEQ ID NO: 6

<211> 19

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<223> Identification of oligonucleotide probes from Trioza outeiensis

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<210> SEQ ID NO: 7

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<223> Identification of oligonucleotide probes from Trioza outeiensis

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<210> SEQ ID NO: 8

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<223> Identification of oligonucleotide probes from Trioza outeiensis

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<210> SEQ ID NO: 9

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<223> Identification of oligonucleotide probes of T. magnicauda

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<223> Identification of oligonucleotide probes of T. magnicauda

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<210> SEQ ID NO: 11

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<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of T. magnicauda

<400> 11

<210> SEQ ID NO: 12

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Paurocephala sauteri

<400> 12

<210> SEQ ID NO: 13

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Paurocephala sauteri

<400> 13

<210> SEQ ID NO: 14

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Paurocephala sauteri

<400> 14

<210> SEQ ID NO: 15

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Paurocephala sauteri

<400> 15

<210> SEQ ID NO: 16

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Microceropsylla nigr

<400> 16

<210> SEQ ID NO: 17

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Microceropsylla nigr

<400> 17

<210> SEQ ID NO: 18

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Microceropsylla nigr

<400> 18

<210> SEQ ID NO: 19

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Microceropsylla nigr

<400> 19

<210> SEQ ID NO: 20

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Diaphorina citri

<400> 20

<210> SEQ ID NO: 21

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Diaphorina citri

<400> 21

<210> SEQ ID NO: 22

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Diaphorina citri

<400> 22

<210> SEQ ID NO: 23

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes for Diaphorina citri

<400> 23

<210> SEQ ID NO: 24

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Chinese genus Cacopsylla chinensis

<400> 24

<210> SEQ ID NO: 25

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Chinese genus Cacopsylla chinensis

<400> 25

<210> SEQ ID NO:26

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Chinese genus Cacopsylla chinensis

<400> 26

<210> SEQ ID NO:27

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Chinese genus Cacopsylla chinensis

<400> 27

<210> SEQ ID NO: 28

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of Oligonucleotide Probes for Cacopsylla qianli

<400> 28

<210> SEQ ID NO: 29

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of Oligonucleotide Probes for Cacopsylla qianli

<400> 29

<210> SEQ ID NO: 30

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of Oligonucleotide Probes for Cacopsylla qianli

<400> 30

<210> SEQ ID NO: 31

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of Oligonucleotide Probes for Cacopsylla qianli

<400> 31

<210> SEQ ID NO: 32

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Cacopsylla pyri

<400> 32

<210> SEQ ID NO: 33

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Cacopsylla pyri

<400> 33

<210> SEQ ID NO: 34

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Cacopsylla pyri

<400> 34

<210> SEQ ID NO: 35

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Cacopsylla pyri

<400> 35

<210> SEQ ID NO: 36

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of Bactericera cockerell oligonucleotide probes

<400> 36

<210> SEQ ID NO: 37

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of Bactericera cockerell oligonucleotide probes

<400> 37

<210> SEQ ID NO: 38

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of Bactericera cockerell oligonucleotide probes

<400> 38

<210> SEQ ID NO: 39

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of Bactericera cockerell oligonucleotide probes

<400> 39

<210> SEQ ID NO: 40

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Cacopsylla oluanpiensis in Taiwan

<400> 40

<210> SEQ ID NO: 41

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Cacopsylla oluanpiensis in Taiwan

<400> 41

<210> SEQ ID NO: 42

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Cacopsylla oluanpiensis in Taiwan

<400> 42

<210> SEQ ID NO: 43

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Cacopsylla oluanpiensis in Taiwan

<400> 43

<210> SEQ ID NO: 44

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Cacopsylla tobirae

<400> 44

<210> SEQ ID NO: 45

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Cacopsylla tobirae

<400> 45

<210> SEQ ID NO: 46

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Cacopsylla tobirae

<400> 46

<210> SEQ ID NO: 47

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Identification of oligonucleotide probes of Cacopsylla tobirae

<400> 47

<210> SEQ ID NO: 48

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> General PCR degenerate primer for the ITS2 DNA fragment of the amplified hibiscus

<400> 48

<210> SEQ ID NO: 49

<211> 20

<212> Primer

<213> Artificial sequence

<220>

<223> General PCR degenerate primer for the ITS2 DNA fragment of the amplified hibiscus

<400> 49

Claims (6)

A method for identifying Neophacopteron euphoriae , Trioza outeiensis , T. magnicauda , Paurocephala sauteri , Microceropsylla nigr , Diaphorina citri), Chinese pear psylla (Cacopsylla chinensis), Qian pear psylla (Cacopsylla qianli), pear psylla (Cacopsylla pyri), Bactericera cockerell, Taiwan Pittosporum psyllids (Cacopsylla oluanpiensis) and Pittosporum psyllids (Cacopsylla tobirae) the method comprising: (a) extracting the deoxyribonucleic acid of the worm to be tested; (b) performing a polymerase chain reaction using the specific primer pair consisting of SEQ ID NO: 48 and SEQ ID NO: 49, increasing the interval between the two regions to be tested (ITS2) a deoxyribonucleic acid fragment; (c) using an oligonucleotide probe for hybridization with the product of step (b), wherein the oligonucleotide probe comprises SEQ ID NO: 1 to SEQ ID NO: At least one of 4, at least one of SEQ ID NO: 5 to SEQ ID NO: 8, at least one of SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 12 to SEQ ID NO : at least one of 15 At least one of SEQ ID NO: 16 to SEQ ID NO: 19, at least one of SEQ ID NO: 20 to SEQ ID NO: 23, at least one of SEQ ID NO: 24 to SEQ ID NO: At least one of SEQ ID NO: 28 to SEQ ID NO: 31, at least one of SEQ ID NO: 32 to SEQ ID NO: 35, and at least one of SEQ ID NO: 36 to SEQ ID NO: 39 a nucleotide sequence of at least one of SEQ ID NO: 40 to SEQ ID NO: 43, at least one of SEQ ID NO: 44 to SEQ ID NO: 47; and (d) an identification step (c) The result of the heterozygous reaction. A method for identifying Neophacopteron euphoriae , Trioza outeiensis , T. magnicauda , Paurocephala sauteri , Microceropsylla nigr , citrus hibiscus Biofilm of ( diaphorina citri ), Cacopsylla chinensis , Cacopsylla qianli , Cacopsylla pyri , Bactericera cockerell , Cacopsylla oluanpiensis and Cacopsylla tobirae , comprising a substrate; and a plurality of oligonucleotide probes immobilized on the substrate, the oligonucleotide probes being the nucleotide sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 47 . For the identification of Neophacopteron euphoriae , Trioza outeiensis , T. magnicauda , Paurocephala sauteri , and Microceropsylla nigr as described in claim 2 , Diaphorina citri , Cacopsylla chinensis , Cacopsylla qianli , Cacopsylla pyri , Bactericera cockerell , Cacopsylla oluanpiensis and Cacopsylla A biological wafer of tobirae , wherein the material of the substrate comprises a nylon film, a polymer material, a slab or a glass. A method for identifying Neophacopteron euphoriae , Trioza outeiensis , T. magnicauda , Paurocephala sauteri , Microceropsylla nigr , citrus hibiscus Biofilm of ( diaphorina citri ), Cacopsylla chinensis , Cacopsylla qianli , Cacopsylla pyri , Bactericera cockerell , Cacopsylla oluanpiensis and Cacopsylla tobirae , comprising a substrate; and a plurality of oligonucleotide probes immobilized on the substrate, the oligonucleotide probes comprising at least one of SEQ ID NO: 1 to SEQ ID NO: 4, At least one of SEQ ID NO: 5 to SEQ ID NO: 8, at least one of SEQ ID NO: 9 to SEQ ID NO: 11, at least one of SEQ ID NO: 12 to SEQ ID NO: At least one of SEQ ID NO: 16 to SEQ ID NO: 19, at least one of SEQ ID NO: 20 to SEQ ID NO: 23, and at least one of SEQ ID NO: 24 to SEQ ID NO: 1. SEQ ID NO: 28 to SEQ ID NO: 3 At least one of 1 , at least one of SEQ ID NO: 32 to SEQ ID NO: 35, at least one of SEQ ID NO: 36 to SEQ ID NO: 39, SEQ ID NO: 40 to SEQ ID NO A nucleotide sequence of at least one of: at least one of SEQ ID NO: 44 to SEQ ID NO: 47. A biochip identified in claim 2 or claim 4 for identifying Neophacopteron euphoriae , Trioza outeiensis , T. magnicauda , Paurocephala sauteri , and lemon Microceropsylla nigr , Diaphorina citri , Cacopsylla chinensis , Cacopsylla qianli , Cacopsylla pyri , Bactericera cockerell , Taiwanese Cacopsylla oluanpiensis And a method of Cacopsylla tobirae comprising: (a) extracting deoxyribonucleic acid of the worm to be tested; (b) using a polymerase chain reaction using SEQ ID NO: 48 and SEQ ID NO: 49 for amplification a spacer 2 region (ITS2) deoxyribonucleic acid fragment; (c) performing a hybrid reaction using the biochip of claim 2 or claim 4; and (d) rendering the color on the biochip Spot position identification step (c) results of the heterozygous reaction. A method for identifying Neophacopteron euphoriae , Trioza outeiensis , T. magnicauda , Paurocephala sauteri , Microceropsylla nigr , Diaphorina citri), Chinese pear psylla (Cacopsylla chinensis), Qian pear psylla (Cacopsylla qianli), pear psylla (Cacopsylla pyri), Bactericera cockerell, Taiwan Pittosporum psyllids (Cacopsylla oluanpiensis), Pittosporum psyllids (Cacopsylla tobirae) of the kit, containing a first oligonucleotide probe combination comprising at least one of SEQ ID NO: 1 to SEQ ID NO: 4; a second oligonucleotide probe combination comprising SEQ ID NO: 5 to SEQ ID NO: At least one of 8; a third oligonucleotide probe combination comprising at least one of SEQ ID NO: 9 to SEQ ID NO: 11; a fourth oligonucleotide probe combination comprising SEQ ID NO: 12 to at least one of SEQ ID NO: 15; a fifth oligonucleotide probe combination comprising at least one of SEQ ID NO: 16 to SEQ ID NO: 19; a sixth oligonucleotide probe combination Containing SEQ ID NO: 20 to At least one of SEQ ID NO: 23; a seventh oligonucleotide probe combination comprising at least one of SEQ ID NO: 24 to SEQ ID NO: 27; an eighth oligonucleotide probe combination comprising At least one of SEQ ID NO: 28 to SEQ ID NO: 31; a ninth oligonucleotide probe combination comprising at least one of SEQ ID NO: 32 to SEQ ID NO: 35; a tenth oligonucleoside An acid probe combination comprising at least one of SEQ ID NO: 36 to SEQ ID NO: 39, an eleventh oligonucleotide probe combination comprising at least one of SEQ ID NO: 40 to SEQ ID NO: And a twelfth oligonucleotide probe combination comprising at least one of SEQ ID NO: 44 to SEQ ID NO: 47.