TWI377256B - Primers and kit of thrips identification and identification method for the same - Google Patents

Description

1377256 VI. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a primer pair, an identification kit and a method thereof for rapidly identifying three species of thrips pests, and in particular to a polymerase-linked reaction A quick method for identifying pests in thrips. [Prior Art] The thrips are common pests on the bulbs of plant flowers, leaves or bulbs. The larvae and adults of the thrips feed on plant tissues, causing damage to the flowers and fruits. The females lay eggs in the plant tissues. Often the plants are unable to grow normally, and the quality and yield of the crops are affected. Most of the thrips that damage flowers are spawning in the younger parts of plants, such as sprouts, young leaves, petals, etc., and the appearance and habits of adults and larvae are similar, and their sucking mouthpieces can suck plant tissues. On the petals, the petals will be discolored and rounded and spotted; on the leaves, the leaves will lose their green color and appear yellow-white, deformed and deformed. In addition, the thrips are also the main vector for diseases such as tomato spotted wiltvirus. The larvae of the thrips are extremely small (about 2 mm). It is extremely difficult to identify the larvae of the thrips by traditional morphological identification. If they want to raise them to adults, they need to be five to seven or more. For example, the western flower pheasant is native to the Americas, and it has many types of host plants that are endangered and adaptable. With the frequent spread of economic and trade exchanges to many countries in the world, it is now present in many countries and often causes serious crop risks. . Western flower thrips are quarantine pests in Taiwan's quarantine regulations and have been detected in imported cut flowers, vegetables and fresh fruits. It has been developed to detect the Western flower thrips by the molecular biology method 1377256 LAMP technique, and the sensitivity of the reaction can reach 100 femtograms, and the identification time can be greatly shortened. However, at present, the research on the identification of Hummer's intensive identification is concentrated in the western flowering horses. The identification techniques of other thrips are still to be developed. Because Hummer is very harmful to cash crops, the current research work on Hummer has continued. However, the Hummer has many kinds of curtains, and the individuals are small and similar in appearance, and the naked eye is difficult to distinguish', which increases the difficulty of research work. _ In view of this, there is a need to develop more and faster and breaking identification methods to identify the main pests of thrips. SUMMARY OF THE INVENTION Accordingly, one aspect of the present invention is to provide a primer pair for identifying three Lima pests, which is selected from the sequences having the sequences shown in SEQ ID NO: 1-14 and the above-described sequences. According to an embodiment of the invention, a first primer pair combination comprises three primer pair combinations, an oligonucleotide primer selected from the sequence of SEQ ID ΝΟ: 1-4, which can be used to identify western flower thrips. Wherein, the primer pair (A) consists of the primer FocclU5 (SEQ ID NO: 1) and the primer Focc2D5 (SEQ ID NO: 2); the primer pair (B) consists of the primer FocclU5 (SEQIDNO: 1) and the primer Focc3D (SEQ ID NO: 3) The composition; the primer pair (C) consists of the primer Focc3U (SEQ ID NO: 4) and the primer Focc3D (SEQ ID NO: 3). In accordance with another embodiment of the present invention, a second primer pair combination comprises three combinations of 1377256 primer pairs, selected from the group consisting of oligonucleotide primers of the sequences set forth in SEQ ID NOs: 5-10, which can be used to identify Taiwan flower thrips. Wherein, the primer pair (D) consists of the primer Fint2U (SEQ ID NO: 5) and the primer Fint2D (SEQ ID NO: 6); the primer pair (E) is derived from the primer Fint3U (SEQIDN0: 7) and the primer, Fint3D (SEQ ID N0:8); the primer pair (F) consists of the primer Fint4U . (SEQIDNO: 9) and the primer Fint4D (SEQIDNO: 10). According to still another embodiment of the present invention, a third primer pair combination comprises three primer pair combinations selected from the group consisting of oligonucleoside φ acid primers of the sequences shown in SEQ ID NOS: 11-14, which can be used to identify onion horses. Wherein, the primer pair (G) consists of the primer Ttab971U (SEQ ID NO: 11) and the primer TtablD (SEQ ID NO: 12); the primer pair (H) consists of the primer Ttab972U (SEQIDNO: 13) and the primer TtablD (SEQ ID NO). : 12) Composition; primer pair (I) consists of the primer Ttab972U (SEQ ID NO: 13) and the primer Ttab974D (SEQ ID NO: 14). Another aspect of the present invention is to provide a kit for identifying three Lima pests, comprising a first primer pair combination, a second primer pair combination, and a third primer pair combination. The first primer pair combination is selected from the group consisting of SEQ ID ΝΟ:1 and SEQ ID NO:2, and/or SEQ ID ΝΟ:1 and SEQ ID NO:3, and/or SEQ ID NO:4 and SEQ ID NO:3; The second primer pair combination is selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 6, and/or SEQ ID NO: 7 and SEQ ID NO: 8, and/or SEQ ID NO: 9 and SEQ ID NO: 10; The third primer pair • The combination is selected from 3 £(}101^0:11 and 3 ugly (310)^0:12, and/or 8£(310

NO: 12 and SEQ ID NO: 13, and/or SEQ ID NO: 13 and SEQ ID NO: 14. Another aspect of the present invention provides a 1377256 method for identifying three species of locust pests, which comprises the steps of: (a) providing a genomic DNA of the worm to be tested as a template, and the concentration of the DNA template is a single a 1/50 ratio of the thrips extracted DNA, about 5 ng; (b) a polymerase chain reaction using at least three pairs of primer pairs or a sequence derived therefrom, the at least three pairs of primer pairs being selected from the first primer pair combination At least one of the primer pair, the second primer, at least one of the combination of at least one of the combination and the third primer pair; and (c) analyzing a fragment of the polymerase chain reaction increase The length, by increasing the molecular weight of the target nucleic acid fragment, can quickly identify three species of cockroach pests. [Embodiment] The embodiments of the present invention use molecular biology techniques to design different specific oligonucleotide primer pairs by using different strains at the molecular level of the genome to quickly and accurately identify different Hummers. Pests. In the embodiment of the present invention, the IRIS2 (intergenic spacer 2) DNA sequence is used as a template, and the primer pair consisting of the primers Pl-2 (SEQ ID NO: 15) and the primer 28SJ2 (SEQ ID NO: 16) is subjected to a polymerase chain reaction amplification increase. The fragment was separated by electrophoresis on agar extract and the template DNA was purified using a recovery reagent. The template DNA is determined by the automatic sequencer to determine the sequence 0. Using the above primer P1-2 and the primer 28SJ2, the Frankliniella occidentalis (Frankliniella / «ίοlike α) and the onion are amplified. Horses (three different hummer's chromosome ITS2 DNA fragments, sequenced by nucleic acid autosequencer, using BioEdit program software to compare the DNA sequences of each taxon with 1377256 and search for multiple species Sequence differences, designing the specific primers of western flower thrips, Taiwan flower thrips, and onion thrips. The first picture shows the ITS2 DNA sequence alignment of three thrips; western flower thrips, Taiwan flower thrips, onion horses The abbreviations are Focc, Fint, and Ttab. Using the difference in sequence of ITS2 DNA fragments of different thrips, a specific pair of primers can be identified that can be identified from different thrips, selected from sequences identified as SEQ ID NO: 1-14. The sequence shown and the sequence derived from the above sequence. According to an embodiment of the invention, a first primer pair combination comprises three combinations of primer pairs selected from the group consisting of SEQ ID NO: 1 -4 Oligonucleotide primer sequences shown promoter, may be used to identify western flower thrips. Wherein primer primer pair (A) of

FocclU5 (SEQ ID NO: 1) and the primer Focc2D5 (SEQ ID NO: 2) can increase the 236 bp fragment (SEQ ID NO: 17) in the deoxyribonucleic acid sample containing the western flowering horse; the introduction of the pair (B) FocclU5 ( SEQ ID NO: 1) and the primer Focc3D (SEQ ID NO: 3) can increase the 243 bp fragment (SEQ ID NO: 18) in the deoxyribonucleic acid sample containing Western flower Lima; the primer pair (C) primer Focc3U ( SEQ ID NO: 4) and the primer Focc3D (SEQ ID NO: 3) can be used to increase a 150 bp fragment (SEQ ID NO: 19) in a DNA sample containing Western flower scorpion. In accordance with another embodiment of the present invention, a second primer pair combination comprises three primer pair combinations selected from the group consisting of oligonucleotide primers of the sequences set forth in SEQ ID NOs: 5-10, which can be used to identify Taiwan flower thrips. Among them, the primer pair Fint2U (SEQ ID NO: 5) and the primer Fint2D (SEQ ID n〇: 6)

A 142 bp fragment (SEQ ID NO: 20) can be added to a DNA sample containing T. chinensis. The primer pair (E) is introduced with Fint3u (SEQ 1377256 ID N〇: 7) and the primer Fint3D (SEQ ID NO). :8) A 303 bp fragment (SEQ ID NO: 21) can be amplified from a DNA sample containing T. chinensis (SEQ ID NO: 9); the introduction (F) is introduced with Fint4U (SEQ ID NO: 9) and the primer Fint4D (SEQ ID NO) :10) A 243 bp fragment (SEQ ID NO: 22) can be amplified in a DNA sample containing the T. sinensis. According to still another embodiment of the present invention, a third primer pair combination comprises three primer pair combinations, and an oligonucleotide primer selected from the sequence of SEQ ID NOS: 11-14 can be used to identify onion horses. Among them, the primer pair (G) primer • Ttab971U ( SEQ iD ΝΟ: 11 ) and the primer TtablD ( SEQ ID NO: 12 ) can increase the 420 bp fragment (SEQ ID) in the DNA sample containing onion thrips NO:23); primer pair (Η) primer Ttab972U (SEQ ID NO: 13) and primer TtablD (SEQ ID NO: 12) can be used to extract a 397 bp fragment in a DNA sample containing Huihui horse ( SEQ ID NO: 24); primer pair (I) primer Ttab972U (SEQ ID NO: 13) and primer Ttab974D (SEQ1〇NO: 14) can increase 346 bp in the DNA sample containing onion thrips Fragment (SEQ ID NO: 25). φ Due to the nature of the polymerase chain reaction itself, even if there is variability in the sequence between the primer and the template to be amplified, a specific DNA fragment can be synthesized by adjusting the reaction temperature of the binding step in the polymerase chain reaction. Thus, in light of the teachings of the present invention, those skilled in the art will be able to design different primers depending on the DNA fragment to be amplified. Therefore, any primers formed by base substitution, addition or reduction for the individual primers described in the embodiments of the present invention can be increased by including the primer pairs corresponding to the embodiments of the present invention. The specific fragments of SEQ ID NO: 17-25 are not intended to be excluded from the scope of the invention. 1377256 Therefore, the above-mentioned individual primer pair may comprise a nucleotide sequence which is modified at the 3' or 5' end of at least one of the primers, and which has a similarity to the original sequence of 7 % or more; and the derivative The oligonucleotide sequence (hereinafter referred to as the derived sequence) can still be amplified together with another primer (the sequence can be the original sequence or the derived sequence) of the corresponding primer pair, and the same specific fragment sequence as described in the embodiment of the present invention is included, and after the amplification The specific fragment sequence having 80% or more similarity with the fragment described in the embodiment of the present invention does not depart from the scope of the present invention.

In accordance with an embodiment of the present invention, a kit for identifying western flower thrips is provided, comprising at least one primer pair selected from the group consisting of primer pair (A), primer pair (B), and primer pair (c). According to an embodiment of the present invention, there is provided a kit for identifying Taiwan comprising at least a pair of primers selected from the group consisting of primer pair (D), = pair (E), and primer pair (F). In accordance with an embodiment of the present invention, a kit for identifying 1|3 horses is provided, comprising at least one pair of primers selected from the group consisting of primer pair (G), primer pair (I). The above-mentioned kit for identifying western flower thrips, Taiwan flower thrips and onion thrips further comprises reagents required for extracting chromosomal DNA of the test worm; and/or reagents required for performing a polymerase chain reaction; and/or The 5 types of agents required for electrophoresis analysis include, but are not limited to, agarose gel electrophoresis or capillary tube gel electrophoresis. A method for identifying a pest pest of an embodiment of the invention comprises the following steps: (a) providing a genomic DNA of the worm to be tested as a template, and extracting chromosomal DNA from the worm body can be obtained by a skilled worker This is achieved by the method of 1377256. The collection of horse-horse specimens is mainly in Taiwan, while the western flower-horse horses are foreign horse specimens. Specimens are immersed in 75% alcohol and stored at -20 °c for later use. For the extraction method of deoxyribonucleic acid from the thrips sample, the method of ^/z (1997) can be slightly modified or a commercially available DNA-purification reagent can be used. The obtained DNA solution was stored at -20 °C for use, and the skeletal specimen of the lysate _DNA was prepared into a slide specimen, and the collection period and location were marked on the slide as a stored specimen. (b) Perform a polymerase chain reaction using (A) - (I) primers. The φ polymerase chain reaction can be achieved by methods well known to those skilled in the art. The polymerase chain reaction usually consists of three steps: (1) denaturation of a template to form two single strands; (2) bonding the two primers to the two strands of step (1); (3) The DNA polymerase extends the primers to obtain two pairs of DNA. The above steps are repeated cyclically, and amplification is obtained by a specific DNA fragment. In general, in order to allow the reaction to proceed sufficiently, a first stage denaturation step is typically performed prior to the recycle reaction. According to one embodiment of the invention, the polymerase chain reaction conditions are raised at $95 ° C for 2 minutes, followed by 35 cycles of 95 ° C for 40 seconds, 60 ° C for 20 seconds, and 72 ° C for 20 seconds, followed by 72 cycles. The reaction was completed in °C for 10 minutes, and the product after the reaction was stored at 4 ° C until use. The polymerase chain reaction product was used for subsequent gel electrophoresis analysis. (c) Analysis of the fragment length of the polymerase chain reaction amplification, which can be rapidly identified by increasing the molecular weight of the target nucleic acid fragment. According to another embodiment of the present invention, before the step (b), the primer pair consisting of the primer P1-2 (SEQ ID NO: 15) and the primer 28SJ2 (SEQ ID NO: 16) is subjected to a polymerase chain reaction to increase the amplitude of the thrips chromosome ITS2 1377256. The DNA fragment is amplified and further purified and used as template DNA. In the example exemplified in the present invention, a total of 17 species of Puma, which are closely related to the western flower thrips, are tested, including the Taiwanese flower horse "Scirtothrips dorsalis", the corn thrips (Frankliniella williamsi), * red With Selenothrips rubrocinctus, TTirips palmi, Rkipiphowthrips cruentatus, Hel Hel (Helionothrips 印·), cotton 蓟 ( (Liu milk·β Μα故) , back of the board. 3⁄4 Equipped with a genus (the 功 汍 . ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 及 及 ) ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( Abdominalis ), in Cthrips hawaiiensis, Bolacothripsgraminis, Bathrips melanicomis, Dichromothrips corto", Beans hummer (Megfl/Eye W5Z· She 5) and Eucalyptus Hummer (ewc/ifln7). Analysis of DNA fragment size by agarose gel electrophoresis after amplification by polymerase chain reaction was negative for all non-Western Thrips and blanks tested. In the example exemplified in the present invention, a total of 12 species of scorpion, which are closely related to the 蓟 蓟 台湾 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 蓟 蓟 蓟 蓟 蓟 蓟 蓟Horses, chrysanthemums, horses, brown-tailed horses, black-horned horses, horses, horses, eucalyptus horses, Tubulifera, and Thrips sp. After amplification by polymerase chain reaction, the size of the DNA fragment was analyzed by agarose gel electrophoresis, and all tested non-Taiwan flower thrips and blank group were negative. + In the example exemplified in the present invention, the test is similar to the onion. The horse is closely related to the horse. There are 17 species of horses, including the western flower hummer, the Taiwan flower hummer, the small yellow pheasant horse, the corn 15 horse, the red belt hummer, Nanhuang, Lai Gj, New Guan Ma, Chrysanthemum Horse, Flower. 15 Horse, Brown Tail Lima, Black-horned Pelican Horse, Orchid Hummer, Eucalyptus Lima, 1377256 Green Horse (Thrips Alliorum), Stendchaetothrips biformis and Beans Thrips. After amplification by polymerase chain reaction, the DNA fragment size was analyzed by agarose gel electrophoresis, and all tested non-green onions and blanks were negative. It can be seen that the specific primer pair of the embodiment of the present invention has high sensitivity and specificity of identification, and can quickly identify western flower thrips, Taiwan flower thrips and onion horses, which is superior to the uncertainty of traditional morphological identification. Embodiments of the present invention also provide a φ set for identifying three damhor pests, comprising a first primer pair combination, a second primer pair combination, and a third primer pair combination. Wherein the first primer pair combination comprises at least one primer pair selected from the group consisting of SEQ ID ΝΟ: 1 and SEQ ID NO: 2, SEQ ID NO: 1 and SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 3 and a pair of primers consisting of the derived sequences of the above individual primers. The second primer pair combination comprises at least one primer pair selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: A pair of bows composed of the derived sequences of the above individual primers. The third primer pair combination includes at least one primer pair selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.

SEQ ID NO: 12 is a primer pair consisting of SEQ ID NO: 13, SEQ ID NO: 13 and SEQ ID NO: 14 and the derived sequences of the above individual primers. According to an embodiment of the present invention, the kit further comprises a reagent required for extracting the chromosomal DNA of the worm to be tested, and/or a reagent required for performing a polymerase chain reaction; and a reagent required for the electrophoresis of the electrophoresis . Electrophoretic analysis includes, but is not limited to, Joan gel electrophoresis or capillary gel electrophoresis. A method for identifying three sets of Lima-like A, comprising polymerase chain reaction using at least three pairs of primer pairs or a sequence derived therefrom, and by rapidly increasing the molecular weight of the dinuclear segment of 12 1377256 = The horses = = : (4) 'the pair of primers are selected from the first to the first ones." Among them, the pair of primers, the pair of second primer pairs, the pair of primers and the third primer are heterozygous. A primer pair. The present invention is not limited by the following examples, but is limited to the disclosure of the following examples.

Example 1 Agar using a specific fragment of the specific specific colorimetric ITS2 of the first primer pair. Referring to Fig. 2A-2C, the primers were used to increase the gel electrophoresis patterns of different types of thrips. 2 'Fig. 2A is an electrophoresis pattern of specific fragments of different species of Lymus color using the primer pair (A). The Μ is a 100 bp DNa ladder flag 'C is the blank control group, and the lane (3) represents the different types of Lima samples (Table 2). The results of Figure 2A show that pCR increases in various horse samples with Western flower thrips specific primer pair (A), and the species tested in addition to lanes 7, 8, 9, 1 and 22 of the Western flower Lima sample 236 There were no obvious target bands, and the other different species (4) horse samples and blank control groups did not increase to the product. Figure 2B is an electrophoresis pattern of specific fragments of different types of Lima chromosome ITS2 amplified by primer pair (8). Material 1〇〇# dna ladder Zen, one c is the blank control group, and the lanes 1-24 represent different types of Lima samples: (Table 1) The result of the 2B figure shows the Western flower 蓟 horse specific primer pair (B ) The PC:R increase was performed on each of the horse-made samples, and the species in the field except the swimming 13 1377256 捭攸=f western flower hummer sample showed obvious eyes at about 243 bp). The horse sample and the blank control group were not ττπΐί maps using the primer pair (C) to increase the electropherogram of specific fragments of different types of 15 horse chromosomes. Μ is a 100 bp DNA ladder

One c is the white control group, and the lane 122 represents different kinds of horses. The results of the 2C chart show that PCR amplification is performed on each horse sample by Western flower Lima specific primer pair jC). The western flower Lima samples of lanes 7, 8, 9, 1 and 22 showed obvious target strips (4) at about 15〇*, and the remaining non-_ma and blank control groups did not increase to the product. According to the above example, it can be seen that the primer pair (A)' primer pair (b) and the primer pair jC) can respectively increase the yield of the sample containing the western flower 15 4 DNA, and the specificity is not obvious. The non-Western flower Lima's sample showed a target segment, showing that the primer pairs (A), (B) and (c) have excellent specificity and can be applied to accurately detect Western flower thrips. ,

Example 2 Referring to Fig. 3A-3C, an agarose gel electrophoresis pattern of a specific fragment of ITS2 of different species of thrips was added to the specificity of the combination of the second primer pair.

Among them, the 3A map uses the primer pair (D) to increase the electropherogram of specific fragments of different types of thrips chromosome ITS2. M is a 1 〇〇 Qi DNA ladder, and lanes 1-12 represent different types of hummer samples (Table 3). The results of Fig. 3A show that PCR amplification of various ^//256 4 horses is carried out by the Taiwan flower 蓟 horse specific primer pair (d), and the tested species of the species of the flower bud horse in the lane 12 are apparent at 142 bp. Outside the target strip, the remaining samples of the non-planted thrips did not increase to the product.帛3B uses the primer pair (E) to increase the electropherogram of different types of Lima chromosomes • specific fragments of ITS2. Μ is a 1 〇〇 bp DNA ladder, and lane M2 represents a different species of hummer (Table 3). The 3B ^ ^ results showed that PCR amplification was performed on various Lima by the Taiwan flower s horse-special-sex scorpion pair (8), and the species tested were in addition to the Western Hualima sample of the lane 12 _ at a target of about 303 bp. Outside the strip, the other different types of 'Qi Ma samples did not increase to the product. Figure 3C (4) Use the primer pair (F) to increase the electropherogram of specific fragments of different types of genomic chromosomes. The _ bp DNA ladder flag 'c is the blank control group' and the lane 丨 (1) represents the different species (four) of the horse sample (Table 3). The results of Figure 3C show that PCR amplification was performed on various 15 Maxian counties with the Taiwanese Pony-specific primer pair (F). The species of the test, except for the Taiwanese® horse sample of the swim lane 12, showed a clear target at about 243 bp. Outside the strip, • The remaining 15 horses and blank control groups of different species did not increase to the product. According to the above examples, it can be seen that the pair of primers (D), the pair of primers (E) and the pair of primers (7) can respectively produce a stable and distinct specialized fragment in the sample containing Taiwan flower _DNA, and will not be in non-Taiwan Huali. The target sample is increased in the sample of the horse, showing that the y subpairs (D), (E) and (f) have excellent specificity and can be applied to accurately detect Taiwan flower Lima. Example 3 Please refer to Section 4A-4C for the specific tube gel electrophoresis pattern of 15 Ma chromosomes using the third primer pair combination specificity 1377256 primer. Only the capillary where '4A is an electropherogram of the specific fragment of the chromosome leg amplified by the primer pair (6). Eight Employees = Flags' lanes represent different types of Lima samples (Table 4). Knife = two onions 15 horses - sexual introduction pairs (6) for a variety of Lima two Ι ΤΤ ΤΤ: ^ ^ 420 bp at the obvious target ^ Samples did not increase to the product. The specificity of each of the Lema (Η) of the other is not shown in Figure I.

This ((4). The first-graph t-tnot ^ not m-special--the scorpion pair (H) on each _ horse PCR =, the species tested except the lane 12 onion Lima sample in about 3 □ 97 = : increase Outside the target strip, the remaining samples of different Laima samples are all sub-paragraphs of 51 pairs. (1) Increased electrophoresis patterns of different species of Laima chromosome: two 2 f specific fragments. Μ is DNA molecular weight:: body ice tract 1 - 24 represents different The main leopard species of the leopard species, the lane 1_24 represents the different sex primer pairs (1) #4) ° The results of the 4C graph show that the onion 15 horses specific species = swimming H species Lima sample to take the increase, tested The standard band showed a significant product at about 346 bp. The same species of thrips and the blank control group did not increase. According to the above example, it can be seen that the increase in the sample of the primer pair (6), the primer pair (8) and the reference 1377256 is stable. The increase in the sample of the hummer and (I) the excellent pair (I) can be distinguished from the specific fragment of the scorpion dna, and will not show the primer pair in the non-onion target segment. (G), (h) One-sex can be applied to accurately detect onion horses. Although the invention has been implemented The above is not intended to limit the invention to any of the skilled artisans. As far as I am within the spirit and scope of the present invention, various modifications and retouchings can be made, and therefore the application of the present invention is attached. The scope defined by the patent scope shall prevail.

BRIEF DESCRIPTION OF THE DRAWINGS In order to make the above and other objects, features, advantages and embodiments of the present invention more obvious, the description of the drawings is as follows: Figure 1 is an alignment of ITS2 DNA sequences of three thrips. . Figure 2A is an electropherogram of specific fragments of different species of Thrips ITS2 amplified by primer pair (A).

Figure 2B is an electrophoresis pattern of specific fragments of different types of thrips chromosome ITS2 amplified by primer pair (B). The 2C figure uses an introduction pair (C) to increase the electropherogram of specific fragments of different species of Thrips chromosome ITS2. Fig. 3A is an electrophoresis pattern of specific fragments of different types of carved chromosome ITS2 amplified by primer pair (D). Figure 3B is an electrophoresis pattern of specific fragments of different types of Lima chromosome ITS2 amplified by primer pair (E). Figure 3C uses an introduction pair (F) to increase the electropherogram of specific fragments of different types of Lima chromosome 17 1377256 ITS2. Figure 4A is an electropherogram of the specific fragment of the different species of the horse chromosome ITS2 using the primer pair (6). Figure 4B is an electrophoresis pattern of specific fragments of different types of thrips using the primer pair (H). • Figure 4C uses an introduction pair (I) to increase the electropherogram of specific fragments of different species of Thrips chromosome ITS2. • [Main component symbol description] No 1377256 Sequence Listing <110> National Chung Hsing University <120> Identification of specific primer pairs, identification kits and methods of 蓟Horse pests <160>10 <210> SEQIDNO: 1 <211>26 <212>DNA <213 > artificial sequence < 220 > primer_bind < 230 > Identification of Western flower thrips specific primer

<400>1 ggtcgcttca ccgct.tcccc cgtaaa 26 <210> SEQEDNO: 2 <211>26 <212>DNA<213>Artificial sequence<220 >primer_bind <230> Identification of Western flower hummer Sexual introduction <400>2 caaagtgcga gaaaataatg caaact 2 6

<210> SEQ ID NO: 3 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > Primerbind <230> Identification of Western flower thrips specificity primer <400>3 cgaaacgcaa agtgcgagaa aataatgc 28 1377256 <210> SEQ ID NO: 4 < 211 > 26 < 212 > DNA < 213 > artificial sequence < 220 >primer_bind < 230 > Identification of Western flower thrips specificity <400 > 4 agcctccaga cgttctgcca primer aaag

<210> SEQ ID NO: 5 < 211 > 27 < 212 > DNA < 213 > artificial sequence < 220 > primerbind < 230 > Identification of Taiwan flower 蓟 horse specificity primer <400 > 5 atcctgtatg gagaaagcac tc tgc Eg

<210> SEQ ID NO: 6 < 211 > 27 < 212 > DNA < 213 > Artificial Sequence < 220 > Primerbind < 230 > Identification of Taiwan Flower Thrips Specificity <400>6 cgagtacgag gacaagaaac gtcacac <210> SEQ ID NO: 7 < 211 > 21 < 212 > DNA < 213 > artificial sequence 1377256 * < 220 > primer_bind < 230 > Identification of Taiwan flower 蓟 horse specific primer <400>7 cagactgttc cgagagaaat c 21 <210> SEQ ID NO: 8 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > primer_bind < 230 > Identification of Taiwan flower 蓟 horse specific primer

<400>8 2 0 ttttgttgca cacttttccg <210> SEQ ID NO: 9 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > Primerbind < 230 > Identification of Taiwan flower 蓟 horse specific primer

<400>9 ccaaactcaa agaccagact gttccgagag 3 0 <210> SEQ ID NO: 10 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > Primer_bind < 230 > Identification of Taiwan Flower Horse Specificity Primer <400>10 cgtcgagtac gaggacaaga aacgtcac 28 3 1377256 <210> SEQ ID NO: 11 <211>25 <212>DNA <213>Artificial Sequence<220 >primer_bind <230> Sexual introduction <400>11 agaaacgatt accagactgc ccaag

<210> SEQ ID NO: 12 < 211 > 25 < 212 > DNA < 213 > artificial sequence < 220 > primer_bind < 230 > Identification of onion and horse specificity primer <400 > 12 agtgatgcag cacaacacat t Ccac

<210> SEQ ID NO: 13 < 211 > 28 < 212 > DNA < 213 > artificial sequence < 220 > primer_bind < 230 > Identification of onion and horse specificity primer <400 > 13 agcgacagca cacacctcgt gtgtgttg <;210>SEQIDNO: 14 <211>26 1377256 <212>DNA<213>Artificial sequence<220>primerbind<230> Identification of onion and horse specificity primer<400>14 cagtgatgca gcacaacaca ttccac 26 <210> SEQ ID NO: 15 < 211 > 19 <212 > DNA < 213 > Artificial Sequence < 220 > Primer_bind And Onion Horse Chromosome ITS2 DNA Fragment Specificity Primer 19 <230> Western Flower Hippo , Taiwan flower 蓟 horse <400>15 gtggatccct gggcttgtg <210> SEQIDNO: 16 <211>17 <212>DNA <213> artificial sequence < 220 > primerbind

<230> Western flower 蓟 horse, Taiwan flower 蓟 horse, and 蓟 蓟 horse chromosome ITS2 DNA fragment specificity primer <400>16 gttagtttct tttcctc 17 <210> SEQ ID NO: 17 <211>236

<212>DNA<2\3> Frankliniella occidentalis <220 > Misc-feature <223> Western flower 蓟 horse chromosome ITS2 DNA characteristic region <400>17 tggtcgcttc accgcttccc ccgtaaagag aaagcactct gcctagcggt 50 1001377256 acgtcttata aaggatagcc agagagcgcc Ctcgtgcgg agacagcctc cagacgttct gccaaaagcg gcgggtgtgt gacgtttctt gttctcggac tcgacgtcgc gccgcccgtc ttgaagactt ggaggtatgc cttacaaaga gcaaccgcgc agtttgcatt attttctcgc actttg

<210> SEQ ID NO: 18 < 211 > 243 < 212 > DNA < 213 > Frankliniella occidentalis < 220 > MisC-feature < 223 > Western flower 蓟 horse chromosome ITS2 DNA characteristic region <400>;18

tggtcgcttc accgcttccc ccgtaaagag aaagcactct gcctagcggt acgtcttata aaggatagcc agagagcgcc ctcgtgcgct agacagcctc cagacgttct gccaaaagcg gcgggtgtgt gacgtttctt gttctcggac tcgacgtcgc gccgcccgtc ttgaagactt ggaggtatgc cttacaaaga gcaaccgcgc agtttgcatt attttctcgc actttgcgtt teg < 210 > SEQE) NO: 19 < 211 > 150

<212>DNA < 213 > Frankliniella occidentalis < 220 > Misc_feature <223 > Western flower 蓟 horse chromosome ITS2 DNA characteristic region

150 200 236 50 100 150 200 243 <400 >19 cagcctccag acgttctgcc aaaagcggcg ggtgtgtgac gtttcttgtt 50 ctcggactcg acgtcgcgcc gcccgtcttg aagacttgga ggtatgeett 100 acaaagagca accgcgcagt ttgeattatt ttctcgcact ttgcgtttcg 150 <210>SEQIDNO:20 <211 >142

< 212 > DNA < 213 > Frankliniella intonsa < 220 > Misc_feature < 223 > Taiwan flower 蓟 horse chromosome ITS2 DNA characteristic region 6 1377256 <400 >20 atcctgtatg gagaaagcac tctgccggta caaagcggta cgtcttataa aggataacca gagagcgccc ccgcgcgc acagccctca gacgttctgc Caaaagttgg cgggtgtgtg acgtttcttg tcctcgtact eg <210>SEQIDNO:21 <211>303

<212>DNA < 213 > Frankliniella intonsa < 220 > Misc a feature <223> Taiwan flower 蓟 horse chromosome ITS2 DNA characteristic region <400>21

50 100 142 50 100 150 200 250 300 303 cagactgttc cgagagaaat ctctggagcg aggttggagt ctccgcgcgc gagcgtggta ctcttaaaat cctcagggag tcacatcctg tatggagaaa gcactctgcc ggtacaaagc ggtaegtett ataaaggata accagagagc gcccccgcgc gctaacagcc ctcagacgtt ctgccaaaag ttggcgggtg tgtgacgttt cttgtcctcg tactcgacgt cgcagcgccc gtettgaaga cttgagggta tgccttataa agagcaaccg ttccggaaaa gtgtgcaaca aaa

<210> SEQ ID NO: 22 < 211 > 243 <212 > DNA < 213 > Frankliniella intonsa < 220 > Misc a feature < 223 > Taiwan flower 蓟 horse chromosome ITS2 DNA characteristic region <400 &gt ; 22 ccaaactcaa agaccagact gttccgagag aaatctctgg agcgaggttg 50 gagtctccgc gegegagegt ggtactctta aaatcctcag ggagtcacat 100 cctgtatgga gaaagcactc tgccggtaca aagcggtacg tettataaag 150 gataaccaga gagcgccccc gcgcgctaac agccctcaga cgttctgcca 200 aaagttggc gggtgtgtgac gtttcttgtc ctcgtactcg aeg 243 < 210 > SEQIDNO: 23 < 211 > 420

<212>DNA 7 50 < 213 > Vnrips tabaci < 220 > Miscfeature <223> The onion horse chromosome ITS2 DNA characteristic region <400 >23 agaaacgatt accagactgc ccaagcgaca gcacacacct cgtgtgtgtt ggagcagggc gagcttgggg ttctgctccc tttgcggagt agttccctta aatgccttga ggcagaggct tctctcagtc ttactgatta taaggaaatc agttcgtcgc aagatgaacc ctgatggact tggtcggttc gacacgaaaa agcacgcaaa cttgagagta cgtctctttg cctcacgcat tctacaatca aacgttcagt tgaaaacaag tagttgggcg tgaagacccc agcgaacggg tgcagtacag gactcgaagc tcgtgcattt agcgtgcgcc tgagtggaat gtgttgtgct gcatcactga ataaaaacga ttcgaatgca ttttgtaatc cgacctcagt tcaggagaga < 210 > SEQIDNO: 24 < 211 > 397

<212>DNA < 213 > Thrips tabaci < 220 > Misc-feature <223> The onion horse chromosome ITS2 DNA characteristic region <400 >24 agcgacagca cacacctcgt gtgtgttgga gcagggcgag cttggggttc tgctcccttt gcggagtagt tcccttaaat gccttgaggc agaggcttct ctcagtctta ctgattataa ggaaatcagt tcgtcgcaag atgaaccctg atggacttgg tcggttcgac acgaaaaagc acgcaaactt gagagtacgt ctctttgcct cacgcattct acaatcaaac gttcagttga aaacaagtag ttgggcgtga agaccccagc gaacgggtgc agtacaggac tcgaagctcg tgcatttagc gtgcgcctga gtggaatgtg ttgtgctgca tcactgaata aaaacgattc gaatgcattt tgtaatccga cctcagttca ggagaga

<210> SEQ ID NO: 25 < 211 > 346 < 212 > DNA < 213 > Thrips tabaci < 220 > Miscfeature < 223 > Onion 蓟 horse chromosome ITS2 DNA characteristic region <400 > 100 150 200 250 300 350 400 420 50 100 150 200 250 300 350 397 1377256 agcgacagca cacacctcgt gtgtgttgga gcagggcgag cttggggttc 50 tgctcccttt gcggagtagt tcccttaaat gccttgaggc agaggcttct 100 ctcagtctta ctgattataa ggaaatcagt tcgtcgcaag atgaaccctg 150 atggacttgg tcggttcgac acgaaaaagc acgcaaactt gagagtacgt 200 ctctttgcct cacgcattct acaatcaaac gttcagttga aaacaagtag 250 ttgggcgtga agaccccagc gaacgggtgc Agtacaggac tcgaagctcg 300 tgcatttagc gtgcgcctga gtggaatgtg ttgtgctgca tcactg 346

Claims (1)

1377256 Announcement Amendment page on August 24, 101. VII. Patent application scope: .哞August 彳日 Revision 1. A kit for identifying three species of scorpion-like pests, Western flower scorpion, Taiwan flower scorpion horse, and onion horse, including: First primer pair combination, including SEQ ID NO: 1 and SEQ ID NO: 2. a primer pair of SEQ ID NO: 4 and SEQ ID NO: 3; a second primer pair combination comprising SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID a primer pair of NO: 10; and a third primer pair combination comprising SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 12 and SEQ ID NO: 13, SEQ ID NO: 13 and SEQ ID NO: 14. The pair of introductions. 2. The kit of claim 1 further comprising the reagent required to extract the DNA of the chromosomal DNA to be tested. 3. The kit as described in claim 1 further contains the reagents required for the polymerase chain reaction. 4. The kit as described in claim 1 further contains the reagents required for electrophoresis analysis. 5. A kit for identifying three species of thrips, Western flower thrips, Taiwan flower thrips, and Hui Lima, comprising: a first primer pair combination comprising SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID ΝΟ: 1 and SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID 1 Aug. 24, 101 revised alternative page 1377256 NO: 3 primer pair; second primer pair combination comprising SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO :7 and the primer pair of SEQ ID NO: 8; and the third primer pair combination, comprising SEQ ID NOill and SEQ ID NO: 12, SEQ ID NO: 12 and SEQ ID NO: 13, SEQ ID NO: 13 and SEQ ID NO: 14 pairs of primers. 6. - Identification of three species of thrips, western flower thrips, and Taiwan flower horses. And a method for the onion horse, comprising: (a) providing a DNA of the chromosome ITS2 gene of the worm to be tested as a template; (b) performing a polymerase chain reaction with the template using three pairs of primer pairs, The third pair of primer pairs comprises: the first primer pair combination comprises: SEQ ID ΝΟ: 1 and SEQ ID NO: 2; and SEQ ID NO: 4 and SEQ ID NO: 3; the second primer pair combination comprises: SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; and SEQ ID NO: 9 and SEQ ID NO: 10; the third primer pair comprises: SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 12 and SEQ ID NO: 13; and SEQ ID NO: 13 and SEQ ID NO: 14; and (c) analysis of the fragment length of the polymerase chain reaction amplification according to the waiting 2 1377256 *.. August 24, 101 The molecular weight of the target nucleic acid fragment amplified by the template of the replacement page test body is greater than the molecular weight of the target worm, wherein: the molecular weight of the target nucleic acid fragment amplified by the template of the test worm is 236 bp Or 150 bp, the worm to be tested is a western flower Lima; the target nucleus of the template to be tested is increased The molecular weight of the fragment is 142 bp, 243 bp or 303 bp, and the worm body to be tested is a Taiwan flower scorpion horse; and the molecular weight of the target nucleic acid fragment amplified by the template of the worm body to be tested is 420 bp, 397 bp or 346 bp, the body to be tested is a green onion horse. 7. A method for identifying three species of thrips, western flower thrips, Taiwan flower thrips, and lynx horses. 'Including · (a) providing the DNA of the chromosome ITS2 gene of the test worm as a template; (b) performing a polymerase chain reaction with the template using three pairs of primer pairs comprising: a first primer pair combination comprising: SEQ ID ΝΟ: 1 and SEQ ID NO: 2; SEQ ID ΝΟ: 1 in combination with SEQ ID NO: 3; and SEQ ID NO: 4 and SEQ ID NO: 3; a second primer pair comprising: SEQ ID NO: 5 and SEQ ID NO: 6; and SEQ ID NO: 7 and SEQ ID NO:8; Modified on August 24, 101, replaces page 1377256. The third primer pair combination comprises: SEQ ID NO: ll and SEQ ID NO: 12; SEQ ID NO: 12 and SEQ ID NO: 13; and SEQ ID NO: 13 and SEQ ID NO: 14 And (c) analyzing the length of the fragment of the amplification of the polymerase chain reaction, according to the molecular weight ratio of the target nucleic acid fragment increased by the template of the test worm, the worm body to be tested, wherein: the worm body to be tested The molecular weight of the target nucleic acid fragment amplified by the template is 236 bp, 243 bp or 150 bp, and the worm body to be tested a western flower thrip horse; the target nucleic acid fragment of the test worm body has a molecular weight of 142 bp or 303 bp, and the test worm body is a Taiwan flower thrip horse; and the template of the test worm body The molecular weight of the amplified target nucleic acid fragment is 420 bp, 397 bp or 346 bp, and the worm is to be stalked.