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WO2013065957A1 - Composition de sonde, puce à adn et kit de classification d'aleurodes, et procédé de classification d'aleurodes les utilisant - Google Patents

Composition de sonde, puce à adn et kit de classification d'aleurodes, et procédé de classification d'aleurodes les utilisant Download PDF

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WO2013065957A1
WO2013065957A1 PCT/KR2012/008113 KR2012008113W WO2013065957A1 WO 2013065957 A1 WO2013065957 A1 WO 2013065957A1 KR 2012008113 W KR2012008113 W KR 2012008113W WO 2013065957 A1 WO2013065957 A1 WO 2013065957A1
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oligonucleotides
seq
nucleotide sequence
probe
powder
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Korean (ko)
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황승용
이원선
김지훈
정진욱
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GENOCHECK CO Ltd
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GENOCHECK CO Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a probe composition, DNA chip and kit for classifying and discriminating whitefly pests belonging to the family Aleyrodidae, and to a method for classifying flours using the same, and more specifically, to classifying tobacco powder and greenhouse powder.
  • the present invention relates to a probe composition, a DNA chip and a kit for determining the biotype of tobacco powder, and a method for classifying flours using the same.
  • the present invention provides a simple, rapid and accurate type and / or type of flours by analyzing genotypes based on single nucleotide polymorphisms (SNPs) present in the COI gene region of mitochondrial DNA of tobacco flour and greenhouse dust.
  • SNPs single nucleotide polymorphisms
  • the present invention relates to a probe composition, a DNA chip and a kit capable of distinguishing and discriminating a biotype, and a method for classifying flours using the same.
  • Agrobacterium is an absorptive insect that occurs in about 600 species belonging to Hemipera Aleyrodidae. Its official name is Bemisia tabaci and Trialeurodes vaporariorum .
  • Tobacco and greenhouse powders are the main pests of greenhouse crops that affect not only gourds, crops, eggplants and flowers, but also strawberries and leafy persimmons. It can cause secondary sooting, lower crop growth, lower commodity value and, in some regions, also infect viral diseases. All of these therefore cause serious problems in the production of vegetables and ornamentals.
  • Tobacco powder in particular, is a pest that causes a wide range of damage, with approximately 1200 species of powder distributed worldwide, among insects. Tobacco powder not only damages crops by direct secretion, but also Begomovirus (Geminiviridae), Crinivirus, Carlavirus (Potyviridae). Over 100 species of plant viruses, including), which can cause significant indirect damage. In addition, the adults and larvae are also parasitic on the back of the leaves, absorbing the juice of the plant, thereby inhibiting the growth of crops, reducing the fading of leaves, falling leaves, and reducing the yield. It reduces the value of the product, and is known to mediate 60 kinds of viral diseases such as tomato sulphide, tobacco leaf, and taro leaf. In fact, it is reported to cause more than $ 500 million in damage per year, acting as a major pest in cotton.
  • Tobacco flour is composed of differentiating populations with different biological characteristics, such as host preference and the type of mediating virus.
  • 24 biotypes have been reported, including B type, Q type, and A type.
  • Type B and Q have the most severe damage worldwide, with strong fertility, mediating severe viruses to crops, broad host range, and resistant to organophosphorus, pyrethroid and carbamate insecticides.
  • the occurrence of tobacco flour is increasing nationwide.
  • Q-type tobacco powder was newly discovered.
  • B and Q types are morphologically similar but differ in several biological properties such as developmental period, host preference, virus mediated efficiency, insecticide resistance, and the like.
  • Republic of Korea Patent Publication No. 10-2011-0104845 has developed a biotype diagnostic method of tobacco powder using the difference between the biotype in the length of the intron region of the carboxyl esterase 2 gene of tobacco powder It was reported. However, this method was not able to discriminate between genus and species levels, including tobacco flour and greenhouse dust.
  • PCR-RFLP PCR-restriction fragment length polymorphism
  • RAPD-PCR random amplified polymorphic DNA analysis
  • microsatelite DNA analysis etc., which uses PCR amplification of random DNA fragments to analyze the patterns. It still has a complicated drawback.
  • the present inventors have intensively researched to develop a method for effectively classifying pests of flour.
  • the present inventors have selected the cytochrome c oxidase subunit I (COI) gene of mitochondria as the most suitable gene group to distinguish genotypes of flour. From this, single nucleotide polymorphisms (SNPs) sites specific to flours were identified.
  • SNPs single nucleotide polymorphisms
  • new primers and probes were prepared using the base sequences to analyze genotypes of flours, and PCR amplification products were specifically hybridized with probes specific to various powders on microarray chips.
  • the present invention has been completed by effectively identifying greenhouse dust and the like and further distinguishing the biotype of tobacco dust with high specificity and sensitivity.
  • An object of the present invention is to provide a probe composition, a DNA chip and a kit, and a method of classifying flours using the same, which classifies tobacco powder and greenhouse powder belonging to family Aleyrodidae and determines the biotype of tobacco powder.
  • the present invention is simple, rapid and accurate by analyzing genotypes based on single nucleotide polymorphism (SNP) present in the cytochrome c oxidase subunit I (COI) gene region of mitochondrial DNA of tobacco flour and greenhouse dust.
  • SNP single nucleotide polymorphism
  • COI cytochrome c oxidase subunit I
  • the purpose of the present invention is to provide a probe composition, a DNA chip, and a kit for classifying tobacco flour and greenhouse dust and determining the biotype of tobacco flour, and a method for classifying flour using the same.
  • the present invention simply classifies tobacco powder and greenhouse powder by simply loading a sample of powdered powder on one slide even for larvae, processed products or powders that cannot be accurately determined by the naked eye.
  • Providing a probe composition, a DNA chip and kit, and a method for classifying flours using the same which can determine a biotype and can significantly test a large amount of a sample in a short time, while significantly reducing the time for analyzing a sample compared to a conventional method. The purpose is.
  • the present invention provides a method for classifying flours using single nucleotide polymorphisms (SNPs) sites of the mitochondrial COI (cytochrome oxidase subunit I) gene.
  • SNPs single nucleotide polymorphisms
  • the inventors have made a number of studies and efforts to confirm that the probe containing the nucleotide sequence belonging to the single nucleotide polymorphisms (SNPs) site among the COI genes of flours is most suitable for distinguishing species or biotypes of flours.
  • the present invention is selected from the group consisting of oligonucleotides having a nucleotide sequence belonging to the single nucleotide polymorphism site, that is, nucleotide sequences of SEQ ID NO: 5 to SEQ ID NO: 11 and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides.
  • pellets is used as a concept including not only adults, but also eggs, larvae, pupa, larvae, processed products, or powdered powder.
  • extraction of DNA from the flour-like sample can be performed by various methods from each tissue of the sample, which is to extract the DNA from any part of the sample because it is to determine the species or biotype by analyzing the extracted DNA Or how to extract is not particularly limited.
  • amplification of the DNA extracted as described above by PCR is also performed to increase the number of test specimens, and the method for obtaining an amplification product is not particularly limited. It is obvious that other DNA extraction methods or amplification methods known to those skilled in the art also belong to the scope of the present invention.
  • polymorphism refers to the generation of two or more replaceable sequences or alleles within a genetically determined population. Polymorphic markers or sites are loci where this phenomenon occurs. Preferred markers have two or more alleles which exhibit a frequency of occurrence of at least 1%, more preferably at least 10% or 20% in the selected population.
  • the polymorphic site may be a single base pair.
  • the oligonucleotide probe for classifying the arachnids of the present invention may be an oligonucleotide comprising one or more polymorphic sites according to the present invention, or a nucleic acid fragment or complement thereof, in addition to the nucleotide sequence described below.
  • the probe composition for classifying flours of one embodiment of the present invention specifically binds to the cytochrome c oxidase subunit I (COI) gene region in the mitochondrial DNA of tobacco powder, and comprises the nucleotide sequence of SEQ ID NO: 9 to the sequence number At least one probe selected from the group consisting of oligonucleotides having a nucleotide sequence of 10 and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides, and Cytochrome c Oxidase subunit I (COI) in mitochondrial DNA of greenhouse powder Oligonucleotides that specifically bind to a gene region and have a nucleotide sequence of SEQ ID NO: 11 or a probe of an oligonucleotide having a nucleotide sequence complementary to such oligonucleotide.
  • COI cytochrome c oxidase subunit I
  • the probe composition for classifying flours of one embodiment of the present invention oligonucleotides having a nucleotide sequence of SEQ ID NO: 5 to the base sequence of SEQ ID NO: 6 and these oligonucleotides as a probe specific to B type of tobacco powder
  • DNA chip for classifying flours of one embodiment of the present invention specifically binds to the cytochrome c Oxidase subunit I (COI) gene region in the mitochondrial DNA of tobacco powder, and the base sequence of SEQ ID NO: 9 to SEQ ID NO: 10
  • Oligonucleotides that bind specifically to and have a nucleotide sequence of SEQ ID NO: 11 or a probe of an oligonucleotide having a nucleotide sequence complementary to such oligonucleotide.
  • the DNA chip for classifying flours of one embodiment of the present invention oligonucleotides having a nucleotide sequence of SEQ ID NO: 5 to the base sequence of SEQ ID NO: 6 and these oligonucleotides as a probe specific to the B type of tobacco powder
  • the probe may be microarrayed on the same substrate as a glass slide.
  • the substrate is preferably a glass substrate or a plastic substrate that is generally used in the manufacture of DNA chips.
  • the method for classifying flours comprises: (a) obtaining a PCR amplification product obtained by amplifying a single nucleotide polymorphism (SNP) region of the mitochondrial COI gene from flours, and (b) converting the PCR amplification product into flours. Hybridizing with the probes specific for the step, and (c) determining whether the tobacco powder or the greenhouse powder and / or the biotype of the tobacco powder according to the hybridization.
  • SNP single nucleotide polymorphism
  • oligonucleotides including the nucleotide sequence of the SNP region of the COI gene of mitochondria for the determination of tobacco powder are selected from the base sequence of SEQ ID NO: 9 to the base sequence of SEQ ID NO: 10
  • At least one probe selected from the group consisting of oligonucleotides having oligonucleotides having a base sequence complementary to these oligonucleotides is hybridized with the PCR amplification product, and the SNP of the COI gene of mitochondria for greenhouse powder determination.
  • An oligonucleotide having a nucleotide sequence of SEQ ID NO: 11 as an oligonucleotide including a nucleotide sequence of a site, or a probe of an oligonucleotide having a nucleotide sequence complementary to such an oligonucleotide is characterized in that it is hybridized with the PCR amplification product.
  • oligonucleotides having a nucleotide sequence of SEQ ID NO: 5 to a base sequence of SEQ ID NO: 6 and oligonucleotides having a nucleotide sequence of SEQ ID NO: 6 for determination of biotype B type of tobacco powder At least one probe selected from the group consisting of oligonucleotides having complementary nucleotide sequences is hybridized with the PCR amplification product, and for determining the biotype Q type of tobacco powder, the nucleotide sequence from SEQ ID NO: 7 to SEQ ID NO: At least one probe selected from the group consisting of oligonucleotides having a nucleotide sequence of 8 and oligonucleotides having a nucleotide sequence complementary to these oligonucleotides is hybridized with the PCR amplification product.
  • PCR amplification kit for classifying flours of an embodiment of the present invention, primer pairs of SEQ ID NO: 1 and 2 for amplifying the SNP site of the mitochondrial COI gene from tobacco powder, amplifying the SNP site of the mitochondrial COI gene from greenhouse powder Primer pairs of SEQ ID NOs: 3 and 4, DNA polymerases, dNTPs, PCR buffers and labeling products of PCR amplification products.
  • the present invention also provides a test kit capable of classifying flours using a single base polymorphic site of the mitochondrial COI gene.
  • the present invention is a test kit that can classify the flour of one embodiment of the present invention, the DNA chip defined as described above, SEQ ID NO: 1 for amplifying the SNP site of mitochondrial COI gene from tobacco powder And primer pairs of 2, and primer pairs of SEQ ID NOs: 3 and 4 for amplifying the SNP site of the mitochondrial COI gene from greenhouse powder, and labeling means for detecting the amplified gene product hybridized with the DNA chip.
  • the labeling material is not limited to a specific fluorescent material, as well as a variety of fluorescent materials that can be identified as a fluorescence detector can be used, enzymatic coloration reaction and isotope labeling is also possible.
  • the label is Cy5, Cy3, biotin-binding material, EDANS (5- (2'-aminoethyl) amino-1-naphthalene sulfate), tetramethyltamine (TMR), tetramethyltamine isocyanate (TMRITC ), x-rhodamine or Texas red and the like, and it is preferable that the phosphor is Cy3 or Cy5.
  • the present invention is not limited thereto, and various labeling materials such as various fluorescent materials, radioactive materials, or luminescent materials known in the art may be used.
  • genotyping is based on a single nucleotide polymorphism present in the COI gene region of the mitochondrial DNA of tobacco and greenhouse dust, and it is possible to classify tobacco dust and greenhouse dust accurately and quickly and determine the biotype of tobacco dust. can do.
  • the present invention is to load a sample of flour on a slide even for larvae, artifacts or powder that can not be accurately determined by the naked eye when produced with a probe composition, DNA chips and kits for classification of flours Simply by classifying tobacco powder and greenhouse powder, it is possible to determine the biotype of tobacco powder.
  • the present invention can accurately genotype multiple flour species or biotypes on one slide according to the microarray DNA chip technology in one experiment at the same time, thereby determining the genotype and species or biotype of flour species. It also opens up the possibility to standardize and automate.
  • the present invention can be easily and quickly, accurately determine the powdered pests can be widely used for effective eradication of pests and improved agricultural productivity.
  • Figure 1 is a schematic diagram showing the gene arrangement on the mitochondrial DNA of flours including a single-base polymorphic site of the COI gene in the mitochondrial DNA of the flours according to the present invention.
  • Figure 2 shows a continuous sequence containing a single base polymorphic site of the COI gene in tobacco flour B type mitochondrial DNA according to the present invention.
  • Figure 3 shows a continuous sequence containing a single base polymorphic site of the COI gene in tobacco powder Q type mitochondrial DNA according to the present invention.
  • Figure 4 shows a continuous sequence containing a single base polymorphic site of the COI gene in mitochondrial DNA of greenhouse dust according to the present invention.
  • Figure 5 shows the layout of the microarray DNA chip according to an embodiment of the present invention.
  • FIG. 6 is a fluorescence image photograph of a DNA chip confirmed after hybridization of the tobacco powder B type PCR amplification product to the microarray DNA chip of the present invention.
  • FIG. 7 is a fluorescence image photograph of a DNA chip confirmed after hybridization of a tobacco agar Q type PCR amplification product to a microarray DNA chip of the present invention.
  • Example 1 Preparation of primers of SEQ ID NO: 1 to SEQ ID NO: 4
  • primers were synthesized to amplify DNA extracted from agari.
  • primers were used according to the respective flours as shown in Table 1 below. These primers were synthesized by requesting Bioneer (Daejeon Metropolitan City).
  • transverse direction (sense) primers and the rearward direction (antisense) primers used for symmetric or asymmetric polymerase chain reaction (PCR) as described in Table 1 are at the ends so that they can be identified by fluorescence after hybridization reaction. It was prepared by attaching rhodamine, cy3, or cy5 or by attaching biotin, and in case of attaching biotin, it was used to bind streptavidin-cyanine after hybridization.
  • This specific primer corresponds to the DNA sequence of the single base polymorphic region of the COI gene in the mitochondrial DNA of the Arachnid, and can further amplify the DNA extracted from the Arachnoid sample.
  • primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 are used, and in order to amplify DNA in greenhouse powder samples, primers of SEQ ID NO: 3 and SEQ ID NO: 4 are described in Table 1. Pairs were used.
  • DNA of powdered species including various biotypes of Bemisia tabaci , Trialeurodes vaporariorum and Tobacco flour were extracted using DNeasy blood & tissue kit of QIAGEN, Germany.
  • PCR amplification reactions were carried out using a DNA engine (Dakara, Japan) under the conditions shown in Table 2 below.
  • the PCR amplification product is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO:
  • SEQ ID NO: 5 When combined with probes containing the base sequence of 10 is classified as tobacco powder B type, when combined with probes comprising the base sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10
  • Tobacco powder is classified as Q type, and when combined with a probe containing the nucleotide sequence of SEQ ID NO: 11 is classified as a greenhouse powder.
  • the probes according to the present invention bind to a single base polymorphic site of the COI gene region in the mitochondrial DNA of the Agari, and hybridization as described below may be performed differentially depending on the species or biotype of the Agari.
  • a probe comprising the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6 specifically binds to the 11-34 or 92-113 nucleotide sequences of the tobacco powder B type mitochondrial COI gene region, respectively, and SEQ ID NO: 7
  • a probe comprising a nucleotide sequence of SEQ ID NO: 8 specifically binds to a 10-34th or 518-542th base sequence of a COI gene region in tobacco powder Q type mitochondrial DNA, and SEQ ID NO: 9 or SEQ ID NO:
  • Probe comprising the nucleotide sequence of 10 is specifically probed with the base sequence of SEQ ID NO: 11 specifically binds to the 56-79 or 406-431 base sequence of the COI gene region of mitochondrial DNA of tobacco
  • the probe is preferably composed of 15 to 30 consecutive base sequences, it is preferable that the step of combining the PCR amplification product from the powder with the probe is preferably repeated at least two times. In this case, the binding of the probe and the PCR amplification product can be further strengthened.
  • Fabrication of microarrayed DNA chips is performed using methods well known to those skilled in the art.
  • the amino link is modified at the 5'-end of the oligonucleotide having each probe sequence of Example 3 on the glass on which the aldehyde functional group is treated, and then spatial interference between the probes integrated on the slide glass during the hybridization reaction.
  • 10 to 20 oligos (dT) were added to minimize the probes used in the examples of the present invention.
  • Probes of the present invention were synthesized by the request of Korea Bioneer (Daejeon Metropolitan City).
  • an amino link was modified at a concentration of 50 ⁇ M on a CSS-100 silylated slide (Silylated Slide, Cell Associate, USA). The same amount of 3X SSC solution was mixed and integrated with the probe, which was reacted at room temperature for 16 hours. After the reaction was completed, the slides were washed once with 0.1% SDS for 5 minutes and twice with distilled water for 5 minutes.
  • the prepared chip was reacted with sodium borohydride (1.3 g NaBH 4 375 ml, PBS 125 ml, 100% EtOH) for 5 minutes, washed twice with distilled water for 5 minutes, and centrifuged at 800 rpm for 5 minutes.
  • sodium borohydride 1.3 g NaBH 4 375 ml, PBS 125 ml, 100% EtOH
  • the fabricated microarray chips were divided into four sub-arrays and covered with a CoverWell perfusion chamber (Grace-Bio Labs, USA) so that the microarray chips prepared in the hybridization experiment could be independently reacted.
  • the configuration (layout) of the finally produced microarray chip is as shown in FIG. 5.
  • PCR amplification products incorporating rhodamine, cy3, cy5 or biotin amplified according to Examples 1 and 2 were mixed with a hybridization solution (3X SSC, 0.25% SDS) in a ratio of 1: 9.
  • a hybridization solution 3X SSC, 0.25% SDS
  • streptavidin-cyanine was added at a concentration of 1 ⁇ g / ml.
  • the prepared hybridization solution was applied to the chamber containing the DNA chip prepared in Example 4 and reacted at 55 ° C. for 1 hour. It was then first washed for 5 minutes in 1X SSC, 0.1% SDS solution and again for 5 minutes in 1X SSC solution. It was then further washed in 0.1X SSC solution for 5 minutes. The washed slides were dried for 5 minutes at a speed of 800 rpm in a centrifuge.
  • the hybridization signal (fluorescence signal) of the reaction microarray chip was used by Genepix 4000B (Axon Instrument, USA), Photomultiplier Tube (PMT) 450, Laser power 100
  • the fluorescence image was stored by scanning at%, 5 ⁇ m resolution, and the positive and negative signals of each probe were determined as fluorescence values.
  • Fluorescence intensity data was analyzed using GenePix Pro 4.1 software (Axon instrument, USA), and the local background value was subtracted from the mean value of each spot. All probes were spotted in duplicate so the signal intensities of the target probes were calculated from the average of the two spots.
  • FIG. 5 is a tobacco powder B type
  • Figure 7 is a tobacco powder Q type
  • Figure 8 is a hybridization reaction results for greenhouse powder.
  • the microarray chip of the present invention had different fluorescent labels depending on the species or biotype of the powdered pest. Therefore, the present invention was confirmed that the powder is effective in discriminating species or biotypes of pests with high specificity and sensitivity.

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Abstract

La présente invention concerne : une composition de sonde, une puce à ADN et un kit servant à distinguer et à identifier de manière simple, rapide et précise les types et/ou les biotypes d'aleurodes, par l'analyse de génotypes, sur la base d'un unique polymorphisme nucléotidique présent dans la région du gène COI dans l'ADN mitochondrial de Bemisia tabaci et de Trialeurodes vaporariorum ; et un procédé de classification d'aleurodes les utilisant. La présente invention permet de classifier Bemisia tabaci et Trialeurodes vaporariorum et d'identifier les biotypes de Bemisia tabaci d'une manière simple, simplement en déposant sur une seule lame des échantillons d'aleurode dont le type ne peut pas être identifié avec précision à l'œil nu, tels que des échantillons de larves, d'artefacts ou de poudre. En outre, la présente invention permet de tester des volumes importants d'échantillon en peu de temps, tout en réduisant sensiblement le temps d'analyse des échantillons par rapport aux procédés classiques.
PCT/KR2012/008113 2011-11-03 2012-10-08 Composition de sonde, puce à adn et kit de classification d'aleurodes, et procédé de classification d'aleurodes les utilisant Ceased WO2013065957A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101546758B1 (ko) 2013-11-19 2015-08-24 대한민국 가루이 매개 토마토 바이러스 다중 진단용 프라이머 세트 및 이를 이용한 진단 방법
CN103898225A (zh) * 2014-04-11 2014-07-02 山东省农业科学院生物技术研究中心 一种鉴别烟粉虱隐种和白粉虱的引物及鉴别方法
CN120060488A (zh) * 2025-03-07 2025-05-30 青岛农业大学 鉴别c-和c+品系烟粉虱单雌系的引物对以及鉴别c-和c+品系烟粉虱单雌系的方法

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