WO2014137196A1 - Dna chip for detecting and quantitatively measuring harmful blue-green algae in korean fresh water system - Google Patents

Dna chip for detecting and quantitatively measuring harmful blue-green algae in korean fresh water system Download PDF

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WO2014137196A1
WO2014137196A1 PCT/KR2014/001911 KR2014001911W WO2014137196A1 WO 2014137196 A1 WO2014137196 A1 WO 2014137196A1 KR 2014001911 W KR2014001911 W KR 2014001911W WO 2014137196 A1 WO2014137196 A1 WO 2014137196A1
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seq
dna chip
oscillatoria
probe
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오희목
김용
안치용
이창수
김희식
이형관
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한국생명공학연구원
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Definitions

  • the present invention relates to a DNA chip capable of simultaneous detection of dangerous microalgae present in freshwater systems in Korea, and a detection and / or quantification method using the same. More specifically, it includes DNA probes designed using gene information specific to six representative risky algae species ( Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc ) that cause green algae and contain toxicity in freshwater in Korea. It relates to a DNA chip, and a detection and / or quantification method using the same.
  • Cyanobacteria blue-green algae, cyanobacteria
  • cyanobacteria are prokaryotes that perform oxygen-producing photosynthesis in the same way as plants, that is, bacteria, living organisms adapted to various environments from hot springs to polar regions. Cyanobacteria have played an important role in creating the present atmosphere by supplying oxygen to the primitive earth, and are now important primary producers, accounting for about 25% of the photosynthesis occurring on Earth.
  • microcystin a toxin produced by cyanobacteria, which inhibits phosphatase type 1 and type 2A, a type of hepatotoxin most often found in lakes and swamps.
  • Microcystin has a heptapeptide structure in which seven amino acids are linked in a ring shape. Arginine), and about 70 species of microcystine are known so far.
  • microcystine has been reported to inhibit not only animals but also plants, zooplankton and phytoplankton, and toxic species that produce microcystine have been found to inhibit nontoxic species that do not produce microcystine within the same cyanobacteria.
  • Microcystine is detected in most large water sources in Korea, which are used as water sources, but so far no cases have exceeded 1 ⁇ g / L, the WHO standard. However, in the summer, it is temporarily close to this, and the increase in water temperature due to global warming is expected to further promote algae and toxin production by algae.
  • the bird warning system is issued in three stages of warning, warning, and outbreaks based on the cell number and chlorophyll concentration of algae, and each water resource management agency and local government take appropriate action.
  • cyanobacteria found mainly in Korea, are found because thousands of cells colonize (e.g., Microcystis) or form filaments similar to those of beads or circumcisions (e.g., Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc).
  • the coefficient is very difficult and it is difficult to expect exact accuracy.
  • toxin analysis is performed after the occurrence of green algae. Toxin analysis requires expensive equipment and the process itself is complicated, and thus, there is a problem that early detection and prophylaxis of cyanobacteria toxins are difficult in the current system.
  • DNA chip technology using the latest molecular biology techniques has been developed in recent decades, and many technical problems are solved, and specific cluster analysis shows high accuracy and practicality.
  • existing DNA chip technology has been developed mainly for the detection of diatoms (e.g., Alexandrium ) that cause marine red tide, and is a genus of cyanobacteria microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc that cause freshwater algae. No specific DNA chip technology has been developed.
  • the present inventors have completed the present invention as a result of diligent efforts to develop a method for rapidly and quantitatively analyzing six species, which are considered major risk cyanobacteria in Korean freshwater, using DNA chips.
  • Another object of the present invention is to provide a DNA chip capable of quantitatively and rapidly measuring quantitatively at the same time in the genus Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc, which are representative of 6 dangerous algae species monitored by a bird alarm.
  • Still another object of the present invention is to detect and / or quantitatively and rapidly and rapidly detect and / or quantitatively measure the genus Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, and Nostoc, which are representative of six dangerous dangerous algae monitored by an algal warning agent . To provide a possible way.
  • the present invention provides a DNA chip for cyanobacteria detection and / or quantification comprising six or more probes consisting of nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 78.
  • the target gene of the probe is characterized in that mcyE, cpcBA, ITS, rbcS, rbcX or nifD .
  • cyanobacteria are genus Microcystis , Anabaena , Oscillatoria , Phormidium , Aphanizomenon and Nostoc .
  • the DNA chip is a probe for detecting and / or quantifying the genus Microcystis , consisting of SEQ ID NOs: 4-6, 13-15, 22-24, 37-39, 46-48 and 73-75 At least one probe having a nucleotide sequence selected from the group;
  • a probe for detecting and / or quantifying the genus Anabaena having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-3, 10-12, 19-21, 34-36, 43-45, 58-60, and 70-72
  • a probe for detection and quantification of the genus Oscillatoria comprising: at least one probe having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 28-30, 52-54, and 64-66;
  • a probe for detection and / or quantification of the genus Phormidium comprising: one or more probes having a nucleotide sequence
  • the genus Microcystis comprises Microcystis aeruginosa
  • the genus Anavena is Anabaena flos-aquae, Anabaena variabilis, Anabaena sp. PCC 7120, Anabaena sp.
  • the genus Oscillatoria is Oscillatoria sancta, Oscillatoria tenuis, Oscillatoria sp. 18R, Oscillatoria sp. 19R
  • the podium is in the Phormidium cf. irriguum, Phormidium ambiguum, Phormidium sp. 4-19b, Phormidium sp.
  • the genus Apanizomenon includes Aphanizomenon flos-aquae
  • the genus Northstock includes Nostoc punctiforme, Nostoc linckia, Nostoc spongiaeforme, Nostoc muscorum .
  • the DNA chip is characterized in that divided into six probe wells (well).
  • the present invention comprises the steps of extracting genomic DNA from cyanobacteria samples; Fragmenting and labeling the extracted genomic DNA; Hybridizing the labeled DNA to a DNA chip; And it provides a method for detecting and / or quantifying southern algae comprising the step of determining the type and amount of southern algae according to the degree of hybridization.
  • the type of cyanobacteria is as described above.
  • sufficient information on six major algae genes that cause green algae in freshwater in Korea can be sufficiently obtained, and it is possible to provide probes and DNA chips capable of specific quantitative analysis using the genetic information.
  • probes and DNA chips capable of specific quantitative analysis using the genetic information.
  • six species of algae to be monitored in the bird alarm can be discriminated simultaneously in one DNA chip, and mass analysis of the sample can be carried out with high efficiency as compared with the conventional microscopy.
  • the gene extracted from the freshwater aquatic sample is not subjected to a polymerase chain reaction (PCR) or other amplification process, it is possible to quantitatively measure the unbiased condition of genetic information in the environment. There is an advantage.
  • PCR polymerase chain reaction
  • the designed probe is manufactured based on a structural gene such as ITS (Internal Transcribed Spacer) and a functional gene such as rpoB , mcyE , cpcBA , nifD , rbcS, rbcX , the structural gene and the functional gene Simultaneous use of the system can provide an extensive assessment of aquatic health, including accurate identification and identification of cyanobacteria species and prediction of toxic substances.
  • ITS Internal Transcribed Spacer
  • the existing DNA chip technology mainly produces diatoms that generate red tide in the ocean.
  • the present invention has the advantage of providing a specific and quantitative measuring method in cyanobacteria Microcystis, Anabaena , Oscillatoria , Phormidium , Aphanizomenon, Nostoc that cause freshwater green algae .
  • FIG. 1 is a photograph showing the DNA chip of the present invention, in which six probes capable of detecting specific and quantitatively dangerous algae ( Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc ) at the same time are arranged in six zones.
  • six probes capable of detecting specific and quantitatively dangerous algae Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc
  • 2A-2E are diagrams showing the specificity of a probe capable of complementarily binding to the genes of Microcystis (a) mcyE , (b) cpcBA , (c) ITS, (d) rbcS , and (e) rbcX .
  • Figures 3a-3e shows the quantification of DNA chips using probes that can complementarily bind to the genes of Microcystis (a) mcyE , (b) cpcBA , (c) ITS, (d) rbcS , (e) rbcX . It is a chart showing
  • 4A-4C are diagrams showing the specificity of a probe capable of complementarily binding to Nostoc genes (a) rpoB , (b) rbcS, (c) rbcX .
  • 5A to 5C are diagrams showing the quantification of DNA chips using probes capable of complementarily binding to Nostoc genes (a) rpoB , (b) rbcS , and (c) rbcX .
  • FIG. 6 is a chart showing the specificity of a probe capable of complementarily binding to the rpoB gene of Anabaena .
  • FIG. 7 is a diagram showing the quantification of the DNA chip using a probe capable of complementarily binding to the rpo B gene of Anabaena .
  • FIG. 8 is a chart showing the specificity of a probe capable of complementarily binding to the ITS gene of Oscillatoria .
  • FIG. 9 is a diagram showing the quantification of the DNA chip using a probe capable of complementarily binding to the ITS gene of Oscillatoria .
  • FIG. 10 is a chart showing the specificity of a probe capable of complementarily binding to mcy E gene of Phormidium .
  • FIG. 11 is a diagram showing the quantification of the DNA chip using a probe capable of complementarily binding to the mcy E gene of Phormidium .
  • FIG. 12 is a diagram showing the quantification of the DNA chip using a probe capable of complementarily binding to the cpc BA gene of Aphanizomenon .
  • Target genes were first selected to produce probes specific to the six major species of green algae that cause green algae in freshwater water systems in Korea.
  • the target gene must be able to provide information on the basic sequence designed by the probe.
  • probes designed based on target genes must have species specificity and for this purpose must have genetic information that distinguishes them from other species. Gene regions that are conserved indistinguishable from other species cannot be candidate genes, and gene regions with too much genetic information between species may not be suitable for probe design.
  • Target genes must also be well grounded enough to have good information in the database.
  • NCBI National Center for Biotechnology Information
  • Geneious program Biomatters Ltd.
  • rbcS is, a gene that blue-green algae is encoding a subunit (small subunit) of an essential RuBisCo composite for fixing the carbon dioxide, by analyzing the rbcS gene to prepare a specific nine probes to Anabaena, Microcystis, Nostoc 3 gae genus (genus) ( SEQ ID NOs: 1 to 9).
  • rbcX is a gene encoding a chaperone protein which is essential for assisting the formation of RuBisCo complex.
  • the rbcX gene was analyzed to produce 9 probes specific to Anabaena, Microcystis and Nostoc genus (SEQ ID NO: 10 to 18).
  • ITS Internal transcribed spacer
  • ITS is a space between coding regions of rRNA, and has various and stable sequences, so that it can be compared between microorganisms having very close associations or microorganisms having a very distant association. Since it can be used as a powerful marker, the ITS gene was analyzed to produce 15 probes specific for 5 genus Anabaena, Microcystis, Nostoc, Oscillatoria, and Phormidium (SEQ ID NOs: 19 to 33).
  • rpoB (RNA polymerase beta subunit) is a gene encoding an RNA polymerase beta subunit.
  • 9 probes specific to Anabaena, Microcystis and Nostoc genus were generated (SEQ ID NOs. 34 to 42). ).
  • mcyE is a gene encoding microcystin, a hepatoxin produced by cyanobacteria, and analyzed by mcyE gene in five genus of Anabaena, Microcystis, Nostoc, Oscillatoria, and Phormidium, which are toxic toxin-producing cyanobacteria. 15 specific probes were prepared (SEQ ID NOs: 43-57). This set of probes can be an important indicator for monitoring algae toxicity, a major problem with water pollution from green algae.
  • nifD is a gene encoding nitrogenase, which is essential for cyanobacteria to fix nitrogen in the air.It analyzes the nifD gene and analyzes twelve probes specific to four genera: Anabaena, Nostoc, Oscillatoria, and Phormidium. Was prepared (SEQ ID NOs: 58-69).
  • cpcBA is a gene encoding phycocyanin, a microalgae photosynthetic auxiliary pigment, and analyzed 9 cpcBA genes to produce 9 probes specific to Anabaena, Microcystis and Aphanizomenon genus (SEQ ID NO: 70 to 78).
  • Probe was fabricated using OligoWiz program (http://www.cbs.dtu.dk/services/OligoWiz/), and a total of 78 probes were prepared as shown in Table 1 below. To this end, we compared the whole genome sequence of Anabaena , Microcystis , Oscillatoria , Phormidium , Nostoc , which were recently registered with NCBI, with the gene base information of each cyanobacteria.
  • Table 1 below shows specific probes for detecting six major cyanobacteria in the Korean freshwater water system monitored by the algae alert.
  • PCC 7120 rbc X AnarbcX2 (SEQ ID NO: 11) GAACATATAGCCGAAGAAGTAGCCGAGTTCTTACCAGAAATGGTTCG Anabaena sp.
  • PCC 7120 rbc X AnarbcX3 (SEQ ID NO: 12) GTTTATCTAACCCCAGTCCTGAATCAGAACAGCAGACAATTTCCGAT Anabaena sp.
  • irriguum ITS PhoITS2 (SEQ ID NO: 32) AACGAGAATGGACAGGCTTTCAAACTATTTTCAGGTTGGGAAAATGG Phormidium cf.
  • irriguum ITS PhoITS3 (SEQ ID NO: 33) GGCTTTCAAACTATTTTCAGGTTGGGAAAATGGGCTATTAGCTCAGG Phormidium cf.
  • AnarpoB1 (SEQ ID NO: 34) CACCTGTTTAAACCAAAAACCATTGGTGAGAATTGGCGAAAAAGTCG Anabaena flos-aquae rpo B AnarpoB2 (SEQ ID NO: 35) GCAGCCAGTGGTAGCCAAGTTATTGAAAAAGGCCAAGAACTTAAATA Anabaena flos-aquae rpo B AnarpoB3 (SEQ ID NO: 36) TATCAACGTTCCAACCAAGACACCTGTTTAAACCAAAAACCATTGGT Anabaena flos-aquae rpo B MicrpoB1 (SEQ ID NO: 37) AAATATCAACGCTCTAACCAAGATACCTGCCTAAATCAGCGTCCTTT Microcystis aeruginosa rpo B MicrpoB2 (SEQ ID NO: 38) GAAATATCAACGCTCTAACCAAGATACCTGCCTAAATCAGCGTCCTT Microcystis a
  • rpo B NosrpoB2 (SEQ ID NO: 41) CCACAGAAATTCGTGTACGTCCCAAAACCAGTACCCAAGAAATTAAG Nostoc sp.
  • 152 rpo B NosrpoB3 (SEQ ID NO: 42) TGTAGCTACTAGTATGATTCCCTTTTTGGAACATGACGACGCTAACC Nostoc sp.
  • 152 rpo B AnamcyE1 (SEQ ID NO: 43) GAACAGCTGAACCATTGAGTCTTGGTACACTATCAGGAATGGTTGAA Anabaena spp.
  • BIR series mcy E AnamcyE2 (SEQ ID NO: 44) CAGCTGCTTTAATTTGTGAAATGACAGGTGTAGAACGAGTCGCTTTT Anabaena spp.
  • BIR series mcy E AnamcyE3 (SEQ ID NO: 45) GGACAAAACGCCAAAAAATTGTGATTTTTGCTGGTTCTTATCACGGT Anabaena spp.
  • mice BIR series mcy E MicmcyE1 (SEQ ID NO: 46) AGAAGATAAAACCACGGCTCAACCCTTAAGTTTAGGCACTCCTTTAG Microcystis aeruginosa mcy E MicmcyE2 (SEQ ID NO: 47) TCATTGAAGCGGTTCAAGAACAAATGAACCGAGGAATAGGTTTAGGA Microcystis aeruginosa mcy E MicmcyE3 (SEQ ID NO: 48) GAACAAATGAACCGAGGAATAGGTTTAGGAATGCAGTCAAATCTGGC Microcystis aeruginosa mcy E NosmcyE1 (SEQ ID NO: 49) TAAAACCACGACTCAACCCTTAAGTTTAGGCACTCCTTTAGGAATGG Nostoc sp.
  • 19R mcy E OscmcyE2 (SEQ ID NO: 53) GGGGTAGAAAGGGTGGCTTTTAGTAATACTGGAACCGAAGCAATTAT Oscillatoria sp. 18R Oscillatoria sp. 19R mcy E OscmcyE3 (SEQ ID NO: 54) GGATTGCTCGTTCTCGCACAAAACGCCCAAAAATTGTTATATTTTCC Oscillatoria sp. 18R Oscillatoria sp. 19R mcy E PhomcyE1 (SEQ ID NO: 55) TGCTATGTTTGCAGGTTCATATCACGGAACCTTTGATGGCATTTTAG Phormidium sp.
  • samples of concentrations of 10 1 to 10 6 cells / ml were prepared for six species of cyanobacteria Cycystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, and Nostoc . The following procedure was performed for the hybridization.
  • each genomic DNA was digested with restriction enzymes AluI (20 units, Promega) and RsaI (20 units, Promega) at 37 ° C. for 2 hours. digestion). After the reaction, the fragmented DNA samples were purified using the QIAQuick PCR clean-up kit (Qiagen) according to the manufacturer's method.
  • Purified DNA was labeled using a Bioprime labeling kit provided by Invitrogen. Add 50 ⁇ l of total reaction solution to the modified dNTP mix (120 uM of dATP, dGTP, dCTP; 60 uM of dTTP) and 60 uM of Cy5-dUTP, add Klenow enzyme and reaction buffer, The labeling reaction was carried out for a time. After the reaction, the labeled DNA was purified using a QIAQuick PCR clean-up kit (Qiagen).
  • Purified DNA was prepared by mixing 50 ug of human Cot-1 DNA (Applied Genetics) / 52 ul of 10X Blocking reagent (Agilent Technology) as competitors to increase the specificity of hybridization, and the mixed sample was prepared by 2X aCGH. Mixed with hybridization buffer (Agilent Technology).
  • the DNA sample mixed well with the hybridization buffer was heated at 95 ° C for 3 minutes, and then reacted at 37 ° C for 30 minutes. After the reaction solution was prepared by centrifugation for 1 minute, it was injected into a gasket slide (Agilent Technology), and the DNA chip was fixed. This DNA chip was placed in a hybridization oven (Agilent Technology) and subjected to a competitive hybridization reaction at 65 ° C. for 18-20 hours. After completion of hybridization, the DNA chip was washed and dried in order according to the manufacturer's method.
  • the dried DNA chip was scanned with an Axon scanner and then quantified the signal intensity of each gene using the GenePix Pro 6.0 (Axon Instruments) program. After calculating the average fluorescence intensity and the local background of each point, the pure fluorescence value was obtained by removing the background value from the mean fluorescence intensity of each point. And for later analysis, the low fluorescence intensity was eliminated.
  • the probe prepared based on rpo B gene specifically reacted with Anabaena .
  • the SNR value was unrelated to the change of Anabaena concentration in the 10 1 ⁇ 10 3 cells / ml concentration range, but as the concentration of Anabaena increased in the 10 3 ⁇ 10 6 cells / ml concentration range.
  • the SNR value also shows a positive correlation (R 2 > 0.95), indicating that Anabaena can be quantitated from 10 3 to 10 6 cells / ml using the manufactured DNA chip.
  • the probe prepared based on the ITS gene specifically reacted with Oscillatoria .
  • the concentration range of 10 1 ⁇ 10 3 cells / ml showed an SNR value unrelated to the change in the concentration of Oscillatoria , but the concentration of Oscillatoria increases in the concentration range of 10 3 ⁇ 10 6 cells / ml
  • the SNR value also increases in proportion (R 2 > 0.95), indicating that Oscillatoria can be quantitatively analyzed from 10 3 to 10 6 cells / ml using the manufactured DNA chip.
  • the probe prepared based on the cpc BA gene showed an SNR value that was not related to the change of Aphanizomenon concentration in the concentration range of 10 1 to 10 3 cells / ml, but was 10 3 to 10 6.
  • the SNR value also increased proportionally (R 2 > 0.99), and Aphanizomenon was converted to 10 3 ⁇ 10 6 cells / It can be seen that quantitative analysis is possible up to ml.

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Abstract

The present invention relates to a DNA chip for detecting and/or quantifying blue-green algae comprising six or more kinds of probes having a base sequence selected from sequence numbers 1 to 78, and a method for detection and/or quantification using the same. According to the present invention, it is possible to obtain enough genetic information of the major six kinds of blue-green algae causing algal bloom in the Korean fresh water system, and to provide a DNA chip capable of specific quantitative analysis by using genetic information and a method for detection and quantification using the DNA chip.

Description

한국 담수 수계의 위해성 남조류의 검출과 정량측정을 위한 ＤＮＡ 칩DNA chip for detection and quantitative determination of risky algae in Korean freshwater
본 발명은 한국의 담수 수계에 존재하는 위해성 미세조류들을 동시검출할 수 있는 DNA 칩 및 이를 이용한 검출 및/또는 정량방법에 관한 것이다. 보다 상세하게는 한국의 담수에서 녹조를 유발하고 독성을 함유하는 대표적인 위해성 남조류 6종(Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc 속)에 특이적인 유전자의 정보를 이용하여 설계한 DNA 프로브를 포함하는 DNA 칩, 및 이를 이용한 검출 및/또는 정량방법에 관한 것이다.The present invention relates to a DNA chip capable of simultaneous detection of dangerous microalgae present in freshwater systems in Korea, and a detection and / or quantification method using the same. More specifically, it includes DNA probes designed using gene information specific to six representative risky algae species ( Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc ) that cause green algae and contain toxicity in freshwater in Korea. It relates to a DNA chip, and a detection and / or quantification method using the same.
남조류(blue-green algae, cyanobacteria)는 식물체와 동일한 방식으로 산소발생 광합성을 수행하는 원핵생물(procaryote), 즉 세균(bacteria)의 일종으로 온천에서부터 극지방까지 다양한 환경에 적응하여 살아가고 있는 생물이다. 남조류는 원시지구에 산소를 공급하여 현재의 대기를 만드는데 중요한 역할을 수행하였으며, 현재 지구상에서 일어나는 광합성의 약 25%를 점유하는 중요한 일차생산자 역할을 담당하고 있다.Cyanobacteria (blue-green algae, cyanobacteria) are prokaryotes that perform oxygen-producing photosynthesis in the same way as plants, that is, bacteria, living organisms adapted to various environments from hot springs to polar regions. Cyanobacteria have played an important role in creating the present atmosphere by supplying oxygen to the primitive earth, and are now important primary producers, accounting for about 25% of the photosynthesis occurring on Earth.
그러나 최근 산업의 급속한 발달과 생활수준의 향상으로 인해 증가하는 하수를 통해 오염물질이 과다 배출되고, 이것이 우선적으로 담수에 흘러들어감에 따라, 많은 호수가 부영양화(eutrophication) 되었다. 또한 기후의 변화로 여름철의 이상 고온과 높은 일조량으로 유속이 느린 강과 호수와 같은 정체된 민물수계의 녹조현상(algal bloom)은 전 세계의 공통적인 환경문제로 부각되었다. 녹조가 발생하면 경관의 심미적 가치가 하락할 뿐만 아니라, 정수처리 비용의 증가, 불쾌한 냄새의 발생, 종 다양성의 감소에 의한 생태계의 파괴, 이후 사멸과정에서의 산소고갈로 인한 어류의 폐사, 남조류 독소에 의한 보건상의 위해 등 다양한 환경문제를 일으킨다.However, due to the recent rapid development of the industry and the improvement of living standards, many lakes have been eutrophicated as the excess of pollutants are released through increasing sewage, which preferentially flows into fresh water. In addition, algal blooms in stagnant freshwater systems, such as rivers and lakes, which are slow due to unusually high temperatures and high sunshine in summer due to climate change, have emerged as a common environmental problem around the world. Green algae not only reduce the aesthetic value of the landscape, but also increase the cost of water treatment, the development of unpleasant odors, the destruction of ecosystems by the reduction of species diversity, and the death of fish due to the depletion of oxygen during the killing process. Causes various environmental problems such as health hazards.
특히 문제가 되는 것은 남조류가 생산하는 독소인 마이크로시스틴(microcystin)인데, 이는 호수나 늪과 같은 호소에서 가장 자주 검출되는 간독소(hepatotoxin)의 일종으로 포스파타제(phosphatase) type 1과 type 2A를 저해한다. 마이크로시스틴은 일곱 개의 아미노산이 고리 모양으로 연결된 헵타펩티드(heptapeptide) 구조를 하고 있으며, 아미노산의 종류에 따라 microcystin-LR (Leucine, Arginine), microcystin-RR (Arginine, Arginine), microcystin-YR (Tyrosine, Arginine) 등으로 나뉘는데, 지금까지 약 70여 종 이상의 마이크로시스틴이 알려져 있다. Of particular concern is the microcystin, a toxin produced by cyanobacteria, which inhibits phosphatase type 1 and type 2A, a type of hepatotoxin most often found in lakes and swamps. . Microcystin has a heptapeptide structure in which seven amino acids are linked in a ring shape. Arginine), and about 70 species of microcystine are known so far.
영국, 호주 등에서는 남조류가 발생한 물을 마신 가축들이 폐사한 사례가 있으며, 브라질에서는 남조류에 오염된 물을 혈액투석에 사용하다 환자가 사망한 사건도 있었다. 마이크로시스틴은 동물뿐만 아니라, 식물, 동물플랑크톤 및 식물플랑크톤에도 저해작용을 나타내는 것으로 보고되었으며, 동일한 남조류 내에서도 마이크로시스틴을 생산하는 독성종이 마이크로시스틴을 생산하지 않는 비독성종을 저해하는 현상이 발견되었다.In the United Kingdom and Australia, livestock that drank water from cyanobacteria died in Brazil, and in Brazil, patients died while using water contaminated with cyanobacteria for hemodialysis. Microcystine has been reported to inhibit not only animals but also plants, zooplankton and phytoplankton, and toxic species that produce microcystine have been found to inhibit nontoxic species that do not produce microcystine within the same cyanobacteria.
마이크로시스틴은 상수원으로 사용되는 국내 대부분의 대형수원지에서 검출되고 있으나, 아직까지는 세계보건기구(WHO)의 기준인 1 ㎍/L을 초과한 사례는 없었다. 그러나 여름철 일시적으로 이에 근접하기도 하며, 지구온난화에 따른 수온 상승이 남조류에 의한 녹조 및 독소 생산을 더욱 촉진할 것으로 예상된다.Microcystine is detected in most large water sources in Korea, which are used as water sources, but so far no cases have exceeded 1 µg / L, the WHO standard. However, in the summer, it is temporarily close to this, and the increase in water temperature due to global warming is expected to further promote algae and toxin production by algae.
녹조발생시 수표면에 부유하는 남조류의 덩어리(scum)가 바람에 의해 국지적으로 농축될 수 있는데, 이때에는 마이크로시스틴의 농도가 일시적으로 매우 상승할 수 있다. 특히 여름철 남조류가 증식하면서 녹조가 발생함에 따라, 남조류 내에서 독성종의 비율이 증가하는 것으로 보고되었으며, 이것은 언제든지 상수원이 위협받을 수 있음을 나타낸다.When algae occurs, a scum of cyanobacteria floating on the surface of the water can be locally concentrated by the wind, where the concentration of microcystine can be very high temporarily. In particular, as the green algae grow as the southern algae proliferate in summer, the proportion of toxic species in the algae has been reported, indicating that the water source can be threatened at any time.
이러한 녹조 및 독소 발생에 대하여 효과적으로 대응하고자, 환경부에서는 1996년 대청호에서의 시범실시를 시작으로 2007년에는 17개 호수로까지 확대하여 조류 경보제를 실시하고 있다. 조류 경보제는 남조류의 세포수와 엽록소의 농도를 기준으로 주의보, 경보, 대발생의 3단계로 발령하며, 각 수자원 관리 담당기관과 지자체는 발령 단계에 맞추어 대응조치를 취한다. To effectively respond to these green algae and toxins, the Ministry of Environment began piloting the Daecheong Lake in 1996 and expanded to 17 lakes in 2007 to provide bird warning. The bird warning system is issued in three stages of warning, warning, and outbreaks based on the cell number and chlorophyll concentration of algae, and each water resource management agency and local government take appropriate action.
그러나, 남조류의 농도를 확인하기 위하여 세포를 현미경으로 동정, 계수하는 것은 숙련된 전공자에게도 까다롭고 시간이 많이 소요되는 과정이다. 특히 국내에서 주로 발견되는 남조류는 수천 개의 세포가 군체를 이루거나(예, Microcystis), 염주알 또는 환형동물의 체절과 유사한 형태의 필라멘트를 형성하기 때문에(예, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc), 계수가 매우 어려울 뿐만 아니라 엄밀한 정확도를 기대하기 어려운 실정이다.However, identifying and counting cells under a microscope to check the concentration of cyanobacteria is a difficult and time-consuming process even for experienced majors. In particular, cyanobacteria, found mainly in Korea, are found because thousands of cells colonize (e.g., Microcystis) or form filaments similar to those of beads or circumcisions (e.g., Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc). In addition, the coefficient is very difficult and it is difficult to expect exact accuracy.
또한, 현행 조류 경보제에서는 녹조 발생 이후에 독소 분석을 실시하는데, 독소분석이 고가의 장비를 필요로 하고 과정 자체가 복잡하여, 현 제도에서는 남조류 독소의 조기 검출과 사전 예방조치가 어려운 문제점이 있다. In addition, in the current algae warning system, toxin analysis is performed after the occurrence of green algae. Toxin analysis requires expensive equipment and the process itself is complicated, and thus, there is a problem that early detection and prophylaxis of cyanobacteria toxins are difficult in the current system.
위와 같은 문제점들을 해결하기 위해서는, 누구나 쉽고 정확하게 측정할 수 있고, 한번에 최대한 많은 항목에 대한 검출을 동시에 할 수 있는 방법이 요구된다. 이와 관련하여, 분자생물학적 최신 기법을 활용한 DNA칩 기술은 최근 10년 사이에 많은 발전을 이루어 상당수의 기술적 문제들이 해결되고 있으며 특정 군집 분석에서는 높은 정확도와 실용성을 보여주고 있다. 그러나, 기존의 DNA 칩 기술은 주로 해양의 적조를 발생하는 규조류 (예, Alexandrium)를 검출하는 것을 목적으로 개발되었을 뿐, 담수의 녹조를 유발하는 남조류 Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc 속에 모두 특이적인 DNA 칩 기술은 개발되어 있지 않았다.In order to solve the above problems, a method is required that anyone can measure easily and accurately, and can simultaneously detect as many items as possible. In this regard, DNA chip technology using the latest molecular biology techniques has been developed in recent decades, and many technical problems are solved, and specific cluster analysis shows high accuracy and practicality. However, existing DNA chip technology has been developed mainly for the detection of diatoms (e.g., Alexandrium ) that cause marine red tide, and is a genus of cyanobacteria microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc that cause freshwater algae. No specific DNA chip technology has been developed.
이에, 본 발명자들은 한국 민물 수계에서 주요 위해성 남조류로 간주되는 6종을, DNA 칩을 이용하여 동시에 빠르고 정량적으로 분석하기 위한 방법을 개발하기 위해 예의 노력한 결과, 본 발명을 완성하게 되었다.Accordingly, the present inventors have completed the present invention as a result of diligent efforts to develop a method for rapidly and quantitatively analyzing six species, which are considered major risk cyanobacteria in Korean freshwater, using DNA chips.
구체적으로, 본 발명의 목적은 조류경보제에서 감시하는 대표적인 위해성 남조류 6종인 Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc 속을 동시에 빠르고 고감도로 정량적 측정이 가능한 프로브를 제공하는 것이다.Specifically, it is an object of the present invention to provide a probe capable of quantitatively and quickly quantitatively measuring the genus Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, and Nostoc, which are representative of six dangerous dangerous algae monitored by a bird alarm.
본 발명의 다른 목적은 조류경보제에서 감시하는 대표적인 위해성 남조류 6종인 Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc 속을 동시에 빠르고 고감도로 정량적 측정이 가능한 DNA 칩을 제공하는 것이다.Another object of the present invention is to provide a DNA chip capable of quantitatively and rapidly measuring quantitatively at the same time in the genus Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc, which are representative of 6 dangerous algae species monitored by a bird alarm.
본 발명의 또 다른 목적은 상기 DNA 칩을 이용하여, 조류 경보제에서 감시하는 대표적인 위해성 남조류 6종인 Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc 속을 동시에 빠르고도 고감도로, 검출 및/또는 정량적 측정이 가능한 방법을 제공하는 것이다.Still another object of the present invention is to detect and / or quantitatively and rapidly and rapidly detect and / or quantitatively measure the genus Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, and Nostoc, which are representative of six dangerous dangerous algae monitored by an algal warning agent . To provide a possible way.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
본 발명은, 서열번호 1 내지 78로 이루어진 군으로부터 선택되는 염기서열로 구성되는 프로브를 6종 이상 포함하는, 남조류 검출 및/또는 정량용 DNA 칩을 제공한다.The present invention provides a DNA chip for cyanobacteria detection and / or quantification comprising six or more probes consisting of nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 to 78.
본 발명의 일 구체예로, 프로브의 표적 유전자는 mcyE, cpcBA, ITS, rbcS, rbcX 또는 nifD인 것을 특징으로 한다.In one embodiment of the invention, the target gene of the probe is characterized in that mcyE, cpcBA, ITS, rbcS, rbcX or nifD .
본 발명의 다른 구체예로, 남조류는 Microcystis 속, Anabaena 속, Oscillatoria 속, Phormidium 속, Aphanizomenon 속 및 Nostoc 속인 것을 특징으로 한다.In another embodiment of the present invention, cyanobacteria are genus Microcystis , Anabaena , Oscillatoria , Phormidium , Aphanizomenon and Nostoc .
본 발명의 또 다른 구체예로, DNA 칩은 Microcystis 속의 검출 및/또는 정량용 프로브로서, 서열번호 4-6, 13-15, 22-24, 37-39, 46-48 및 73-75로 이루어진 군에서 선택되는 염기서열을 갖는 하나 이상의 프로브; Anabaena 속의 검출 및/또는 정량용 프로브로서, 서열번호 1-3, 10-12, 19-21, 34-36, 43-45, 58-60 및 70-72 로 이루어진 군에서 선택되는 염기서열을 갖는 하나 이상의 프로브; Oscillatoria 속의 검출 및 정량용 프로브로서, 서열번호 28-30, 52-54 및 64-66으로 이루어진 군에서 선택되는 염기서열을 갖는 하나 이상의 프로브; Phormidium 속의 검출 및/또는 정량용 프로브로서, 서열번호 31-33, 55-57 및 67-69로 이루어진 군에서 선택되는 염기서열을 갖는 하나 이상의 프로브; Aphanizomenon 속의 검출 및/또는 정량용 프로브로서, 서열번호 76-78로 이루어진 군에서 선택되는 염기서열을 갖는 하나 이상의 프로브; 및 Nostoc 속의 검출 및/또는 정량용 프로브로서, 서열번호 7-9, 16-18, 25-27, 40-42, 49-51 및 61-63으로 이루어진 군에서 선택되는 염기서열을 갖는 하나 이상의 프로브를 포함하는 것을 특징으로 한다.In another embodiment of the invention, the DNA chip is a probe for detecting and / or quantifying the genus Microcystis , consisting of SEQ ID NOs: 4-6, 13-15, 22-24, 37-39, 46-48 and 73-75 At least one probe having a nucleotide sequence selected from the group; A probe for detecting and / or quantifying the genus Anabaena , having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-3, 10-12, 19-21, 34-36, 43-45, 58-60, and 70-72 One or more probes; A probe for detection and quantification of the genus Oscillatoria , comprising: at least one probe having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 28-30, 52-54, and 64-66; A probe for detection and / or quantification of the genus Phormidium , comprising: one or more probes having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31-33, 55-57, and 67-69; A probe for detection and / or quantification of the genus Aphanizomenon , comprising: at least one probe having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 76-78; And one or more probes having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7-9, 16-18, 25-27, 40-42, 49-51, and 61-63, as probes for detecting and / or quantifying the genus Nostoc . Characterized in that it comprises a.
본 발명의 또 다른 구체예로, Microcystis 속은 Microcystis aeruginosa을 포함하고, 아나베나 속은 Anabaena flos-aquae, Anabaena variabilis, Anabaena sp. PCC 7120, Anabaena sp. BIR series를 포함하고, 오실라토리아 속은 Oscillatoria sancta, Oscillatoria tenuis, Oscillatoria sp. 18R, Oscillatoria sp. 19R을 포함하고, 포미디움 속은 Phormidium cf. irriguum, Phormidium ambiguum, Phormidium sp. 4-19b, Phormidium sp. 1-6c, Phormidium sp. 2-26b3을 포함하고, 아파니조메논 속은 Aphanizomenon flos-aquae을 포함하고, 노스톡 속은 Nostoc punctiforme, Nostoc linckia, Nostoc spongiaeforme, Nostoc muscorum을 포함하는 것을 특징으로 한다.In another embodiment of the invention, the genus Microcystis comprises Microcystis aeruginosa , and the genus Anavena is Anabaena flos-aquae, Anabaena variabilis, Anabaena sp. PCC 7120, Anabaena sp. Including the BIR series, the genus Oscillatoria is Oscillatoria sancta, Oscillatoria tenuis, Oscillatoria sp. 18R, Oscillatoria sp. 19R, and the podium is in the Phormidium cf. irriguum, Phormidium ambiguum, Phormidium sp. 4-19b, Phormidium sp. 1-6c, Phormidium sp. It includes 2-26b3, the genus Apanizomenon includes Aphanizomenon flos-aquae , and the genus Northstock includes Nostoc punctiforme, Nostoc linckia, Nostoc spongiaeforme, Nostoc muscorum .
본 발명의 또 다른 구체예로, DNA 칩은 프로브 집적부위(well)가 6개로 구획되어 있는 것을 특징으로 한다.In another embodiment of the present invention, the DNA chip is characterized in that divided into six probe wells (well).
본 발명은 남조류 샘플에서 게놈 DNA를 추출하는 단계; 상기 추출된 게놈 DNA를 절편화 및 표지하는 단계; 상기 표지된 DNA를 DNA 칩에 혼성화 반응시키는 단계; 및 상기 혼성화 정도에 따라 남조류의 종류 및 양을 판별하는 단계를 포함하여 이루어지는, 남조류의 검출 및/또는 정량방법을 제공한다. 이때, 남조류의 종류는 상기한 바와 같다.The present invention comprises the steps of extracting genomic DNA from cyanobacteria samples; Fragmenting and labeling the extracted genomic DNA; Hybridizing the labeled DNA to a DNA chip; And it provides a method for detecting and / or quantifying southern algae comprising the step of determining the type and amount of southern algae according to the degree of hybridization. At this time, the type of cyanobacteria is as described above.
본 발명에 의하면, 한국의 민물 수계에서 녹조를 유발하는 주요 6종의 남조류 유전자 정보가 충분히 확보가능하며, 이 유전자 정보를 이용하여 특이적 정량적 분석이 가능한 프로브 및 DNA 칩의 제공이 가능하며, 1종의 남조류당 3개 이상의 프로브를 설계함으로써 남조류의 종간 구별을 빠르고도 쉽게 통계적으로 가능하게 할 수 있다.According to the present invention, sufficient information on six major algae genes that cause green algae in freshwater in Korea can be sufficiently obtained, and it is possible to provide probes and DNA chips capable of specific quantitative analysis using the genetic information. By designing three or more probes per species of algae, one can quickly and easily statistically distinguish between species of algae.
또한, 본 발명에 의하면, 조류 경보제에서 감시대상이 되는 6종의 남조류를 하나의 DNA 칩에서 동시에 판별할 수 있고, 기존의 현미경법에 비해 시료를 고효율로 대량 분석이 가능하다.In addition, according to the present invention, six species of algae to be monitored in the bird alarm can be discriminated simultaneously in one DNA chip, and mass analysis of the sample can be carried out with high efficiency as compared with the conventional microscopy.
또한, 본 발명에 의하면, 담수 수계 샘플로부터 추출한 유전자에 대하여 중합효소 연쇄 반응(polymerase chain reaction, PCR)이나 기타 증폭과정을 거치지 않기 때문에, 환경 내 유전정보가 편향되지 않은 상태의 정량적 측정이 가능하다는 장점이 있다.In addition, according to the present invention, since the gene extracted from the freshwater aquatic sample is not subjected to a polymerase chain reaction (PCR) or other amplification process, it is possible to quantitatively measure the unbiased condition of genetic information in the environment. There is an advantage.
또한, 본 발명에 의하면, 설계된 프로브가 ITS(Internal Transcribed Spacer)와 같은 구조 유전자와, rpoB, mcyE, cpcBA, nifD, rbcS, rbcX와 같은 기능성 유전자에 기반을 두어 제작되기 때문에, 구조 유전자와 기능성 유전자를 동시에 이용하여 남조류 종류의 정확한 판별 및 동정과 독성물질의 예측 등 수계 건강성을 광범위하게 평가할 수 있다. In addition, according to the present invention, since the designed probe is manufactured based on a structural gene such as ITS (Internal Transcribed Spacer) and a functional gene such as rpoB , mcyE , cpcBA , nifD , rbcS, rbcX , the structural gene and the functional gene Simultaneous use of the system can provide an extensive assessment of aquatic health, including accurate identification and identification of cyanobacteria species and prediction of toxic substances.
또한, 기존의 DNA 칩을 이용한 특정 생물종의 검출방법이 단일 미생물종을 목적으로 개발되었다면, 본 발명에 의하면, 수계에 우점하는 광범위한 위해성 남조류 6종을 동시에 빠르고도 고감도로, 정량적인 검출이 가능하다는 장점이 있다.In addition, if a conventional method for detecting a specific species using a DNA chip was developed for the purpose of a single microbial species, according to the present invention, it is possible to quantitatively detect a wide range of dangerous seaweeds that are dominant in water, at the same time, quickly and sensitively. Has the advantage.
또한, 기존의 DNA 칩 기술은 주로 해양의 적조를 발생하는 규조류 (예, Alexandrium)를 검출하는 것을 목적으로 개발되었다면, 본 발명은 담수의 녹조를 유발하는 남조류 Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc 속에 특이적이고 정량적인 측정방법을 제공한다는 장점이 있다.In addition, the existing DNA chip technology mainly produces diatoms that generate red tide in the ocean. If developed for the purpose of detecting (eg, Alexandrium ), the present invention has the advantage of providing a specific and quantitative measuring method in cyanobacteria Microcystis, Anabaena , Oscillatoria , Phormidium , Aphanizomenon, Nostoc that cause freshwater green algae .
도 1은 본 발명의 DNA 칩을 나타내는 사진으로서, 위해성 남조류 6종(Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc)을 동시에 특이적이고 정량적으로 검출할 수 있는 프로브가 6개의 구역에 배열되어 있다. 1 is a photograph showing the DNA chip of the present invention, in which six probes capable of detecting specific and quantitatively dangerous algae ( Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc ) at the same time are arranged in six zones.
도 2a 내지 도 2e는 Microcystis의 유전자 (a) mcyE, (b) cpcBA, (c) ITS, (d) rbcS, (e) rbcX 에 상보적으로 결합할 수 있는 프로브의 특이성을 나타내는 도표이다.2A-2E are diagrams showing the specificity of a probe capable of complementarily binding to the genes of Microcystis (a) mcyE , (b) cpcBA , (c) ITS, (d) rbcS , and (e) rbcX .
도 3a 내지 도 3e는 Microcystis의 유전자 (a) mcyE, (b) cpcBA, (c) ITS, (d) rbcS, (e) rbcX 에 상보적으로 결합할 수 있는 프로브를 이용한 DNA 칩의 정량성을 나타내는 도표이다.Figures 3a-3e shows the quantification of DNA chips using probes that can complementarily bind to the genes of Microcystis (a) mcyE , (b) cpcBA , (c) ITS, (d) rbcS , (e) rbcX . It is a chart showing
도 4a 내지 도 4c는 Nostoc의 유전자 (a) rpoB, (b) rbcS, (c) rbcX에 상보적으로 결합할 수 있는 프로브의 특이성을 나타내는 도표이다.4A-4C are diagrams showing the specificity of a probe capable of complementarily binding to Nostoc genes (a) rpoB , (b) rbcS, (c) rbcX .
도 5a 내지 도 5c는 Nostoc의 유전자 (a) rpoB, (b) rbcS, (c) rbcX에 상보적으로 결합할 수 있는 프로브를 이용한 DNA 칩의 정량성을 나타내는 도표이다.5A to 5C are diagrams showing the quantification of DNA chips using probes capable of complementarily binding to Nostoc genes (a) rpoB , (b) rbcS , and (c) rbcX .
도 6은 Anabaena의 rpoB 유전자에 상보적으로 결합할 수 있는 프로브의 특이성을 나타내는 도표이다.6 is a chart showing the specificity of a probe capable of complementarily binding to the rpoB gene of Anabaena .
도 7은 Anabaena의 rpoB 유전자에 상보적으로 결합할 수 있는 프로브를 이용한 DNA 칩의 정량성을 나타내는 도표이다.7 is a diagram showing the quantification of the DNA chip using a probe capable of complementarily binding to the rpo B gene of Anabaena .
도 8은 Oscillatoria의 ITS 유전자에 상보적으로 결합할 수 있는 프로브의 특이성을 나타내는 도표이다.8 is a chart showing the specificity of a probe capable of complementarily binding to the ITS gene of Oscillatoria .
도 9는 Oscillatoria의 ITS 유전자에 상보적으로 결합할 수 있는 프로브를 이용한 DNA 칩의 정량성을 나타내는 도표이다.9 is a diagram showing the quantification of the DNA chip using a probe capable of complementarily binding to the ITS gene of Oscillatoria .
도 10은 Phormidium의 mcyE 유전자에 상보적으로 결합할 수 있는 프로브의 특이성을 나타내는 도표이다.10 is a chart showing the specificity of a probe capable of complementarily binding to mcy E gene of Phormidium .
도 11은 Phormidium의 mcyE 유전자에 상보적으로 결합할 수 있는 프로브를 이용한 DNA 칩의 정량성을 나타내는 도표이다.11 is a diagram showing the quantification of the DNA chip using a probe capable of complementarily binding to the mcy E gene of Phormidium .
도 12는 Aphanizomenon의 cpcBA 유전자에 상보적으로 결합할 수 있는 프로브를 이용한 DNA 칩의 정량성을 나타내는 도표이다.12 is a diagram showing the quantification of the DNA chip using a probe capable of complementarily binding to the cpc BA gene of Aphanizomenon .
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 일례일 뿐, 본 발명이 하기 실시예에 한정된 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only examples of the present invention, and the present invention is not limited to the following examples.
[실시예]EXAMPLE
실시예 1. 타겟 유전자 선정 및 프로브 제작Example 1. Target Gene Selection and Probe Construction
한국의 민물 수계에서 녹조를 유발하는 주요 6종의 남조류에 특이적인 프로브를 제작하기 위하여 먼저 타겟 유전자를 선정하였다. 타겟이 되는 유전자는 프로브로 설계되는 기본적인 염기서열의 정보를 제공할 수 있어야 한다. 또한 타겟 유전자를 기반으로 설계된 프로브는 종 특이성을 보유하고 있어야 하며, 이를 위하여 다른 종과 구별이 되는 유전정보를 갖고 있어야 한다. 다른 종과 구별이 안될 정도로 보전된 유전자 부위는 후보 유전자가 될 수 없으며 종간 유전정보가 너무 다른 유전자 부위도 프로브 설계에 적합하지 않을 수 있다. 타겟 유전자는 또한 데이터베이스에 양질의 정보를 갖고 있을 만큼 충분한 기반 연구가 진행되어 있어야 한다. Target genes were first selected to produce probes specific to the six major species of green algae that cause green algae in freshwater water systems in Korea. The target gene must be able to provide information on the basic sequence designed by the probe. In addition, probes designed based on target genes must have species specificity and for this purpose must have genetic information that distinguishes them from other species. Gene regions that are conserved indistinguishable from other species cannot be candidate genes, and gene regions with too much genetic information between species may not be suitable for probe design. Target genes must also be well grounded enough to have good information in the database.
대표적인 데이터베이스인 미국 국립생물정보센터(National Center for Biotechnology Information, NCBI)에서 유전자 정보를 얻은 후, Geneious 프로그램 (Biomatters Ltd.)으로 다중정렬방법(multiple alignment)을 수행하였다. 그 결과, 본 발명의 타겟인 6종의 남조류를 동시에 특이적으로 구별하기 위한 최적 후보 유전자로서 아래와 같은 유전자를 선정하였다. After obtaining genetic information from the National Center for Biotechnology Information (NCBI), a representative database, multiple alignments were performed with the Geneious program (Biomatters Ltd.). As a result, the following genes were selected as the best candidate genes for specifically distinguishing six species of cyanobacteria that are the targets of the present invention.
rbcS는, 남조류가 이산화탄소를 고정하는데 필수적인 RuBisCo 복합체의 소단위(small subunit)를 코딩하는 유전자로, rbcS 유전자를 분석하여 Anabaena, Microcystis, Nostoc 3개 속(genus)에 특이적인 9개의 프로브를 제작하였다(서열번호 1 내지 9). rbcS is, a gene that blue-green algae is encoding a subunit (small subunit) of an essential RuBisCo composite for fixing the carbon dioxide, by analyzing the rbcS gene to prepare a specific nine probes to Anabaena, Microcystis, Nostoc 3 gae genus (genus) ( SEQ ID NOs: 1 to 9).
rbcX는, RuBisCo 복합체의 형성을 보조하는데 필수적인 샤프론(chaperone) 단백질을 코딩하는 유전자로, rbcX 유전자를 분석하여 Anabaena, Microcystis, Nostoc 3개 속(genus)에 특이적인 9개의 프로브를 제작하였다(서열번호 10 내지 18). rbcX is a gene encoding a chaperone protein which is essential for assisting the formation of RuBisCo complex. The rbcX gene was analyzed to produce 9 probes specific to Anabaena, Microcystis and Nostoc genus (SEQ ID NO: 10 to 18).
ITS(Internal transcribed spacer)는 rRNA의 코딩부위 사이의 공간으로서, 다양하고 안정된 서열을 가지고 있어서 매우 가까운 연관관계에 있는 미생물이나 매우 먼 연관관계를 갖는 미생물 사이에서도 비교가 가능하여 DNA 칩의 프로브 제작에 강력한 마커로 사용될 수 있기 때문에, ITS 유전자를 분석하여 Anabaena, Microcystis, Nostoc, Oscillatoria, Phormidium 5개 속(genus)에 특이적인 15개의 프로브를 제작하였다(서열번호 19 내지 33). ITS (Internal transcribed spacer) is a space between coding regions of rRNA, and has various and stable sequences, so that it can be compared between microorganisms having very close associations or microorganisms having a very distant association. Since it can be used as a powerful marker, the ITS gene was analyzed to produce 15 probes specific for 5 genus Anabaena, Microcystis, Nostoc, Oscillatoria, and Phormidium (SEQ ID NOs: 19 to 33).
rpoB(RNA polymerase beta subunit)는 RNA 중합효소 베타 소단위를 코딩하는 유전자로, rpoB 유전자를 분석하여 Anabaena, Microcystis, Nostoc 3개 속(genus)에 특이적인 9개의 프로브를 제작하였다(서열번호 34 내지 42). rpoB (RNA polymerase beta subunit) is a gene encoding an RNA polymerase beta subunit. By analyzing the rpoB gene, 9 probes specific to Anabaena, Microcystis and Nostoc genus were generated (SEQ ID NOs. 34 to 42). ).
mcyE는 남조류가 생산하는 간독소인 마이크로시스틴(microcystin)을 코딩하는 유전자로, mcyE 유전자를 분석하여 독성 남조류(toxin-producing cyanobacteria)인 Anabaena, Microcystis, Nostoc, Oscillatoria, Phormidium 5개 속(genus)에 특이적인 15개의 프로브를 제작하였다(서열번호 43 내지 57). 이 프로브 세트는 녹조로 인한 수질 오염의 큰 문제인 남조류 독성의 감시에 있어 중요한 지표가 될 수 있다. mcyE is a gene encoding microcystin, a hepatoxin produced by cyanobacteria, and analyzed by mcyE gene in five genus of Anabaena, Microcystis, Nostoc, Oscillatoria, and Phormidium, which are toxic toxin-producing cyanobacteria. 15 specific probes were prepared (SEQ ID NOs: 43-57). This set of probes can be an important indicator for monitoring algae toxicity, a major problem with water pollution from green algae.
nifD는 남조류가 공기중의 질소를 고정하는 데에 필수적인 질소고정효소(nitrogenase)를 코딩하는 유전자로, nifD 유전자를 분석하여 Anabaena, Nostoc, Oscillatoria, Phormidium 4개 속(genus)에 특이적인 12개의 프로브를 제작하였다(서열번호 58 내지 69). nifD is a gene encoding nitrogenase, which is essential for cyanobacteria to fix nitrogen in the air.It analyzes the nifD gene and analyzes twelve probes specific to four genera: Anabaena, Nostoc, Oscillatoria, and Phormidium. Was prepared (SEQ ID NOs: 58-69).
cpcBA는 미세조류의 광합성기구 보조색소인 피코시아닌(phycocyanin)을 코딩하는 유전자로, cpcBA 유전자를 분석하여 Anabaena, Microcystis, Aphanizomenon 3개 속(genus)에 특이적인 9개의 프로브를 제작하였다(서열번호 70 내지 78). cpcBA is a gene encoding phycocyanin, a microalgae photosynthetic auxiliary pigment, and analyzed 9 cpcBA genes to produce 9 probes specific to Anabaena, Microcystis and Aphanizomenon genus (SEQ ID NO: 70 to 78).
프로브의 제작은 OligoWiz 프로그램(http://www.cbs.dtu.dk/services/OligoWiz/)을 이용하였으며, 하기 [표 1]에 나타낸 바와 같이 총 78개의 프로브를 제작하였다. 이를 위해 최근 NCBI에 등록된 Anabaena, Microcystis, Oscillatoria, Phormidium, Nostoc 의 전체 유전자 염기정보(whole genome sequence)와 각 남조류의 유전자 염기정보를 비교하였다.Probe was fabricated using OligoWiz program (http://www.cbs.dtu.dk/services/OligoWiz/), and a total of 78 probes were prepared as shown in Table 1 below. To this end, we compared the whole genome sequence of Anabaena , Microcystis , Oscillatoria , Phormidium , Nostoc , which were recently registered with NCBI, with the gene base information of each cyanobacteria.
하기 표 1은, 조류 경보제로 감시하는 한국 민물 수계의 주요 남조류 6종을 검출하기 위한 특이적인 프로브를 나타낸 것이다.Table 1 below shows specific probes for detecting six major cyanobacteria in the Korean freshwater water system monitored by the algae alert.
표 1
프로브 명칭 염기서열 타겟 남조류 종명 타겟 유전자
AnarbcS1(서열번호 1) AATATTAAGCAGTGCCAAATTCTGAGCTTCATCGTTCACAAACCCAG Anabaena sp. PCC 7120 rbcS
AnarbcS2(서열번호 2) TATTGGCAGAAGTTCAATCTTGCCGTTCTCAATATCCTGGTCACTAC Anabaena sp. PCC 7120 rbcS
AnarbcS3(서열번호 3) TTGTTTGGTGCTAAAACATCCCGTGAAGTATTGGCAGAAGTTCAATC Anabaena sp. PCC 7120 rbcS
MicrbcS1(서열번호 4) CCCCCCCTCACCGATCAACAAATTGCTAAACAAATTCAATACATGAT Microcystis aeruginosa rbcS
MicrbcS2(서열번호 5) TTCTGATTGCTACATCCGCGTTATCGCTTTTGACAACATCAAACAAT Microcystis aeruginosa rbcS
MicrbcS3(서열번호 6) GTGGAAACTGCCTTTATTCTCTGTTTCTGGTCCCCAAGAAGTTCTTA Microcystis aeruginosa rbcS
NosrbcS1(서열번호 7) TGATAACATCAAGCAGTGCCAAGTTCTCAGCTTTCTTGTTCACAAAC Nostoc punctiforme rbcS
NosrbcS2(서열번호 8) TCCAGCGATCGAGTTCAACGAAACTTCTGAGCCAACAGAATTATATT Nostoc punctiforme rbcS
NosrbcS3(서열번호 9) AGAAGTATTGAGCGAAGTTCAAGGATGCCGTTCTCAATTTAACGGTA Nostoc punctiforme rbcS
AnarbcX1(서열번호 10) TAGCCTTGCGGATAATGACTGTCAGAGAACATATAGCCGAAGAAGTA Anabaena sp. PCC 7120 rbcX
AnarbcX2(서열번호 11) GAACATATAGCCGAAGAAGTAGCCGAGTTCTTACCAGAAATGGTTCG Anabaena sp. PCC 7120 rbcX
AnarbcX3(서열번호 12) GTTTATCTAACCCCAGTCCTGAATCAGAACAGCAGACAATTTCCGAT Anabaena sp. PCC 7120 rbcX
MicrbcX1(서열번호 13) AACGGGAATTATTAATGACAATACCGAACATCGTCGGCAACTTTTGG Microcystis aeruginosa rbcX
MicrbcX2(서열번호 14) TATTGCGGGGTTGATGACTGAGAATAAAGAGTTAGTTTTGCGGATCA Microcystis aeruginosa rbcX
MicrbcX3(서열번호 15) AGTCTTACAAAGTTACCTAACTTACCAAGCGGTTCGCACGATTATCG Microcystis aeruginosa rbcX
NostrbcX1(서열번호 16) CGAATCATGACTGTCAGAGAACACATTGCGGAAGAAATTGCAGAATT Nostoc punctiforme rbcX
NostrbcX2(서열번호 17) AAAAACCAGATTTGGCTTTGCGAATCATGACTGTCAGAGAACACATT Nostoc punctiforme rbcX
NostrbcX3(서열번호 18) AACACATTGCGGAAGAAATTGCAGAATTTTTACCAGAAATGGTTCGC Nostoc punctiforme rbcX
AnaITS1(서열번호 19) CTCCACATGAAAAAGCGAAAAGCCAGAAAGGAAGAATTCAGCAACTA Anabaena flos-aquae ITS
AnaITS2(서열번호 20) GCAGGATAAGCCAATGAAAAAGTGGTCAAGCTAATAAGGGCTAATGG Anabaena flos-aquae ITS
AnaITS3(서열번호 21) GAACCTTGAAAACTGCATAAAAACGCGATTAGATTAGCAGGCAGACA Anabaenaflos- aquae ITS
MicITS1(서열번호 22) AGGGAGACCTAATTCAGGTAGGAGACGAAAAAAAAGTAGTCCCTACC Microcystis aeruginosa ITS
MicITS2(서열번호 23) CAAAATTGGCTTTCAAACTAGGTTCTGGGTTCACACAAGACCTGAAT Microcystis aeruginosa ITS
MicITS3(서열번호 24) ACGAAAAAAAAGTAGTCCCTACCAAGAATCAATCCCAAAAGGTCGGA Microcystis aeruginosa ITS
NosITS1(서열번호 25) TCCTTTTTAGGGAGACCTACCCAACTTTAATATCGAGGACACACAGT Nostoc linckia ITS
NosITS2(서열번호 26) GGATCACCTCCTTTTTAGGGAGACCTACCCAACTTTAATATCGAGGA Nostoc linckia ITS
NosITS3(서열번호 27) ATCACCTCCTTTTTAGGGAGACCTACCCAACTTTAATATCGAGGACA Nostoc linckia ITS
OscITS1(서열번호 28) AGTCAATAGCTAACCATTTCATGAGGTGCAGAAAGTGGTCAAGTGAT Oscillatoria sancta ITS
OscITS2(서열번호 29) ACTAATGTCCAGCCAGAACCTTGAAAACTGCATAGTCAATCAGTCAA Oscillatoria sancta ITS
OscITS3(서열번호 30) AGAGGTTAGTCAATAGCTAACCATTTCATGAGGTGCAGAAAGTGGTC Oscillatoria sancta ITS
PhoITS1(서열번호 31) ACCTAGCCCAACTGATGACCGAAAAGAAAGTTAATAGGTCACTGTTG Phormidium cf. irriguum ITS
PhoITS2(서열번호 32) AACGAGAATGGACAGGCTTTCAAACTATTTTCAGGTTGGGAAAATGG Phormidium cf. irriguum ITS
PhoITS3(서열번호 33) GGCTTTCAAACTATTTTCAGGTTGGGAAAATGGGCTATTAGCTCAGG Phormidium cf. irriguum ITS
AnarpoB1(서열번호 34) CACCTGTTTAAACCAAAAACCATTGGTGAGAATTGGCGAAAAAGTCG Anabaena flos-aquae rpoB
AnarpoB2(서열번호 35) GCAGCCAGTGGTAGCCAAGTTATTGAAAAAGGCCAAGAACTTAAATA Anabaena flos-aquae rpoB
AnarpoB3(서열번호 36) TATCAACGTTCCAACCAAGACACCTGTTTAAACCAAAAACCATTGGT Anabaena flos-aquae rpoB
MicrpoB1(서열번호 37) AAATATCAACGCTCTAACCAAGATACCTGCCTAAATCAGCGTCCTTT Microcystis aeruginosa rpoB
MicrpoB2(서열번호 38) GAAATATCAACGCTCTAACCAAGATACCTGCCTAAATCAGCGTCCTT Microcystis aeruginosa rpoB
MicrpoB3(서열번호 39) GGCAAGAGCGAGATCGAGTACGAAATTCAGAAATATCAACGCTCTAA Microcystis aeruginosa rpoB
NosrpoB1(서열번호 40) TATCAACGATCCAACCAGGATACCTGTTTAAATCAAAAACCCCTCGT Nostoc sp. 152 rpoB
NosrpoB2(서열번호 41) CCACAGAAATTCGTGTACGTCCCAAAACCAGTACCCAAGAAATTAAG Nostoc sp. 152 rpoB
NosrpoB3(서열번호 42) TGTAGCTACTAGTATGATTCCCTTTTTGGAACATGACGACGCTAACC Nostoc sp. 152 rpoB
AnamcyE1(서열번호 43) GAACAGCTGAACCATTGAGTCTTGGTACACTATCAGGAATGGTTGAA Anabaena spp. BIR series mcyE
AnamcyE2(서열번호 44) CAGCTGCTTTAATTTGTGAAATGACAGGTGTAGAACGAGTCGCTTTT Anabaena spp. BIR series mcyE
AnamcyE3(서열번호 45) GGACAAAACGCCAAAAAATTGTGATTTTTGCTGGTTCTTATCACGGT Anabaena spp. BIR series mcyE
MicmcyE1(서열번호 46) AGAAGATAAAACCACGGCTCAACCCTTAAGTTTAGGCACTCCTTTAG Microcystis aeruginosa mcyE
MicmcyE2(서열번호 47) TCATTGAAGCGGTTCAAGAACAAATGAACCGAGGAATAGGTTTAGGA Microcystis aeruginosa mcyE
MicmcyE3(서열번호 48) GAACAAATGAACCGAGGAATAGGTTTAGGAATGCAGTCAAATCTGGC Microcystis aeruginosa mcyE
NosmcyE1(서열번호 49) TAAAACCACGACTCAACCCTTAAGTTTAGGCACTCCTTTAGGAATGG Nostoc sp. 74.2 mcyE
NosmcyE2(서열번호 50) TAGGAGAAGATAAAACCACGACTCAACCCTTAAGTTTAGGCACTCCT Nostoc sp. 74.2 mcyE
NosmcyE3(서열번호 51) TAATTAGTGAAATGGGCCGGGTCGAAAGAGTCGCTTTTAGTAATACG Nostoc sp. 74.2 mcyE
OscmcyE1(서열번호 52) CCGTAAGTCTGGGTACACCATCAGGAATGGTTGAAGATGTAATTGTT Oscillatoria sp. 18ROscillatoria sp. 19R mcyE
OscmcyE2(서열번호 53) GGGGTAGAAAGGGTGGCTTTTAGTAATACTGGAACCGAAGCAATTAT Oscillatoria sp. 18ROscillatoria sp. 19R mcyE
OscmcyE3(서열번호 54) GGATTGCTCGTTCTCGCACAAAACGCCCAAAAATTGTTATATTTTCC Oscillatoria sp. 18ROscillatoria sp. 19R mcyE
PhomcyE1(서열번호 55) TGCTATGTTTGCAGGTTCATATCACGGAACCTTTGATGGCATTTTAG Phormidium sp. 4-19bPhormidium sp. 1-6cPhormidium sp. 2-26b3 mcyE
PhomcyE2(서열번호 56) AGCTGCTTTAATTAGTGAAATGGGAGGAGTAGAACGGGTTGCTTTTA Phormidium sp. 4-19bPhormidium sp. 1-6cPhormidium sp. 2-26b3 mcyE
PhomcyE3(서열번호 57) ACAAAACGCCAAAAAATTGCTATGTTTGCAGGTTCATATCACGGAAC Phormidium sp. 4-19bPhormidium sp. 1-6cPhormidium sp. 2-26b3 mcyE
AnanifD1(서열번호 58) ATCCGAGACTGGATTTTCCCAGAATACGACAAGCTGAAGAAAGAAAA Anabaena variabilis nifD
AnanifD2(서열번호 59) TGGATTTTCCCAGAATACGACAAGCTGAAGAAAGAAAACAGACTCGA Anabaena variabilis nifD
AnanifD3(서열번호 60) TTTCCCAGAATACGACAAGCTGAAGAAAGAAAACAGACTCGACTTCG Anabaena variabilis nifD
NosnifD1(서열번호 61) AGAATACGACAAGCTCAAGAAAGAAAACAGACTTGACTTCGAGCCAA Nostoc spongiaeforme Nostoc punctiforme Nostoc muscorum nifD
NosnifD2(서열번호 62) TCCCAGAATACGACAAGCTCAAGAAAGAAAACAGACTTGACTTCGAG Nostoc spongiaeforme Nostoc punctiforme Nostoc muscorum nifD
NosnifD3(서열번호 63) ATCCGTGACTGGATTTTCCCAGAATACGACAAGCTCAAGAAAGAAAA Nostoc spongiaeforme Nostoc punctiforme Nostoc muscorum nifD
OscnifD1(서열번호 64) GACGACGTTTCAGCCTACGAGTTTGAGGAGTTCATTAAAGAACTCAA Oscillatoria tenuis nifD
OscnifD2(서열번호 65) TTTCAGCCTACGAGTTTGAGGAGTTCATTAAAGAACTCAAACCCGAC Oscillatoria tenuis nifD
OscnifD3(서열번호 66) GGAGTTCATTAAAGAACTCAAACCCGACTTAGTAGCTTCTGGCATCA Oscillatoria tenuis nifD
PhonifD1(서열번호 67) GTTAATTGGTACAGGCTACGAGTTCGGTCACAACGACGATTACAAAC Phormidium ambiguum nifD
PhonifD2(서열번호 68) AAAGTTATCGCCAAATACGAAGCGCAAACGAAAGCTGTAATTGAAAA Phormidium ambiguum nifD
PhonifD3(서열번호 69) AAGCTGAATCTAGTTCACTGCTATCGCTCCATGAACTATATCAGCCG Phormidium ambiguum nifD
AnacpcBA1(서열번호 70) GACCTATCTCCTAGCTGGTATGTTGAAGCTCTAAAGCACATCAAAGC Anabaena variabilis cpcBA
AnacpcBA2(서열번호 71) GTTTAAGTGGTCAAGCTGCTAACGAAGCTAACACCTACATCGACTAC Anabaena variabilis cpcBA
AnacpcBA3(서열번호 72) AAAGCACATCAAAGCTAATCATGGTTTAAGTGGTCAAGCTGCTAACG Anabaena variabilis cpcBA
MiccpcBA1(서열번호 73) CTAGCTGGTACATCGAAGCTCTCAAATATATCAAAGCCAACCATGGT Microcystis aeruginosa cpcBA
MiccpcBA2(서열번호 74) CTTCGACCTGTCTCCTAGCTGGTACATCGAAGCTCTCAAATATATCA Microcystis aeruginosa cpcBA
MiccpcBA1(서열번호 75) CATCGAAGCTCTCAAATATATCAAAGCCAACCATGGTTTAAGTGGCG Microcystis aeruginosa cpcBA
AphcpcBA1(서열번호 76) AATTCCCTTACACCACATCTACACCAGGTAATCAATACGCATCCGAT Aphanizomenon flos-aquae cpcBA
AphcpcBA2(서열번호 77) CTAGTGTTCTTGATGACCGTTGCTTAAATGGTTTGCGCGAAACATAC Aphanizomenon flos-aquae cpcBA
AphcpcBA3(서열번호 78) GCCTAACAATTACAAATAGCTCGAATCTTAATTGAGCTTCTTCCAAC Aphanizomenon flos-aquae cpcBA
Table 1
Probe Name Sequence Target cyanobacteria species Target genes
AnarbcS1 (SEQ ID NO: 1) AATATTAAGCAGTGCCAAATTCTGAGCTTCATCGTTCACAAACCCAG Anabaena sp. PCC 7120 rbc S
AnarbcS2 (SEQ ID NO: 2) TATTGGCAGAAGTTCAATCTTGCCGTTCTCAATATCCTGGTCACTAC Anabaena sp. PCC 7120 rbc S
AnarbcS3 (SEQ ID NO: 3) TTGTTTGGTGCTAAAACATCCCGTGAAGTATTGGCAGAAGTTCAATC Anabaena sp. PCC 7120 rbc S
MicrbcS1 (SEQ ID NO: 4) CCCCCCCTCACCGATCAACAAATTGCTAAACAAATTCAATACATGAT Microcystis aeruginosa rbc S
MicrbcS2 (SEQ ID NO: 5) TTCTGATTGCTACATCCGCGTTATCGCTTTTGACAACATCAAACAAT Microcystis aeruginosa rbc S
MicrbcS3 (SEQ ID NO: 6) GTGGAAACTGCCTTTATTCTCTGTTTCTGGTCCCCAAGAAGTTCTTA Microcystis aeruginosa rbc S
NosrbcS1 (SEQ ID NO: 7) TGATAACATCAAGCAGTGCCAAGTTCTCAGCTTTCTTGTTCACAAAC Nostoc punctiforme rbc S
NosrbcS2 (SEQ ID NO: 8) TCCAGCGATCGAGTTCAACGAAACTTCTGAGCCAACAGAATTATATT Nostoc punctiforme rbc S
NosrbcS3 (SEQ ID NO: 9) AGAAGTATTGAGCGAAGTTCAAGGATGCCGTTCTCAATTTAACGGTA Nostoc punctiforme rbc S
AnarbcX1 (SEQ ID NO: 10) TAGCCTTGCGGATAATGACTGTCAGAGAACATATAGCCGAAGAAGTA Anabaena sp. PCC 7120 rbc X
AnarbcX2 (SEQ ID NO: 11) GAACATATAGCCGAAGAAGTAGCCGAGTTCTTACCAGAAATGGTTCG Anabaena sp. PCC 7120 rbc X
AnarbcX3 (SEQ ID NO: 12) GTTTATCTAACCCCAGTCCTGAATCAGAACAGCAGACAATTTCCGAT Anabaena sp. PCC 7120 rbc X
MicrbcX1 (SEQ ID NO: 13) AACGGGAATTATTAATGACAATACCGAACATCGTCGGCAACTTTTGG Microcystis aeruginosa rbc X
MicrbcX2 (SEQ ID NO: 14) TATTGCGGGGTTGATGACTGAGAATAAAGAGTTAGTTTTGCGGATCA Microcystis aeruginosa rbc X
MicrbcX3 (SEQ ID NO: 15) AGTCTTACAAAGTTACCTAACTTACCAAGCGGTTCGCACGATTATCG Microcystis aeruginosa rbc X
NostrbcX1 (SEQ ID NO: 16) CGAATCATGACTGTCAGAGAACACATTGCGGAAGAAATTGCAGAATT Nostoc punctiforme rbc X
NostrbcX2 (SEQ ID NO: 17) AAAAACCAGATTTGGCTTTGCGAATCATGACTGTCAGAGAACACATT Nostoc punctiforme rbc X
NostrbcX3 (SEQ ID NO: 18) AACACATTGCGGAAGAAATTGCAGAATTTTTACCAGAAATGGTTCGC Nostoc punctiforme rbc X
AnaITS1 (SEQ ID NO: 19) CTCCACATGAAAAAGCGAAAAGCCAGAAAGGAAGAATTCAGCAACTA Anabaena flos-aquae ITS
AnaITS2 (SEQ ID NO: 20) GCAGGATAAGCCAATGAAAAAGTGGTCAAGCTAATAAGGGCTAATGG Anabaena flos-aquae ITS
AnaITS3 (SEQ ID NO: 21) GAACCTTGAAAACTGCATAAAAACGCGATTAGATTAGCAGGCAGACA Anabaenaflos- aquae ITS
MicITS1 (SEQ ID NO: 22) AGGGAGACCTAATTCAGGTAGGAGACGAAAAAAAAGTAGTCCCTACC Microcystis aeruginosa ITS
MicITS2 (SEQ ID NO: 23) CAAAATTGGCTTTCAAACTAGGTTCTGGGTTCACACAAGACCTGAAT Microcystis aeruginosa ITS
MicITS3 (SEQ ID NO: 24) ACGAAAAAAAAGTAGTCCCTACCAAGAATCAATCCCAAAAGGTCGGA Microcystis aeruginosa ITS
NosITS1 (SEQ ID NO: 25) TCCTTTTTAGGGAGACCTACCCAACTTTAATATCGAGGACACACAGT Nostoc linckia ITS
NosITS2 (SEQ ID NO: 26) GGATCACCTCCTTTTTAGGGAGACCTACCCAACTTTAATATCGAGGA Nostoc linckia ITS
NosITS3 (SEQ ID NO: 27) ATCACCTCCTTTTTAGGGAGACCTACCCAACTTTAATATCGAGGACA Nostoc linckia ITS
OscITS1 (SEQ ID NO: 28) AGTCAATAGCTAACCATTTCATGAGGTGCAGAAAGTGGTCAAGTGAT Oscillatoria sancta ITS
OscITS2 (SEQ ID NO: 29) ACTAATGTCCAGCCAGAACCTTGAAAACTGCATAGTCAATCAGTCAA Oscillatoria sancta ITS
OscITS3 (SEQ ID NO: 30) AGAGGTTAGTCAATAGCTAACCATTTCATGAGGTGCAGAAAGTGGTC Oscillatoria sancta ITS
PhoITS1 (SEQ ID NO: 31) ACCTAGCCCAACTGATGACCGAAAAGAAAGTTAATAGGTCACTGTTG Phormidium cf. irriguum ITS
PhoITS2 (SEQ ID NO: 32) AACGAGAATGGACAGGCTTTCAAACTATTTTCAGGTTGGGAAAATGG Phormidium cf. irriguum ITS
PhoITS3 (SEQ ID NO: 33) GGCTTTCAAACTATTTTCAGGTTGGGAAAATGGGCTATTAGCTCAGG Phormidium cf. irriguum ITS
AnarpoB1 (SEQ ID NO: 34) CACCTGTTTAAACCAAAAACCATTGGTGAGAATTGGCGAAAAAGTCG Anabaena flos-aquae rpo B
AnarpoB2 (SEQ ID NO: 35) GCAGCCAGTGGTAGCCAAGTTATTGAAAAAGGCCAAGAACTTAAATA Anabaena flos-aquae rpo B
AnarpoB3 (SEQ ID NO: 36) TATCAACGTTCCAACCAAGACACCTGTTTAAACCAAAAACCATTGGT Anabaena flos-aquae rpo B
MicrpoB1 (SEQ ID NO: 37) AAATATCAACGCTCTAACCAAGATACCTGCCTAAATCAGCGTCCTTT Microcystis aeruginosa rpo B
MicrpoB2 (SEQ ID NO: 38) GAAATATCAACGCTCTAACCAAGATACCTGCCTAAATCAGCGTCCTT Microcystis aeruginosa rpo B
MicrpoB3 (SEQ ID NO: 39) GGCAAGAGCGAGATCGAGTACGAAATTCAGAAATATCAACGCTCTAA Microcystis aeruginosa rpo B
NosrpoB1 (SEQ ID NO: 40) TATCAACGATCCAACCAGGATACCTGTTTAAATCAAAAACCCCTCGT Nostoc sp. 152 rpo B
NosrpoB2 (SEQ ID NO: 41) CCACAGAAATTCGTGTACGTCCCAAAACCAGTACCCAAGAAATTAAG Nostoc sp. 152 rpo B
NosrpoB3 (SEQ ID NO: 42) TGTAGCTACTAGTATGATTCCCTTTTTGGAACATGACGACGCTAACC Nostoc sp. 152 rpo B
AnamcyE1 (SEQ ID NO: 43) GAACAGCTGAACCATTGAGTCTTGGTACACTATCAGGAATGGTTGAA Anabaena spp. BIR series mcy E
AnamcyE2 (SEQ ID NO: 44) CAGCTGCTTTAATTTGTGAAATGACAGGTGTAGAACGAGTCGCTTTT Anabaena spp. BIR series mcy E
AnamcyE3 (SEQ ID NO: 45) GGACAAAACGCCAAAAAATTGTGATTTTTGCTGGTTCTTATCACGGT Anabaena spp. BIR series mcy E
MicmcyE1 (SEQ ID NO: 46) AGAAGATAAAACCACGGCTCAACCCTTAAGTTTAGGCACTCCTTTAG Microcystis aeruginosa mcy E
MicmcyE2 (SEQ ID NO: 47) TCATTGAAGCGGTTCAAGAACAAATGAACCGAGGAATAGGTTTAGGA Microcystis aeruginosa mcy E
MicmcyE3 (SEQ ID NO: 48) GAACAAATGAACCGAGGAATAGGTTTAGGAATGCAGTCAAATCTGGC Microcystis aeruginosa mcy E
NosmcyE1 (SEQ ID NO: 49) TAAAACCACGACTCAACCCTTAAGTTTAGGCACTCCTTTAGGAATGG Nostoc sp. 74.2 mcy E
NosmcyE2 (SEQ ID NO: 50) TAGGAGAAGATAAAACCACGACTCAACCCTTAAGTTTAGGCACTCCT Nostoc sp. 74.2 mcy E
NosmcyE3 (SEQ ID NO: 51) TAATTAGTGAAATGGGCCGGGTCGAAAGAGTCGCTTTTAGTAATACG Nostoc sp. 74.2 mcy E
OscmcyE1 (SEQ ID NO: 52) CCGTAAGTCTGGGTACACCATCAGGAATGGTTGAAGATGTAATTGTT Oscillatoria sp. 18R Oscillatoria sp. 19R mcy E
OscmcyE2 (SEQ ID NO: 53) GGGGTAGAAAGGGTGGCTTTTAGTAATACTGGAACCGAAGCAATTAT Oscillatoria sp. 18R Oscillatoria sp. 19R mcy E
OscmcyE3 (SEQ ID NO: 54) GGATTGCTCGTTCTCGCACAAAACGCCCAAAAATTGTTATATTTTCC Oscillatoria sp. 18R Oscillatoria sp. 19R mcy E
PhomcyE1 (SEQ ID NO: 55) TGCTATGTTTGCAGGTTCATATCACGGAACCTTTGATGGCATTTTAG Phormidium sp. 4-19b Phormidium sp. 1-6c Phormidium sp. 2-26b3 mcy E
PhomcyE2 (SEQ ID NO: 56) AGCTGCTTTAATTAGTGAAATGGGAGGAGTAGAACGGGTTGCTTTTA Phormidium sp. 4-19b Phormidium sp. 1-6c Phormidium sp. 2-26b3 mcy E
PhomcyE3 (SEQ ID NO: 57) ACAAAACGCCAAAAAATTGCTATGTTTGCAGGTTCATATCACGGAAC Phormidium sp. 4-19b Phormidium sp. 1-6c Phormidium sp. 2-26b3 mcy E
AnanifD1 (SEQ ID NO: 58) ATCCGAGACTGGATTTTCCCAGAATACGACAAGCTGAAGAAAGAAAA Anabaena variabilis nif D
AnanifD2 (SEQ ID NO: 59) TGGATTTTCCCAGAATACGACAAGCTGAAGAAAGAAAACAGACTCGA Anabaena variabilis nif D
AnanifD3 (SEQ ID NO: 60) TTTCCCAGAATACGACAAGCTGAAGAAAGAAAACAGACTCGACTTCG Anabaena variabilis nif D
NosnifD1 (SEQ ID NO: 61) AGAATACGACAAGCTCAAGAAAGAAAACAGACTTGACTTCGAGCCAA Nostoc spongiaeforme Nostoc punctiforme Nostoc muscorum nif D
NosnifD2 (SEQ ID NO: 62) TCCCAGAATACGACAAGCTCAAGAAAGAAAACAGACTTGACTTCGAG Nostoc spongiaeforme Nostoc punctiforme Nostoc muscorum nif D
NosnifD3 (SEQ ID NO: 63) ATCCGTGACTGGATTTTCCCAGAATACGACAAGCTCAAGAAAGAAAA Nostoc spongiaeforme Nostoc punctiforme Nostoc muscorum nif D
OscnifD1 (SEQ ID NO: 64) GACGACGTTTCAGCCTACGAGTTTGAGGAGTTCATTAAAGAACTCAA Oscillatoria tenuis nif D
OscnifD2 (SEQ ID NO: 65) TTTCAGCCTACGAGTTTGAGGAGTTCATTAAAGAACTCAAACCCGAC Oscillatoria tenuis nif D
OscnifD3 (SEQ ID NO: 66) GGAGTTCATTAAAGAACTCAAACCCGACTTAGTAGCTTCTGGCATCA Oscillatoria tenuis nif D
PhonifD1 (SEQ ID NO: 67) GTTAATTGGTACAGGCTACGAGTTCGGTCACAACGACGATTACAAAC Phormidium ambiguum nif D
PhonifD2 (SEQ ID NO: 68) AAAGTTATCGCCAAATACGAAGCGCAAACGAAAGCTGTAATTGAAAA Phormidium ambiguum nif D
PhonifD3 (SEQ ID NO: 69) AAGCTGAATCTAGTTCACTGCTATCGCTCCATGAACTATATCAGCCG Phormidium ambiguum nif D
AnacpcBA1 (SEQ ID NO: 70) GACCTATCTCCTAGCTGGTATGTTGAAGCTCTAAAGCACATCAAAGC Anabaena variabilis cpc BA
AnacpcBA2 (SEQ ID NO: 71) GTTTAAGTGGTCAAGCTGCTAACGAAGCTAACACCTACATCGACTAC Anabaena variabilis cpc BA
AnacpcBA3 (SEQ ID NO: 72) AAAGCACATCAAAGCTAATCATGGTTTAAGTGGTCAAGCTGCTAACG Anabaena variabilis cpc BA
MiccpcBA1 (SEQ ID NO: 73) CTAGCTGGTACATCGAAGCTCTCAAATATATCAAAGCCAACCATGGT Microcystis aeruginosa cpc BA
MiccpcBA2 (SEQ ID NO: 74) CTTCGACCTGTCTCCTAGCTGGTACATCGAAGCTCTCAAATATATCA Microcystis aeruginosa cpc BA
MiccpcBA1 (SEQ ID NO: 75) CATCGAAGCTCTCAAATATATCAAAGCCAACCATGGTTTAAGTGGCG Microcystis aeruginosa cpc BA
AphcpcBA1 (SEQ ID NO: 76) AATTCCCTTACACCACATCTACACCAGGTAATCAATACGCATCCGAT Aphanizomenon flos-aquae cpc BA
AphcpcBA2 (SEQ ID NO: 77) CTAGTGTTCTTGATGACCGTTGCTTAAATGGTTTGCGCGAAACATAC Aphanizomenon flos-aquae cpc BA
AphcpcBA3 (SEQ ID NO: 78) GCCTAACAATTACAAATAGCTCGAATCTTAATTGAGCTTCTTCCAAC Aphanizomenon flos-aquae cpc BA
실시예 2. 프로브의 특이성 및 DNA 칩의 정량성 평가Example 2. Evaluation of Specificity of Probes and Quantitative Quantities of DNA Chips
제작된 프로브를 DNA 칩에 부착시키기 위해 먼저 프로브의 염기서열 5' 말단에 아민기를 붙이고 시토신 5 분자(CCCCC)를 연결한 뒤, 이 수정된 프로브를 숙신산 무수물(succinic anhydride)로 처리한 아민기 기능성 글라스 슬라이드(amine functional glass side, Nanosc Inc.)에 Proteogen CM-1000을 이용하여 부착시켰다(도 1 참조). In order to attach the fabricated probe to the DNA chip, first, an amine group was attached to the 5 'end of the probe, the cytosine 5 molecule (CCCCC) was connected, and the modified probe was treated with succinic anhydride. A glass slide (amine functional glass side, Nanosc Inc.) was attached using Proteogen CM-1000 (see FIG. 1).
상기 DNA 칩의 특이성과 정량성을 평가하기 위하여, 먼저 6종의 남조류 Microcystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, Nostoc에 대하여 101~106 cells/ml 농도의 샘플을 준비한 후, DNA 칩의 혼성화반응(hybridization)을 위해 다음 과정을 수행하였다.In order to evaluate the specificity and quantitativeness of the DNA chip , samples of concentrations of 10 1 to 10 6 cells / ml were prepared for six species of cyanobacteria Cycystis, Anabaena, Oscillatoria, Phormidium, Aphanizomenon, and Nostoc . The following procedure was performed for the hybridization.
각 농도별 남조류 샘플 50 ml에서 게놈 DNA를 추출한 후, 각각의 게놈 DNA 1 ㎍에 제한효소 AluI (20 units, Promega)과 RsaI (20 unit, Promega)을 37℃에서 2시간 동안 처리하여 절편화(digestion) 시켰다. 반응이 끝난 후, 절편화된 DNA 샘플은 QIAQuick PCR clean-up kit (Qiagen)를 이용하여 제조사의 방법에 따라 정제하였다. After extracting genomic DNA from 50 ml of algal samples for each concentration, 1 μg of each genomic DNA was digested with restriction enzymes AluI (20 units, Promega) and RsaI (20 units, Promega) at 37 ° C. for 2 hours. digestion). After the reaction, the fragmented DNA samples were purified using the QIAQuick PCR clean-up kit (Qiagen) according to the manufacturer's method.
정제한 DNA는 Invitrogen사에서 제공되는 Bioprime 표지 키트(labeling kit)을 사용하여 표지화 반응을 수행하였다. 총 반응액의 양을 50 ul로 하여 modified dNTP mix (각각 120 uM의 dATP, dGTP, dCTP; 60 uM의 dTTP)와 60 uM의 Cy5-dUTP를 첨가하고 Klenow 효소와 반응 버퍼를 넣고 37℃에서 2시간동안 표지화 반응을 수행하였다. 반응이 끝난 후, 표지화된 DNA는 QIAQuick PCR clean-up kit(Qiagen)을 사용하여 정제하였다. 정제된 DNA는 혼성화반응의 특이성을 높이기 위해, 50 ug의 인간 Cot-1 DNA (Applied Genetics)/ 52 ul 의 10X Blocking reagent (Agilent Technology)를 경쟁자로 각각 혼합하여 준비하고, 이 혼합 샘플은 2X aCGH 혼성화 버퍼(Agilent Technology)와 섞었다.Purified DNA was labeled using a Bioprime labeling kit provided by Invitrogen. Add 50 μl of total reaction solution to the modified dNTP mix (120 uM of dATP, dGTP, dCTP; 60 uM of dTTP) and 60 uM of Cy5-dUTP, add Klenow enzyme and reaction buffer, The labeling reaction was carried out for a time. After the reaction, the labeled DNA was purified using a QIAQuick PCR clean-up kit (Qiagen). Purified DNA was prepared by mixing 50 ug of human Cot-1 DNA (Applied Genetics) / 52 ul of 10X Blocking reagent (Agilent Technology) as competitors to increase the specificity of hybridization, and the mixed sample was prepared by 2X aCGH. Mixed with hybridization buffer (Agilent Technology).
혼성화 버퍼와 잘 섞은 DNA 샘플을 95℃에서 3분간 가열한 후, 37℃에서 30분간 반응시켰다. 반응이 끝난 용액을 1분간 원심 분리하여 준비한 후, 게스켓 슬라이드(gasket slide, Agilent Technology)에 주입하고, DNA 칩을 고정하였다. 이 DNA 칩을 혼성화 오븐 (Agilent Technology)에 넣고, 65℃에서 18-20시간 동안 경쟁적 혼성화 반응을 진행하였다. 혼성화 반응이 끝난 DNA 칩은 제조사의 방법에 따라 순서대로 세척하여 건조시켰다. The DNA sample mixed well with the hybridization buffer was heated at 95 ° C for 3 minutes, and then reacted at 37 ° C for 30 minutes. After the reaction solution was prepared by centrifugation for 1 minute, it was injected into a gasket slide (Agilent Technology), and the DNA chip was fixed. This DNA chip was placed in a hybridization oven (Agilent Technology) and subjected to a competitive hybridization reaction at 65 ° C. for 18-20 hours. After completion of hybridization, the DNA chip was washed and dried in order according to the manufacturer's method.
건조시킨 DNA 칩은 Axon 스캐너를 이용하여 이미지 스캔을 한 후, GenePix Pro 6.0 (Axon Instruments) 프로그램을 이용하여 각 유전자의 시그널 강도(signal intensity)를 정량화하였다. 각 점의 평균 형광세기와 로컬 백그라운드(local background)의 값을 계산한 후, 각 점의 평균 형광세기에서 백그라운드 값을 제거하여 순수 형광 값을 구하였다. 그리고 이후 진행되는 분석을 위해서 낮은 형광세기를 지니는 점은 제거했다. The dried DNA chip was scanned with an Axon scanner and then quantified the signal intensity of each gene using the GenePix Pro 6.0 (Axon Instruments) program. After calculating the average fluorescence intensity and the local background of each point, the pure fluorescence value was obtained by removing the background value from the mean fluorescence intensity of each point. And for later analysis, the low fluorescence intensity was eliminated.
반응시 나타나는 다양성을 보정해주기 위해 강도 기준 표준화 방법(Locally Weighted Scatterplot Smoothing, LOWESS)을 사용하였다. 신호 대 잡음비 (Signal to Noise Ratio, SNR)값은 평균 표준화 신호 채널 강도(normalized signal channel intensity)를 평균 표준화 통제 채널 강도(normalized control channel intensity)로 나누어 계산하였다. 이 비율의 값이 2.0 이상이거나 0.5 이하의 값을 나타내는 유전자를 유전자 카피(copy)수가 유의하게 차이가 나는 것으로 결정하였다.In order to compensate for the variability in the reaction, a strength-based standardization method (Locally Weighted Scatterplot Smoothing, LOWESS) was used. The signal to noise ratio (SNR) value was calculated by dividing the average normalized signal channel intensity by the average normalized control channel intensity. Genes showing a value of this ratio above 2.0 or below 0.5 were determined to have significant differences in the number of gene copies.
분석 결과, Microcystis의 경우, 도 2에 나타낸 바와 같이, mcyE, cpcBA, ITS, rbcS, rbcX 유전자를 기반으로 제작된 프로브는 Microcystis와 특이적으로 반응하였다. 또한, 도 3에 나타낸 바와 같이, 101~103 cells/ml 농도구간에서는 Microcystis의 농도 변화와 상관없는 SNR값을 보였으나, 104~106 cells/ml 농도구간에서 Microcystis의 농도가 증가함에 따라 SNR값도 비례적으로 증가하는 양의 상관관계(R2>0.95)를 보여, 제작된 DNA 칩을 이용하여 Microcystis를 104~106 cells/ml까지 정량분석이 가능함을 알 수 있다.As a result, in the case of Microcystis , as shown in Figure 2, the probes based on mcy E, cpc BA, ITS, rbc S, rbc X genes specifically reacted with Microcystis . In addition, as shown in Figure 3, in the concentration range of 10 1 ~ 10 3 cells / ml showed an SNR value unrelated to the change in the concentration of Microcystis , but the concentration of Microcystis increases in the concentration range of 10 4 ~ 10 6 cells / ml As a result, the SNR value also shows a positive correlation (R 2 > 0.95), indicating that Microcystis can be quantitated from 10 4 to 10 6 cells / ml using the manufactured DNA chip.
Nostoc의 경우, 도 4에 나타낸 바와 같이, rpoB, rbcX, rbcS 유전자를 기반으로 제작된 프로브는 Nostoc와 특이적으로 반응하였다. 또한, 도 5에 나타낸 바와 같이, 101~103 cells/ml 농도구간에서는 Nostoc의 농도 변화와 상관없는 SNR값을 보였으나, 104~106 cells/ml 농도구간(rpoB)과 103~106 cells/ml 농도구간(rbcX, rbcS)에서 Nostoc의 농도가 증가함에 따라 SNR값도 비례적으로 증가하는 양의 상관관계(R2>0.90)를 보여, 제작된 DNA 칩을 이용하여 Nostoc을 103 또는 104~106 cells/ml까지 정량분석이 가능함을 알 수 있다.For Nostoc, Fig. 4, rpo B, a probe made of rbc X, rbc S gene was based on the reaction as Nostoc and specifically as shown in Fig. In addition, as shown in Figure 5, 10 1 ~ 10 3 cells / ml concentration range showed an SNR value unrelated to the change in the concentration of Nostoc , but 10 4 ~ 10 6 cells / ml concentration range ( rpo B) and 10 3 Nostoc at ~ 10 6 cells / ml concentration interval ( rbc X, rbc S) As the concentration increased, the SNR value also increased proportionally (R 2 > 0.90), indicating that Nostoc was quantitated up to 10 3 or 10 4 ~ 10 6 cells / ml using the manufactured DNA chip. It can be seen that.
Anabaena의 경우, 도 6에 나타낸 바와 같이, rpoB 유전자를 기반으로 제작된 프로브는 Anabaena와 특이적으로 반응하였다. 또한, 도 7에 나타낸 바와 같이, 101~103 cells/ml 농도구간에서는 Anabaena의 농도 변화와 상관없는 SNR값 보였으나, 103~106 cells/ml 농도구간에서 Anabaena의 농도가 증가함에 따라 SNR값도 비례적으로 증가하는 양의 상관관계(R2>0.95)를 보여, 제작된 DNA 칩을 이용하여 Anabaena를 103~106 cells/ml까지 정량분석이 가능함을 알 수 있다. In the case of Anabaena , as shown in Figure 6, the probe prepared based on rpo B gene specifically reacted with Anabaena . In addition, as shown in FIG. 7, the SNR value was unrelated to the change of Anabaena concentration in the 10 1 ~ 10 3 cells / ml concentration range, but as the concentration of Anabaena increased in the 10 3 ~ 10 6 cells / ml concentration range. The SNR value also shows a positive correlation (R 2 > 0.95), indicating that Anabaena can be quantitated from 10 3 to 10 6 cells / ml using the manufactured DNA chip.
Oscillatoria의 경우, 도 8에 나타낸 바와 같이, ITS 유전자를 기반으로 제작된 프로브는 Oscillatoria와 특이적으로 반응하였다. 또한, 도 9에 나타낸 바와 같이, 101~103 cells/ml 농도구간에서는 Oscillatoria의 농도 변화와 상관없는 SNR값을 보였으나, 103~106 cells/ml 농도구간에서 Oscillatoria의 농도가 증가함에 따라 SNR값도 비례적으로 증가하는 양의 상관관계(R2>0.95)를 보여, 제작된 DNA 칩을 이용하여 Oscillatoria를 103~106 cells/ml까지 정량분석이 가능함을 알 수 있다. In the case of Oscillatoria , as shown in FIG. 8, the probe prepared based on the ITS gene specifically reacted with Oscillatoria . In addition, as shown in Figure 9, the concentration range of 10 1 ~ 10 3 cells / ml showed an SNR value unrelated to the change in the concentration of Oscillatoria , but the concentration of Oscillatoria increases in the concentration range of 10 3 ~ 10 6 cells / ml As a result, the SNR value also increases in proportion (R 2 > 0.95), indicating that Oscillatoria can be quantitatively analyzed from 10 3 to 10 6 cells / ml using the manufactured DNA chip.
Phormidium의 경우, 도 10에 나타낸 바와 같이, mcyE 유전자를 기반으로 제작된 프로브는 Phormidium와 특이적으로 반응하였다. 또한, 도 11에 나타낸 바와 같이, 101~103 cells/ml 농도구간에서는 Phormidium의 농도 변화와 상관없는 SNR값을 보였으나, 103~106 cells/ml 농도구간에서 Phormidium의 농도가 증가함에 따라 SNR값도 비례적으로 증가하는 양의 상관관계(R2>0.95)를 보여, 제작된 DNA 칩을 이용하여 Phormidium를 104~106 cells/ml까지 정량분석이 가능함을 알 수 있다. In the case of Phormidium, as shown in FIG. 10, a probe prepared based on the mcy E gene specifically reacted with Phormidium . In addition, as shown in Figure 11, the concentration range of 10 1 ~ 10 3 cells / ml showed an SNR value unrelated to the change in the concentration of Phormidium , but the concentration of Phormidium increased in the 10 3 ~ 10 6 cells / ml concentration range As a result, the SNR value also shows a positive correlation (R 2 > 0.95), indicating that Phormidium can be quantitated from 10 4 to 10 6 cells / ml using the manufactured DNA chip.
Aphanizomenon의 경우, 도 12에 나타낸 바와 같이, cpcBA 유전자를 기반으로 제작된 프로브는 101~103 cells/ml 농도구간에서는 Aphanizomenon의 농도 변화와 상관없는 SNR값을 보였으나, 103~106 cells/ml 농도구간에서 Aphanizomenon의 농도가 증가함에 따라 SNR값도 비례적으로 증가하는 양의 상관관계(R2>0.99)를 보여, 제작된 DNA 칩을 이용하여 Aphanizomenon를 103~106 cells/ml까지 정량분석이 가능함을 알 수 있다. In the case of Aphanizomenon , as shown in FIG. 12, the probe prepared based on the cpc BA gene showed an SNR value that was not related to the change of Aphanizomenon concentration in the concentration range of 10 1 to 10 3 cells / ml, but was 10 3 to 10 6. As the concentration of Aphanizomenon increased in the cells / ml concentration range, the SNR value also increased proportionally (R 2 > 0.99), and Aphanizomenon was converted to 10 3 ~ 10 6 cells / It can be seen that quantitative analysis is possible up to ml.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해하여야만 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (13)

  1. 서열번호 1 내지 서열번호 78로 이루어진 군으로부터 선택되는 염기서열로 구성되는 프로브를 6종 이상 포함하는, 남조류 검출 및/또는 정량용 DNA 칩.A DNA chip for detecting and / or quantifying cyanobacteria comprising six or more probes consisting of nucleotide sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 78.
  2. 제 1 항에 있어서, 상기 프로브의 타겟 유전자는 mcyE, cpcBA, ITS, rbcS, rbcX 또는 nifD인 것을 특징으로 하는, 남조류 검출 및/또는 정량용 DNA 칩.The method of claim 1, wherein the target gene is, blue-green algae detection and / or quantitative DNA chips, characterized in that mcyE, cpcBA, ITS, rbcS, rbcX or nifD of the probe.
  3. 제 1 항 있어서, 상기 남조류는 마이크로시스티스(Microcystis)속, 아나베나(Anabaena)속, 오실라토리아(Oscillatoria)속, 포미디움(Phormidium)속, 아파니조메논(Aphanizomenon)속 또는 노스톡(Nostoc)속인 것을 특징으로 하는, 남조류 검출 및/또는 정량용 DNA 칩. According to claim 1, wherein the cyanobacteria are Microcystis genus, Anabaena genus, Oscillatoria genus Oscillatoria genus Phormidium genus Aphanizomenon genus or Nostoc. DNA chip for detecting and / or quantifying algae , characterized in that the genus.
  4. 제 3 항에 있어서, 상기 DNA 칩은The method of claim 3, wherein the DNA chip
    마이크로시스티스속의 검출 및/또는 정량용 프로브로서, 서열번호 4-6, 13-15, 22-24, 37-39, 46-48 및 73-75로 이루어진 군으로부터 선택되는 염기서열로 구성되는 하나 이상의 프로브;A probe for detection and / or quantification of the genus microcistis, comprising one nucleotide sequence selected from the group consisting of SEQ ID NOs: 4-6, 13-15, 22-24, 37-39, 46-48 and 73-75 More than one probe;
    아나베나속의 검출 및/또는 정량용 프로브로서, 서열번호 1-3, 10-12, 19-21, 34-36, 43-45, 58-60 및 70-72로 이루어진 군으로부터 선택되는 염기서열로 구성되는 하나 이상의 프로브;A probe for detecting and / or quantifying Anabena genus, the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-3, 10-12, 19-21, 34-36, 43-45, 58-60, and 70-72 One or more probes configured;
    오실라토리아속의 검출 및/또는 정량용 프로브로서, 서열번호 28-30, 52-54 및 64-66로 이루어진 군으로부터 선택되는 염기서열로 구성되는 하나 이상의 프로브;A probe for detection and / or quantification of the genus Oscillaria, comprising: at least one probe consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 28-30, 52-54, and 64-66;
    포미디움속의 검출 및/또는 정량용 프로브로서, 서열번호 31-33, 55-57 및 67-69로 이루어진 군으로부터 선택되는 염기서열로 구성되는 하나 이상의 프로브;A probe for detecting and / or quantifying indium form, comprising: one or more probes consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31-33, 55-57, and 67-69;
    아파니조메논속의 검출 및/또는 정량용 프로브로서, 서열번호 76-78로 이루어진 군으로부터 선택되는 염기서열로 구성되는 하나 이상의 프로브; 및A probe for detecting and / or quantifying apanizomenone, comprising: at least one probe consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 76-78; And
    노스톡속의 검출 및/또는 정량용 프로브로서, 서열번호 7-9, 16-18, 25-27, 40-42, 49-51 및 61-63로 이루어진 군으로부터 선택되는 염기서열로 구성되는 하나 이상의 프로브;At least one probe consisting of a base sequence selected from the group consisting of SEQ ID NOs: 7-9, 16-18, 25-27, 40-42, 49-51, and 61-63 Probes;
    를 포함하는 것을 특징으로 하는, 남조류 검출 및/또는 정량용 DNA 칩.DNA chip for detecting and / or quantifying algae, comprising a.
  5. 제 3 항에 있어서, 상기 마이크로시스티스 속은 마이크로시스티스 애루기노사(Microcystis aeruginosa)를 포함하는 것을 특징으로 하는, 남조류 검출 및/또는 정량용 DNA 칩.The DNA chip for detecting and / or quantifying algae according to claim 3, wherein the microcistis genus comprises Microcystis aeruginosa .
  6. 제 3 항에 있어서, 상기 아나베나 속은 아나베나 프로스-아퀴(Anabaena flos-aquae), 아나베나 바리아빌리스(Anabaena variabilis), 아나베나 sp.PCC 7120 (Anabaena sp. PCC 7120), 및 아나베나 BIR 시리즈(Anabaena sp. BIR series)를 포함하는 것을 특징으로 하는, 남조류 검출 및/또는 정량용 DNA 칩.4. The method of claim 3 wherein the analog vena genus Ana vena Prosper-Aquitania (Anabaena flos-aquae), Ana vena Barrier borrowed switch (Anabaena variabilis), Ana vena sp.PCC 7120 (. Anabaena sp PCC 7120 ), and analog vena DNA chip for detecting and / or quantifying algae, comprising a BIR series (Anabaena sp. BIR series ).
  7. 제 3 항에 있어서, 상기 오실라토리아 속은 오실라토리아 산크타(Oscillatoria sancta), 오실라토리아 테누이스(Oscillatoria tenuis), 오실라토리아 sp. 18R(Oscillatoria sp. 18R), 및 오실라토리아 sp. 19R(Oscillatoria sp. 19R)을 포함하는 것을 특징으로 하는, 남조류 검출 및/또는 정량용 DNA 칩.The method of claim 3, wherein the genus Oscillatoria is Oscillatoria sancta , Oscillatoria tenuis , Oscillatoria sp. 18R (Oscillatoria sp. 18R) , and oscillatoria sp. 19R (Oscillatoria sp. 19R) comprising, DNA chip for cyanobacteria detection and / or quantification.
  8. 제 3 항에 있어서, 상기 포미디움 속은 포미디움 cf. 이리굼(Phormidium cf. irriguum), 포미디움 암비굼(Phormidium ambiguum), 포미디움 sp. 4-19b(Phormidium sp. 4-19b), 포미디움 sp. 1-6c(Phormidium sp. 1-6c), 및 포미디움 sp. 2-26b3(Phormidium sp. 2-26b3)을 포함하는 것을 특징으로 하는, 남조류 검출 및/또는 정량용 DNA 칩. The method of claim 3, wherein the podium is in the podium cf. Irigum (Phormidium cf. irriguum) , Phodiumdium ambiguum , Podium sp. 4-19b (Phormidium sp. 4-19b), podium sp. 1-6c (Phormidium sp. 1-6c) , and podium sp. DNA chip for detecting and / or quantifying cyanobacteria, comprising 2-26b3 (Phormidium sp. 2-26b3) .
  9. 제 3 항에 있어서, 상기 아파니조메논 속은 아파니조메논 프로스-아퀴(Aphanizomenon flos-aquae)를 포함하는 것을 특징으로 하는, 남조류 검출 및/또는 정량용 DNA 칩.According to claim 3, wherein the Aphanizomenon Menon genus Aphanizomenon Menon flosses in-Aquitania, cyanobacteria detection and / or quantitative DNA chip comprising the (Aphanizomenon flos-aquae).
  10. 제 3 항에 있어서, 상기 노스톡 속은 노스톡 펑크티포르메(Nostoc punctiforme), 노스톡 링키아(Nostoc linckia), 노스톡 스폰지포르메(Nostoc spongiaeforme), 및 노스톡 무스코룸(Nostoc muscorum)을 포함하는 것을 특징으로 하는, 남조류 검출 및/또는 정량용 DNA 칩.4. The method of claim 3 wherein the furnace stock genus furnace Stock puncture tea formate methoxy (Nostoc punctiforme), no stock ring Escherichia (Nostoc linckia), no stock sponge formate methoxy (Nostoc spongiaeforme), and no stock mousse Corum (Nostoc muscorum) the DNA chip for detecting and / or quantifying algae, characterized in that it comprises.
  11. 제 1 항 내지 제 10 항 중 어느 한 항에 있어서, 상기 DNA 칩은 프로브 집적부위(well)가 6개로 구획되어 있는 것을 특징으로 하는, 남조류 검출 및/또는 정량용 DNA 칩.The DNA chip according to any one of claims 1 to 10, wherein the DNA chip is divided into six probe wells.
  12. 하기의 단계를 포함하는, 남조류의 검출 및/또는 정량방법:A method for detecting and / or quantifying cyanobacteria, comprising the following steps:
    남조류 샘플에서 게놈 DNA를 추출하는 단계; Extracting genomic DNA from cyanobacteria samples;
    상기 추출된 게놈 DNA를 절편화 및 표지하는 단계; Fragmenting and labeling the extracted genomic DNA;
    상기 표지된 DNA를 제 1 항 내지 제 10 항 중 어느 한 항의 DNA 칩에 혼성화 반응시키는 단계; 및 Hybridizing the labeled DNA to the DNA chip of any one of claims 1 to 10; And
    상기 혼성화 정도에 따라 남조류의 종류 및 양을 판별하는 단계. Determining the type and amount of cyanobacteria according to the degree of hybridization.
  13. 제 12 항에 있어서, 상기 남조류는 마이크로시스티스(Microcystis)속, 아나베나(Anabaena)속, 오실라토리아(Oscillatoria)속, 포미디움(Phormidium)속, 아파니조메논(Aphanizomenon)속 또는 노스톡(Nostoc)속인 것을 특징으로 하는, 남조류의 검출 및/또는 정량방법.The genus of claim 12, wherein the cyanobacteria are Microcystis , Anabaena , Oscillatoria , Phormidium , Aphanizomenon , or Northstock . Nostoc genus ), characterized in that the detection and / or quantification method of cyanobacteria.
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