WO2016098953A1 - Marqueur génétique pour la détermination de la pathogénicité de streptococcus iniae et trousse d'examen de marqueurs déterminants de la pathogénicité en utilisant un acide nucléique peptidique - Google Patents

Marqueur génétique pour la détermination de la pathogénicité de streptococcus iniae et trousse d'examen de marqueurs déterminants de la pathogénicité en utilisant un acide nucléique peptidique Download PDF

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WO2016098953A1
WO2016098953A1 PCT/KR2015/002344 KR2015002344W WO2016098953A1 WO 2016098953 A1 WO2016098953 A1 WO 2016098953A1 KR 2015002344 W KR2015002344 W KR 2015002344W WO 2016098953 A1 WO2016098953 A1 WO 2016098953A1
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iniae
probe
nucleic acid
genotype
streptococcus iniae
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PCT/KR2015/002344
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Korean (ko)
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김명석
최혜승
정승희
한현자
도정완
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대한민국(관리부서:국립수산과학원)
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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
    • 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
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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

Definitions

  • the present invention relates to a probe for genotyping of Streptococcus iniae that can hybridize under severe conditions with a sequence fragment containing a single nucleotide polymorphism of T368C or T385G in the cpsD sequence of Streptococcus iniae of SEQ ID NO: 1.
  • Streptococcus inia is Gram-positive cocci, first isolated from the Amazon River dolphin (Inia geoffrensis) in 1972, and is highly infected with tilapia, yellowtail, rainbow trout, and flounder. S. iniae can infect humans and cause cellulitis, and more than 20 cases of S. iniae infections in humans have been reported in the United States, Canada, Hong Kong, Taiwan, and Singapore. This is becoming important.
  • Streptococcus iniae vaccines were used to prevent streptococosis , a bacterial disease in fish, in the rainbow trout farms of Israel.
  • the new strain negative for Arginine dihydrolase in rainbow trout injected with the vaccine . iniae has been separated.
  • the first isolated S. iniae was named serotype I, after which the other serologically isolated S. iniae was named serotype II, and the S. iniae was divided into two serotypes.
  • Random amplified polymorphic DNA (RAPD) analysis is a method that can quickly differentiate between and between genetic species by obtaining an unspecific pattern of DNA based on PCR.
  • Two serotypes of Streptococcus iniae were divided into RAPD (Bachrach et al., Appl Environ Microbiol ., 67: 3756-3758, 2001), and 94 Streptococcus inia e isolated from Korea using this method had three genotypes. (genotypes), but studies on the pathogenicity of these genotypes are lacking (Jung YU and Heo MS, Kor J Life Sci ., 16: 345-351, 2006).
  • Streptococcal species include extracellular capsules, and capsule operons contain capsular polysaccharides (Cps) A, B, C, D, and E genes.
  • the cpsD gene encodes an autophosphorylating protein tyrosine kinase in Streptococcus pneumoniae , which is considered to be essential for the formation of capsules.
  • the ⁇ cpsD Streptococcus iniae from which the cpsD gene has been removed is encapsulated compared to wild type S. iniae . This decrease and no pathogenicity in hybrid bass, the cpsD gene was found to play an important role in inducing the formation of capsules of Streptococcal species and the death of fish.
  • peptide nucleic acid is a similar DNA in which nucleic acid bases are linked by peptide bonds rather than phosphate bonds, and was first synthesized in 1991 by Nielsen et al.
  • Peptide nucleic acids are not found in nature but are artificially synthesized by chemical methods.
  • Peptide nucleic acids form double strands through hybridization with native nucleic acids of complementary base sequences.
  • PNA / DNA double strands are more stable than DNA / DNA double strands and PNA / RNA double strands are more stable than DNA / RNA double strands.
  • peptide nucleic acids have a greater ability to detect single nucleotide polymorphisms (SNPs) than natural nucleic acids because of their high degree of double strand instability due to single base mismatch.
  • SNPs single nucleotide polymorphisms
  • Peptide nucleic acids are not only chemically stable but also biologically stable because they are not degraded by nucleases or proteases.
  • peptide nucleic acid is one of gene recognition materials, such as LNA (Locked nucleic acid), MNA (Mopholino nucleic acid), the basic skeleton is composed of polyamide.
  • Peptide nucleic acids have the advantages of very good affinity and selectivity, high physical and chemical properties, and easy storage and easy decomposition.
  • the present inventors have made efforts to develop a method for determining the pathogenicity of Streptococcus iniae in a quick and simple manner, using a genetic marker for determining the pathogenicity of S. iniae and a peptide nucleic acid (PNA) excellent in binding to the DNA.
  • PNA peptide nucleic acid
  • An object of the present invention is to provide a probe that can determine the nucleotide polymorphism and genotype of the target gene of Streptococcus iniae .
  • Another object of the present invention to provide a polymorphism kit of S. iniae containing the probe.
  • Another object of the present invention to provide a method for determining genotype of S. iniae containing the probe.
  • the present invention provides a probe for determining the genotype of Streptococcus iniae that can hybridize under severe conditions with a sequence fragment containing a single nucleotide polymorphism of T368C or T385G in the cpsD sequence of Streptococcus iniae of SEQ ID NO: 1.
  • the present invention also provides a kit for determining single nucleotide polymorphism (SNP) for determining the genotype of Streptococcus iniae comprising the probe.
  • SNP single nucleotide polymorphism
  • the present invention also provides a single nucleotide polymorphism (SNP) determination method for determining the genotype of Streptococcus iniae using the melting curve analysis of the probe.
  • SNP single nucleotide polymorphism
  • FIG. 2 is a gene position diagram for explaining a base mutation portion included in the peptide nucleic acid on the cpsD gene that determines the genotype of S. iniae .
  • Figure 3 is a schematic diagram illustrating the different degree of hybridization for each peptide nucleic acid according to the binding position in the cpsD gene and genotype of the single nucleic acid polymorphism (SNP) for determining the genotype of S. iniae .
  • SNP single nucleic acid polymorphism
  • Figure 4 is a schematic diagram for explaining the hybridization step and the step of obtaining the melting curve of the single nucleotide polymorphism (SNP) determination method for determining the genotype of S. iniae using peptide nucleic acid.
  • SNP single nucleotide polymorphism
  • FIG. 5 is a graph illustrating a step of obtaining a melting peak curve from a melting curve in a single base polymorphism (SNP) determination method for determining genotype of S. iniae using peptide nucleic acids.
  • SNP single base polymorphism
  • FIG. 6 is a conceptual diagram for explaining the technical characteristics of the peptide nucleic acid for determining the single base polymorphism (SNP) to determine the genotype of S. iniae .
  • SNP single base polymorphism
  • Figure 7 is a result of comparing the results of nucleotide sequence analysis of the same sample and the single nucleotide polymorphism (SNP) determination to determine the genotype of S. iniae .
  • SNP single nucleotide polymorphism
  • FIG. 8 is a table summarizing the melting temperature (Tm) obtained from a melting curve graph of peptide nucleic acids for genotypes of S. iniae .
  • a single nucleotide polymorphism marker that determines the genotype of S. iniae was selected for quick and easy analysis of the pathogenicity according to the genotype of Streptococcus iniae .
  • the present inventors isolated 29 S. iniae DNA from infected flounder in Korea, and these S. iniae are divided into two genotypes through repetitive sequence-based PCR (rep-PCR) and random amplified polymorphic DNA (RAPD). It was confirmed that the results were consistent with their phylogenetic results using the nucleotide sequence of the cpsD gene of S. iniae . Compare the two genotypes of S. iniae with the known functional domains of the cpsD protein and, via rep-PCR, there is a single nucleotide polymorphism (SNP) at 368 and 385 in the cpsD gene between these two genotypes In case of C or G, it was confirmed that S. iniae is pathogenic.
  • rep-PCR repetitive sequence-based PCR
  • RAPD random amplified polymorphic DNA
  • the single nucleotide polymorphism site can determine the genotype of S. iniae , and the binding strength of peptide nucleic acid to DNA is much better than that of DNA (DNA-DNA ⁇ PNA-DNA), and peptide nucleic acid is 1 nucleotide.
  • Melt temperature (Tm) differs by about 10 ⁇ 15 °C even with incomplete mismatch.
  • Tm single nucleotide polymorphism
  • SNP single nucleotide polymorphism
  • the present invention relates to a probe for genotyping of Streptococcus iniae that can hybridize under severe conditions with a sequence fragment containing a single nucleotide polymorphism of T368C or T385G in the cpsD sequence of Streptococcus iniae of SEQ ID NO: 1 .
  • peptide nucleic acid probes can be analyzed using a hybridization method different from the hydrolysis method of TaqMan probe. Probes that play a similar role include molecular beacon probes and scorpion probes.
  • the probe is preferably a PNA characterized by having a reporter and a quencher at the sock end. That is, in the peptide nucleic acid probe according to the present invention, a reporter and a quencher capable of quenching reporter fluorescence may be coupled to both ends.
  • the reporter in the group consisting of FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7', -tetrachloro-6-carboxy -4,7-dichlorofluorescein) and CY5
  • the quencher may be one or more selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, but is not limited thereto, preferably using Dabcyl It features.
  • the base sequence length of the peptide nucleic acid according to the present invention is not particularly limited, but may be prepared in a length of 5 to 30 mer including a single nucleotide polymorphism (SNP) that determines the genotype of Streptococcus iniae , preferably 9 to 17mer. Can be produced in length.
  • SNP single nucleotide polymorphism
  • the length of the peptide nucleic acid probe can be adjusted to produce a probe having a desired Tm value, and even a peptide nucleic acid probe of the same length can be adjusted to change the nucleotide sequence.
  • peptide nucleic acids have a higher binding force than DNA and have a higher basic Tm value
  • the peptide nucleic acid can be produced in a shorter length than DNA, so that even adjacent peptide nucleic acids can be discriminated.
  • the difference in Tm value is about 0.5 ° C., which is very small and requires additional analysis program or detailed temperature change or correction. Therefore, when two or more peptide nucleic acids appear, the analysis was difficult. According to the peptide nucleic acid probe is not affected by the probe sequence and peptide nucleic acid can be easily analyzed.
  • the peptide nucleic acid set of the present invention may include one tube including two peptide nucleic acids, and the peptide nucleic acids included in the tube may be configured with different reporters.
  • the peptide nucleic acid probe according to the present invention is designed such that the base mutation position of the target nucleic acid is in the center position of the peptide nucleic acid probe for the difference in melting temperature (Tm) of the target nucleic acid and the target nucleic acid having a base mutation It is preferable.
  • the base mutation is located in the center position of the probe to make the structural difference of the probe, the peptide nucleic acid probe is bound to form a loop (Tm) difference due to this structural difference It may appear large.
  • the peptide nucleic acid probe according to the present invention includes 13 to 17 base sequences
  • the peptide nucleic acid probe has a sequence corresponding to a single nucleotide polymorphism (SNP) site at at least one of the 6th to 9th positions. It is preferable.
  • SNP single nucleotide polymorphism
  • Such peptide nucleic acids may have structural modifications, including sequences corresponding to single nucleotide polymorphisms (SNPs) that determine the genotype of Streptococcus iniae at the center of the nucleotide sequence, thereby resulting in complete hybridization of target nucleic acids (perfect hybridization). match with the melting temperature (Tm) can be made larger.
  • SNPs single nucleotide polymorphisms
  • the peptide nucleic acid for determining the single nucleotide polymorphism (SNP) to determine the genotype of Streptococcus iniae shows a different melting temperature (Tm) depending on the binding sequence (or SNP), so that one peptide nucleic acid ( Alternatively, two or more base sequences can be detected by a probe), and two or more peptide nucleic acids can be included and used in one tube.
  • Tm melting temperature
  • Peptide nucleic acid of the present invention relates to a technique for determining genotyping of Streptococcus iniae , and hybridizes the peptide nucleic acid of the present invention with the target nucleic acid of S. iniae and analyzes the melting curve to simplify, rapid and accurate single nucleotide polymorphism of S. iniae . Can be determined.
  • the "strict conditions" used to achieve stringent specific levels vary depending on the nature of the nucleic acid being hybridized. For example, the length of the nucleic acid region to be hybridized, degree of homology, nucleotide sequence composition (eg, GC / AT composition ratio), and nucleic acid type (eg, RNA, DNA) select hybridization conditions. Is considered. Further considerations are whether the nucleic acid is immobilized, for example, in a filter or the like.
  • Examples of very stringent conditions are as follows: 2X SSC / 0.1% SDS at room temperature (hybridization conditions); 0.2X SSC / 0.1% SDS at room temperature (low stringency conditions); 0.2X SSC / 0.1% SDS at 42 ° C. (conditions with moderate stringency); 0.1X SSC at 68 ° C. conditions with high stringency.
  • the washing process can be carried out using one of these conditions, for example a condition with high stringency, or each of the above conditions, each of 10-15 minutes in the order described above, all or all of the conditions described above. Some iterations can be done. However, as described above, the optimum conditions vary with the particular hybridization reaction involved and can be determined experimentally. In general, conditions of high stringency are used for hybridization of critical probes.
  • the present invention relates to a method for determining the genotype of Streptococcus iniae by single nucleotide polymorphism (SNP) determination.
  • SNP single nucleotide polymorphism
  • SNP single nucleotide polymorphism
  • the hybridization step is to react the peptide nucleic acid according to the present invention with the target nucleic acid of S. iniae .
  • This step may include a PCR procedure, and it is possible to use a forward / reverse primer set for PCR.
  • Such hybridization steps and PCR conditions may include any of a variety of methods that are well known to those of ordinary skill in the art (hereinafter, referred to as “the skilled person”).
  • the skilled person After the PCR is completed, it is also possible to include a melting process.
  • the target nucleic acid of S. iniae contains the nucleotide polymorphism (SNP) site present in the cpsD gene of the present invention described above. Genetic variation of the cpsD gene which is expressed on the surface of S. iniae was confirmed in the present invention to affect the infectivity and virulence, it is effective to determine the infectivity and virulence of S. iniae.
  • SNP nucleotide polymorphism
  • the step of obtaining the melting curve for each temperature is to obtain the melting temperature (Tm) of S. iniae target nucleic acid.
  • Tm melting temperature
  • the hybridization method analysis method may use FMCA (Fluorescence Melting Curve Analysis), the FMCA is to analyze the difference between the binding force between the product produced after the PCR reaction and put the probe by Tm.
  • the Tm value can be obtained by measuring the intensity of fluorescence each time it is increased by 1 ° C.
  • the step of determining the single base polymorphism (SNP) for determining the genotype of the Streptococcus iniae is to determine the single base polymorphism (SNP) for determining the genotype of S. iniae from the melting temperature of the obtained melting curve. For example, a single nucleotide polymorphism (SNP) as compared to the melting temperature of the single-nucleotide polymorphism (SNP) to determine the genotype of the S. iniae the known melting temperature of the obtained melting curve for determining the genotype of the S. iniae Can be determined.
  • the melting curves of the peptide nucleic acids according to the present invention have different melting temperatures (Tm) for each single nucleotide polymorphism (SNP) that determines the genotype of S. iniae .
  • Tm melting temperatures
  • SNP single nucleotide polymorphism
  • SNP Base polymorphism
  • FIG. 5 is a graph for explaining an example of the step of obtaining the melting peak curve from the melting curve in the single base polymorphism (SNP) determination method for determining the genotype of Streptococcus iniae using the peptide nucleic acid according to the present invention, as shown here
  • the melting peak curve for each temperature can be obtained using the slope value of the melting curve, and this melting peak curve is easy to determine the melting temperature (Tm) of the single base polymorphism (SNP) that determines the genotype of S. iniae . .
  • the step of obtaining a melting curve for each temperature comprising the step of obtaining a melting peak curve for each temperature from the obtained melting curve for each temperature, to determine a single base polymorphism (SNP) to determine the genotype of the S. iniae
  • SNP single base polymorphism
  • the present invention also relates to a kit for determining single nucleotide polymorphism (SNP) for determining the genotype of Streptococcus iniae comprising a probe.
  • SNP single nucleotide polymorphism
  • the kit is a kit characterized in that for the base polymorphism analysis of multiple target DNA or a single target DNA.
  • the probe of the kit is a single nucleotide polymorphism (SNP) determination probe for determining the genotype of Streptococcus iniae , characterized in that the PNA.
  • SNP single nucleotide polymorphism
  • the kit may optionally include reagents required for conducting target amplification PCR reactions (eg, PCR reactions) such as buffers, DNA polymerase cofactors and deoxyribonucleotide-5-triphosphate, and may be used for various polynucleotide molecules, reverse transcription. It is also possible to include enzymes, various buffers and reagents, and antibodies that inhibit DNA polymerase activity.
  • reagents required for conducting target amplification PCR reactions such as buffers, DNA polymerase cofactors and deoxyribonucleotide-5-triphosphate
  • enzymes eg, various buffers and reagents, and antibodies that inhibit DNA polymerase activity.
  • single nucleotide variations of target nucleic acids and mutations caused by deletions or insertions of base nucleic acids can be effectively detected through dissolution curve analysis by peptide nucleic acid probes, and thus single nucleotide polymorphisms that determine the genotype of Streptococcus iniae . (SNP) can be determined and pathogenicity can be determined.
  • SNP single nucleotide polymorphisms that determine the genotype of Streptococcus iniae .
  • Rep-RCR (repetitive sequence-based PCR) was performed on 29 known strains of Streptococcus iniae (ATCC 29178) and cultured flounder in Korea to identify two genotypes.
  • the genotypes 1 and 2 were named as genotype 1 (86.2%).
  • BoxA primer of SEQ ID NO: 2 targets the cpsD gene of S. iniae, and the base sequence and rep-RCR condition of the BoxA primer used are as follows.
  • Streptococcus iniae genotype 1 and genotype 2 a single nucleotide polymorphism (SNP) was found. Specifically, 368bp and 385bp base sequence of the cpsD gene (NCBI database: JF795257.1) of S. iniae strain DGX07 was found to be different between the two genotypes. Streptococcus iniae genotype 1 was C (368bp), G (385bp) And genotype 2 is T (368 bp), T (385 bp) (FIG. 2).
  • PBS sterile saline
  • 0.1 mL of normal saline was intraperitoneally injected.
  • the mortality was examined for 7 days at 23.1 °C, and the dead meat was dissected and the Streptococcus iniae was re-isolated.
  • Hemi-bacterial bacterial counts (LD50) were calculated using Finney's probit method, and hemi-accumulated concentrations (LD50) for flounder of S.
  • iniae genotype 1 ranged from 7.0 to 7.2 ⁇ 10 5 cfu / fish and half of flounder for genotype 2
  • the lethal concentration ranged from 3.4 to 3.6 ⁇ 10 8 cfu / fish, indicating that genotype 1 was about 2000 times more pathogenic for flounder than two.
  • the gene-specific nucleotide sequence involved in each genotype was selected based on the single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • the single nucleotide polymorphism (SNP) that determines the genotype of Streptococcus iniae is 368 (C / T), and thus the nucleotide sequence (SEQ ID NO: 3) centering the position is the nucleotide sequence of the peptide nucleic acid according to the present invention. It consisted of.
  • nucleotide sequence of the peptide nucleic acid according to the present invention was determined and shown in Table 1 below.
  • O means a linker and K means lysine.
  • Peptide nucleic acid probes according to the present invention were prepared with the nucleotide sequences, reporters and quencher as shown in Table 1 above. Peptide nucleic acid probes were designed using Peptide Nucleic Acid Probe Designer (Appliedbiosystems, USA). All peptide nucleic acid probes used in the present invention were synthesized by HPLC purification method in Panagene (Panagene, Korea), and the purity of all synthesized probes was confirmed by mass spectrometry. Unnecessary secondary structures were avoided.
  • Figure 3 is a gene position map for explaining an example of the base mutant portion contained in the peptide nucleic acid on the S. iniae cpsD gene according to the present invention, the number represents a single nucleotide polymorphism site according to the present invention.
  • a dissolution curve was derived for each DNA having two genotypes of Streptococcus iniae , and analyzed to determine the single nucleotide polymorphism to determine the genotype of S. iniae . (SNP) was determined.
  • PCR was performed using a CFX96 TM Real-Time system (BIO-RAD, USA), and all experimental conditions used asymmetric PCR (asymmetric PCR) to generate a single stranded target nucleic acid.
  • the conditions of asymmetric PCR are as follows; 1X EyeBio Real-Time FMCA TM Buffer (SeaSunBio Real-Time FMCA TM Buffer, Eye Bio, Korea), 2.5mM MgCl 2 , 200 ⁇ M dNTPs, 1.0 U Taq polymerase, 0.05 ⁇ M forward primer (SEQ ID NO: 4) and 0.5 ⁇ M reverse primer (SEQ ID NO: 5, asymmetric PCR, Table 2) were added 0.5 ⁇ l probe (peptide nucleic acid prepared in Example 2), 0.5 ⁇ l S. iniae DNA followed by real-time PCR Was carried out.
  • the real-time PCR process was denatured at 95 ° C. for 5 minutes, and then reacted at 95 ° C. for 30 seconds, 58 ° C. for 45 seconds, and 74 ° C. for 45 seconds. This was repeated 38 cycles, and fluorescence was measured in real time.
  • the melting curve analysis was performed for 1 minute at 95 ° C, followed by hybridization at 35 ° C for 5 minutes, and then the melting curve analysis was performed by increasing the fluorescence by increasing the temperature from 35 ° C to 85 ° C in 1 ° C increments. The suspension was held for 10 seconds between each step (FIG. 4).
  • Figure 6 is a conceptual diagram for explaining the technical characteristics of the peptide mononucleotide polymorphism (SNP) for determining the genotype of Streptococcus iniae genotype according to an embodiment of the present invention, as shown here, the peptide according to the present invention
  • the nucleic acid may generate a fluorescent signal after hybridization with the target nucleic acid, and rapidly melts from the target nucleic acid at an appropriate melting temperature (Tm) of the peptide nucleic acid as the temperature increases, thereby extinguishing the fluorescent signal.
  • Tm melting temperature
  • the present invention is to detect the presence or absence of the base mutation of the target nucleic acid through a high-resolution fluorescence melting curve analysis (FMCA) obtained from the fluorescence signal according to the temperature change.
  • FMCA fluorescence melting curve analysis
  • Peptide nucleic acid according to the present invention shows the expected melting temperature (Tm) value when the complete hybridization (perfect match) with the target nucleic acid sequence, but incomplete hybridization (mismatch) with the target nucleic acid in which the base mutation is present than expected It may be characterized by showing a low melting temperature (Tm) value.
  • the base polymorphism in the case of using the peptide nucleic acid according to the present invention, can be analyzed in the same manner as in the sequencing analysis. Through this, the peptide base nucleic acid according to the present invention determines the genotype of S. iniae . SNP) can be discriminated.
  • the Tm value obtained by performing the melting curve analysis was designated as a perfect match for the peak of 60 °C or more, and the peak below 55 °C as an incomplete mismatch (mismatch), Using the analysis result, genotype 1 was assigned a perfect match and genotype 2 was mismatched so that each genotype had a unique value (FIG. 8).
  • a single base polymorphism that determines the genotype of S. iniae by using a peptide nucleic acid (PNA) having excellent binding ability to DNA has a different melting temperature (Tm) for each species having a genotype of Streptococcus iniae .
  • SNP can be used to determine the pathogenicity of S. iniae simply, quickly and accurately.

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Abstract

La présente invention concerne une sonde de détermination d'un génotype de Steptococcus iniae, la sonde étant capable de s'hybrider, sous des conditions strictes, à un fragment de séquence contenant une variation de polymorphisme à nucléotide unique de T368C ou T385G dans la séquence cpsD, qui est représentée par SEQ ID no : 1, de Streptococcus iniae et, plus particulièrement, une trousse qui permet à chaque génotype influençant la pathogénicité de présenter une température de fusion différente en utilisant un acide nucléique peptidique (PNA) ayant une excellente capacité de liaison à l'ADN, permettant de là une détermination simple, rapide, et précise de la pathogénicité de S. iniae.
PCT/KR2015/002344 2014-12-15 2015-03-11 Marqueur génétique pour la détermination de la pathogénicité de streptococcus iniae et trousse d'examen de marqueurs déterminants de la pathogénicité en utilisant un acide nucléique peptidique WO2016098953A1 (fr)

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