KR102293200B1 - PCR primer set and probe for detection of Gobiobotia naktongensis and real-time PCR method using the same - Google Patents

Abstract

The present invention relates to a method for discriminating white horseradish from other freshwater fish by amplifying a target sequence specific for white horsetail using a primer set comprising an oligonucleotide of SEQ ID NO: 3 and an oligonucleotide of SEQ ID NO: 4 as PCR primers. .
In addition, the present invention uses a mixture containing the oligonucleotide of SEQ ID NO: 3, the oligonucleotide of SEQ ID NO: 4, and a probe for detecting oligonucleotides derived from SEQ ID NO: 5 It relates to a method for rapid detection with specificity and sensitivity.

Description

PCR primer set and probe for detection of Gobiobotia naktongensis and real-time PCR method using the same

The present invention relates to a method for discriminating white horseradish from other freshwater fish by amplifying a target sequence specific for white horsetail using a primer set comprising an oligonucleotide of SEQ ID NO: 3 and an oligonucleotide of SEQ ID NO: 4 as PCR primers. .

In addition, the present invention uses a mixture containing the oligonucleotide of SEQ ID NO: 3, the oligonucleotide of SEQ ID NO: 4 and a probe for detecting oligonucleotides derived from SEQ ID NO: 5 It relates to a method for rapid detection with specificity and sensitivity.

White horsetail ( Gobiobotia) naktongensis ) is a small benthic fish belonging to Genus Gobiobotia, endemic to Korea. Habitat is divided into Imjin River, Han River, Geum River, and Nakdong River. eat and live

The number of white squirrels has recently declined sharply due to environmental pollution, dam construction, and river development.

Even after being designated as an endangered species, the size of the population is continuously decreasing due to large-scale river civil engineering projects such as the Four Major Rivers project, river maintenance projects, and water pollution. In particular, in the case of the white horse mackerel living in the Imjin River and the Han River, the population is so small that it is recognized as extinct because it has not been captured for the past 10 years.

Recently, a technology that can predict which species inhabit by analyzing environmental DNA (eDNA) in water has been developed and is being used by many researchers. Environmental DNA is not DNA extracted from living organisms, but organic matter collected from the surrounding environment in which it lives. In the case of fish, it refers to a method of identifying through sequencing by securing genomes from mucus, secretions, scales, etc. For example, it is a method that can find out which organisms live in a river by amplifying and analyzing a specific gene from a DNA fragment found by sampling environmental water. Therefore, it is possible to find out whether a target organism is inhabited without directly collecting it, so it does not depend on the skills of the collector, and it is a very effective method for identifying breeding sites or habitats of rare species in terms of cost and reliability compared to labor-intensive methods. However, in the case of rare species with a very small population size or low biomass, a highly sensitive analysis method should be used.

Existing methods for identifying white horsetail include PCR and purification of the mitochondrial cytochrome b (COB) region or cytochrome c oxidase I (COX I) region to secure the nucleotide sequence, compare and analyze, or draw a phylogenetic tree to confirm. . In addition, by comparing and analyzing the mitochondrial cytochrome b (COB) region, the specific marker for each water system based on the specific nucleotide sequence for each water system is amplified through the PCR method. And there is a method of classifying by size (Reference: Korea Patent Publication No. 10-2012-0136201 No.).

However, both methods require a lot of time and effort for analysis, and have disadvantages in that quantitative analysis is difficult. In the first method, it takes at least 2 days for genetic sample purification, PCR, PCR product purification, base sequencing, and analysis, and in the second method, it takes 7 hours for genetic sample identity, PCR, and electrophoretic analysis of PCR products. It takes longer than expected, and since various steps are required for analysis, there is a high possibility of sample contamination. In addition, quantitative analysis is not possible in the first method, and the second method is not accurate because quantitative analysis is performed by the thickness and brightness of the band in electrophoresis.

The present invention solves the above problems and at the same time develops a specific primer set and probe for the detection of white sagebrush in order to improve the specificity and sensitivity of the detection of white sagebrush in environmental water, and establishes a real-time PCR method using them. It was applied to the analysis of environmental DNA of white horseradish from males.

Korean Patent Publication No. 10-2012-0136201

An object of the present invention is to provide a specific primer set and probe having excellent specificity and sensitivity, capable of isolating DNA from fish or environmental water to detect white horsetail.

In addition, an object of the present invention is to provide a real-time PCR method specific to white serrata that can reduce the hassle and process of sequencing or electrophoresis by using a specific primer set and a probe and can perform quantitative analysis.

In addition, an object of the present invention is to provide a method capable of maximizing identification efficiency and practicality by providing a detection kit that accurately detects a white snail sample within a short time through real-time PCR.

In order to achieve the above object, the present invention provides a primer composition for detecting white horseradish comprising a primer of SEQ ID NO: 3 and a primer of SEQ ID NO: 4.

In addition, the present invention provides a probe for detecting white horseradish containing the oligonucleotide of SEQ ID NO: 5.

In addition, the present invention provides a composition for detecting white horseradish comprising the primer of SEQ ID NO: 3, the primer of SEQ ID NO: 4, and the probe.

In addition, the present invention comprises the steps of isolating DNA from fish or environmental water;

the isolated DNA; And mixing the primer composition comprising the primer of SEQ ID NO: 3 and the primer of SEQ ID NO: 4 and amplifying by PCR; and

Provided is a method for detecting white horsetail, comprising the step of confirming the amplified product by electrophoresis.

The present invention also includes the steps of isolating DNA from fish or environmental water;

the isolated DNA; and amplifying by PCR by mixing a composition for detecting white horseradish containing the primer of SEQ ID NO: 3, the primer of SEQ ID NO: 4, and the probe of claim 2; and

There is provided a method for detecting white horsetail, comprising the step of confirming _{the C T} value of the amplified product.

In one embodiment of the present invention, the step of confirming by electrophoresis is characterized in that when the 196 ~ 198 bp band is detected, it is determined as a white squirrel.

In one embodiment of the present invention, the _{step of determining the C T value of the amplified product is characterized in that when C T} appears at or below the maximum number of cycles, it is determined as a white horse.

The present invention can provide a specific primer set and probe having excellent specificity and sensitivity, capable of isolating DNA from fish or environmental water to detect white horsetail.

In addition, by using a specific primer set and a probe, the present invention can reduce the cumbersome and process of sequencing or electrophoresis, and can provide a specific real-time PCR method for succulents capable of quantitative analysis.

In addition, the present invention provides a method for accurately and accurately detecting a white horsetail sample within a short period of time through real-time PCR, so that it can be quickly and accurately detected.

1 shows the PCR amplification reaction of the mitochondrial COB gene region of 24 carp fish of the present invention.
2 to 13 show the COB gene nucleotide sequence of a total of 1,140 bp of 34 individuals.
14 shows a calibration curve of the real-time PCR amplification reaction for each concentration of genomic DNA of the present invention.
15 shows the results of a real-time PCR amplification reaction using a primer set and a probe for detecting white snails for 24 koi fish of the present invention. Genomic DNA (20ng) of individual Gna01~H. Genomic DNA (20ng) of individual Gna02~White squirrel #2.

Hereinafter, the present invention will be described in detail based on examples and drawings. Terms, examples, drawings, etc. used in the present invention are merely exemplified to explain the present invention in more detail and help those of ordinary skill in the art to understand, and the scope of the present invention should not be construed as being limited thereto. .

Technical terms and scientific terms used in the present invention represent meanings commonly understood by those of ordinary skill in the art to which this invention belongs, unless otherwise defined.

[0001] The present invention relates to a primer composition for detecting white horseradish comprising a primer of SEQ ID NO: 3 and a primer of SEQ ID NO: 4.

In addition, the present invention relates to a probe for detecting white horseradish containing the oligonucleotide of SEQ ID NO: 5.

In addition, the present invention relates to a composition for detecting white horseradish comprising the primer of SEQ ID NO: 3, the primer of SEQ ID NO: 4, and the probe.

In addition, the present invention comprises the steps of isolating DNA from fish or environmental water;

the isolated DNA; And mixing the primer composition comprising the primer of SEQ ID NO: 3 and the primer of SEQ ID NO: 4 and amplifying by PCR; and

The present invention relates to a method for detecting white horsetail, comprising the step of confirming the amplified product by electrophoresis.

The present invention also includes the steps of isolating DNA from fish or environmental water;

the isolated DNA; and amplifying by PCR by mixing a composition for detecting white horseradish containing the primer of SEQ ID NO: 3, the primer of SEQ ID NO: 4, and the probe of claim 2; and

The present invention relates to a method for detecting white horsetail, comprising the step of confirming _{the C T} value of the amplified product.

The step of confirming by electrophoresis is characterized in that when a band of 196 to 198 bp is detected, it is determined as a white squirrel.

_{The step of confirming the C T} value of the amplified product _{is characterized in that when C T} appears below the maximum number of cycles, it is determined as a white horse.

(Example 1) Securing a genetic sample of carp and fish

As shown in the table below, 24 samples of carpidae, including the subdidae family, were obtained (Table 1). The nucleotide sequences of other similar species such as squirrelfish, stony shark, dolphins, persimmons, and sand porcini were obtained from the GenBank, and a total of 34 samples were obtained and the subsequent process was carried out.

series
number sample number scientific name country name classification One Gob-001 Misgurnus anguillicaudatus loach carp order loach 2 Gob-002 Cyprinus carpio carp carp order carp subfamily 3 Gob-003 Pseudogobio esocinus sand plain Carpoptera 4 Gob-004 Pseudogobio esocinus sand plain Carpoptera 5 Gob-005 Zacco platypus pyramid carp order pyramidae 6 Gob-006 Rhynchocypris oxycephalus willow carpidae 7 Gob-007 Microphysogobio jeoni ?瘟歷早? Carpoptera 8 Gob-008 Microphysogobio jeoni ?瘟歷早? Carpoptera 9 Gob-009 Gobiobotia naktongensis white horse Carpoptera 10 Gob-010 Gobiobotia naktongensis white horse Carpoptera 11 Gob-011 Gobiobotia naktongensis white horse Carpoptera 12 Gob-012 Gobiobotia naktongensis white horse Carpoptera 13 Gob-013 Gobiobotia naktongensis white horse Carpoptera 14 Gob-014 Microphysogobio rapidus as soon as the rapids Carpoptera 15 Gob-015 Microphysogobio rapidus as soon as the rapids Carpoptera 16 Gob-016 Microphysogobio rapidus as soon as the rapids Carpoptera 17 Gob-017 Microphysogobio jeoni ?瘟歷早? Carpoptera 18 Gob-018 Squalidus japonicus koreanus scoundrel Carpoptera 19 Gob-019 Microphysogobio yaluensis as soon as you turn Carpoptera 20 Gob-020 Coreoleuciscus aeruginos Chamsiri Carpoptera 21 Gob-021 Gobiobotia macrocephala kuguri Carpoptera 22 Gob-022 Sarcocheilichthys variegatus wakiyae tuna fish Carpoptera 23 Gob-023 Ladislavia taczanowskii Sammy Carpoptera 24 Gob-024 Saurogobio dabryi cowboy Carpoptera

A genetic sample was prepared by mixing 600 μl of DNA extraction solution (10 mM Tris-HCl pH 8.0; 125 mM NaCl; 10 mM EDTA pH 8.0; 1% SDS; 8 M Urea) (Asahida et al. 1996) and 6 μl of Proteinase K solution (20 mg/ml). secured.

The concentration and purity of the extracted genomic DNA were confirmed using a spectrophotometer.

The concentration of extracted genomic DNA varied from 302.9 to 4907.9 ng/μl.

The A260/280 value, which can evaluate the quality of genomic DNA, was generally good, ranging from 1.83 to 2.13 (Table 2).

turn sample name Concentration (ng/μl) Absorbance (A260/280) One Gob-001 3761.6 2.05 2 Gob-002 1885.5 1.95 3 Gob-003 302.9 1.83 4 Gob-004 2026.6 1.92 5 Gob-005 842.3 1.93 6 Gob-006 1996.5 1.88 7 Gob-007 2185.3 2.04 8 Gob-008 3446.7 2.03 9 Gob-009 819.7 1.85 10 Gob-010 760.6 1.89 11 Gob-011 2456.7 1.93 12 Gob-012 3144.6 2.02 13 Gob-013 1594.4 1.90 14 Gob-014 3793.7 1.98 15 Gob-015 4906.3 2.09 16 Gob-016 601.8 1.89 17 Gob-017 2329.0 2.13 18 Gob-018 4907.9 2.00 19 Gob-019 4212.4 2.10 20 Gob-020 839.2 2.03 21 Gob-021 2137.4 2.06 22 Gob-022 934.3 1.96 23 Gob-023 752.6 1.95 24 Gob-024 2329.0 2.13

(Example 2) General PCR amplification and purification

The nucleotide sequence of the front primer used to amplify the entire or specific region of the mitochondrial COB gene region of 24 carpidae fish using the designed PCR primer combinations is 5'-GATCCTRCAAAYTCYTAGTTAACAGCTA-3' (SEQ ID NO: 1, PCR primer 1 ), and the nucleotide sequence of the rear primer was 5'-TGRAATCCTARTTGHGWGGG-3' (SEQ ID NO: 2, PCR primer 2). Including this ^{, a PCR reaction solution was prepared as follows using AccuPower ®} Taq PCR PreMix & Master Mix (Bioneer, Korea) (Table 3).

Reagent AccuPower ^® Taq PCR PreMix & Master Mix 10 μl Genomic DNA (20 ng/μl) 1 μl Forward primer (5 pmol) 1 μl Reverse primer (5 pmol) 1 μl Nuclease-free water 7 μl Total 20 μl

PCR amplification was performed under the following conditions using the ProFlex PCR System (Life Technologies, USA) (Table 4). At this time, when the PCR machine reaches the temperature (95℃) where the initial denaturation occurs, put the PCR tube containing all the PCR reaction solutions into the PCR amplifier to start the PCR amplification reaction, that is, the hot-start PCR amplification reaction. was performed.

Temperature Time No. cycles Initial denaturation 95℃ 5 min One Denaturation 95℃ 30 s 35 Annealing 55℃ 30 s Elongation 72℃ 1 min final elongation 72℃ 5 min One Storage 4℃ ∞ One

The PCR amplification product was purified according to the user's manual of the AccuPrep PCR Purification Kit (Bioneer, Korea).

As a result of performing a PCR amplification reaction of the mitochondrial COB gene region on the extracted genomic DNA, PCR amplification products of the expected size were successfully confirmed in all samples (FIG. 1).

(Example 3) DNA sequence information decoding and analysis

We requested Macrogen Co., Ltd. to decipher the DNA sequence information of PCR amplification products. The decoded DNA sequence information is edited using BioEdit 7.2.5 (http://www.mbio.ncsu.edu/bioedit/bioedit.html) to correct the error, properly trimming, and then creating a contig proceeded.

The matrix including the nucleotide sequence obtained from the gene bank is as follows. A total of 1,140bp of the COB gene sequence was successfully obtained in all individuals.

By comparative analysis of the COB sequence matrix, primer sets (SEQ ID NOs: 3 and 4) and probes (SEQ ID NO: 5) for detecting white sagebrush, which can only be amplified from white sage, were prepared as follows (Table 5).

name Base sequence (5'→3') note Gna-2f ctcttcttcacctRctgttt SEQ ID NO: 3 Gna-n1r gggttactatRgggtttgca SEQ ID NO: 4 Gna-3p aacaacccagccggccta TaqMan probe
(SEQ ID NO: 5)

The probe may have a reporter (reporter) and a fluorescent substance of a quencher (quencher) capable of quenching the reporter fluorescence may be bound to both ends.

The reporter is composed of FAM (6-carboxyfluorescein), Texas red, HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, Cy3 and Cy5. It may be at least one selected from the group being It is preferred that Cy5 is used and BHQ2 is used as the quencher.

For example, the probe (Gna-3p) may be defined as cy5-aacaacccagccggccta-BHQ2.

The present invention provides a probe for detecting white horseradish using the specific oligonucleotide of SEQ ID NO: 5 derived from the nucleotide sequence of white horsetail.

The probe is a TaqMan probe, and the 5' end is a reporter fluorescent labeling factor (FAM, Cy5, etc.), and the 3' end is a suppressor material (TAMRA, BHQ2, etc.), SEQ ID NO: 3 and the sequence Preference is given to use with the oligonucleotide of number 4.

The probe specifically hybridizes to the template DNA in the annealing step during the PCR process, and only the probe hybridized to the template is degraded by the 5'->3' exonuclease activity of the polymerase in the extension step. The fluorescent labeling factor is released from the probe, and the inhibition by the inhibitor is released to emit fluorescence.

(Example 4) Identification of white horsetail

A PCR reaction was performed by separating DNA from the obtained fish or environmental water.

The present invention provides a set of PCR primers for the detection of white horsetail, capable of amplifying a specific target sequence of white horsetail using the oligonucleotides of SEQ ID NO: 3 and SEQ ID NO: 4.

In the electrophoresis step after performing PCR using the PCR primer set for detection of white sage, a band of 196 to 198 bp was detected in the case of white sage, but the band was not observed in other fish.

(Example 5) Securing environmental water samples

(Sample name: eDNA Test)

On June 3, 2019, at the downstream of Sejongbo in the Geumgang River, the white horseradish collected using a foot stick was soaked in river water for 10 minutes, then 1000ml was collected and filtered using disposable Celluose Nitrate filter paper (pore size 0.45㎛). sealed and refrigerated.

(Sample name: breeding water)

500 ml of breeding water in a 200 L PVC tank for breeding 10 Nakdonggang white snails in the Endangered Species Restoration Center of the National Institute of Ecology was collected and stored in the same manner as above.

(Example 6) Environmental DNA extraction and real-time PCR verification

Environmental DNA was extracted using the DNeasy Tissue & Blood Kit (Qiagen, USA) according to the user's manual.

To prepare a calibration curve for the real-time PCR amplification reaction, a set of primers (Gna-2f and Gna-n1r) and a species-specific probe (Gna-3p; cy5-aacaacccagccggccta-) BHQ2) was used to carry out real-time PCR amplification reactions for each concentration of pure sage genomic DNA. At this time, the genomic DNA of the genital warts was diluted 7 times by 10 times at a concentration of 100 ng/μl and used for the real-time PCR amplification reaction.

To perform real-time PCR amplification, a PCR reaction solution was prepared as follows using AccuPower® Plus DualStar™ qPCR PreMix & Master Mix (Bioneer, Korea) (Table 6).

Reagent AccuPower® Plus DualStar™ qPCR PreMix & Master Mix 10 μl CXR 0.2 μl Genomic DNA (20 ng/μl) 1 μl Forward primer (5 pmol) 1 μl Reverse primer (5 pmol) 1 μl Probe (5 pmol) 1 μl SYTO™ 9 (50 μM) 1 ul Nuclease-free water 4.8 μl Total 20 μl

PCR amplification was performed under the following conditions using QuantStudio5 (Life Technologies, USA) (Table 7).

Temperature Time No. cycles Initial denaturation 95℃ 2 min One Denaturation 95℃ 15 sec 40 Annealing/elongation 65℃ 1 min Melt curve stage 95℃ 15 sec One 60℃ 1 min ↓ 0.05℃/sec 95℃ 15 sec

As a result, it was confirmed that the C _T value confirmed through the real-time PCR amplification reaction was increased in inverse proportion to the concentration of genomic DNA of the genus (Fig. 14, r ² =0.999), and therefore it was determined that quantitative analysis was possible.

The present invention relates to the PCR primer set (primer of SEQ ID NO: 3 and primer of SEQ ID NO: 4) for detecting white squash and C, which means a significant cycle when performing real-time PCR using a probe including the oligonucleotide of SEQ ID NO: 5 _{When T} (critical threshold) is detected below the maximum cycle, it is characterized in that it is judged as a white squirrel.

In addition, when performing real-time PCR using the oligonucleotide mixture, it is characterized in that quantitative analysis can be performed by performing relative quantification according to the _{C T value.}

Therefore, according to the present invention, the PCR primer set and probe for detecting genus serrata, and the real-time PCR method using the same are fast and accurate, and have excellent sensitivity, so that the efficiency and practicality of detecting a trace amount of sagebrush are very high.

The results of verifying the specificity of the primer set (Gna-2f and Gna-n1r) and the probe (Gna-3p; cy5-aacaacccagccggccta-BHQ2) for the detection of white sage were as follows (Fig. 15). The real-time PCR amplification reaction was confirmed only in white snails, and the specificity was verified, and the base line was stable up to a _{C T value of 40.} Therefore, it can be seen that the species specificity of white horsetail was very high, and there were no errors that could occur due to false-negative.

As a result of performing real-time PCR amplification reaction using pure white sage genomic DNA (10ng/μl), environmental DNA sample for sage test (eDNA test), and environmental DNA sample extracted from fermented sagebrush (breeding water), the result of the real-time PCR amplification The DNA was _{confirmed at a C T} value of 22.965~22.966, and the environmental DNA sample for the test was found at 32.176 and the number of breeding at 37.601. When converted by substituting it into the prepared calibration curve, the environmental DNA sample for the test was equivalent to 0.053 ng, and the number of breeding corresponded to 0.024 ng (Table 8).

number sample name Ct value Genomic DNA conversion value (ng) detection ^** One Gna eDNA Test 32.176 0.053 ○ 2 Gna breeding water 37.659 0.024 ○ 3 positive control
(10 ng of DNA as soon as possible) 22.966 10.000 ○ 4 negative control
(tertiary distilled water) ND ^* 0.000 -

^* ND: Not detected, ^** Criteria for detection: Detected: ○, Not detected: -

<110> National Institute of Ecology <120> PCR primer set and probe for detection of Gobiobotia naktongensis and real-time PCR method using the same <130> PNC-2019-001 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 1 gatcctrcaa aytcytagtt aacagcta 28 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 2 tgraatccta rttghgwggg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 3 ctcttcttca cctrctgttt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 4 gggttactat rgggtttgca 20 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 5 aacaacccag ccggccta 18

Claims (7)

It comprises a primer of SEQ ID NO: 3 and a primer of SEQ ID NO: 4,
A primer composition for the detection of whitefish that can discriminate it from other freshwater fish.
delete A probe comprising the primer of SEQ ID NO: 3, the primer of SEQ ID NO: 4 and the oligonucleotide of SEQ ID NO: 5,
A composition for the detection of whitefish, which can discriminate it from other freshwater fish.
isolating DNA from fish or environmental water;
the isolated DNA; and amplifying by PCR by mixing the primer composition for detecting white squash of claim 1; and
In the method of detecting white horseradish, comprising the step of confirming the amplified product by electrophoresis,
The step of confirming by electrophoresis is a method of detecting white horsetail, characterized in that when a band of 196 to 198 bp is detected, it is determined as a white horsetail.
isolating DNA from fish or environmental water;
the isolated DNA; and amplifying by PCR by mixing the composition for detecting white horseradish according to claim 3; and
In the method for detecting white horseradish comprising the step of confirming _{the C T} value of the amplified product,
_{In the step of determining the C T} value of the amplified product, it _{is determined that the C T} is displayed at or below the maximum number of cycles.
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