KR20220071072A - PCR primer set and probe for environmental DNA detection of Kichulchoia brevifasciata and real-time PCR method using the same - Google Patents

Abstract

The present invention relates to a method for distinguishing a kichulchoia from other freshwater fish by amplifying a target sequence specific to the kichulchoia, using a primer set including an oligonucleotide of SEQ ID NO: 3 and an oligonucleotide of SEQ ID NO: 4, which are PCR primers. In addition, the present invention relates to a method for rapidly detecting a kichulchoia from fish or environmental water with excellent specificity and sensitivity through real-time PCR, using a composition including an oligonucleotide of SEQ ID NO: 3, an oligonucleotide of SEQ ID NO: 4, and a probe for detecting an oligonucleotide of SEQ ID NO: 5, derived from the kichulchoia.

Description

PCR primer set and probe for environmental DNA detection of Kichulchoia brevifasciata and real-time PCR method using the same

The present invention relates to a method for discriminating between a small number and other freshwater fish by amplifying a target sequence specific for the small number by using a primer set including the oligonucleotide of SEQ ID NO: 3 and the oligonucleotide of SEQ ID NO: 4 as PCR primers. will be.

In addition, the present invention uses a composition comprising the oligonucleotide of SEQ ID NO: 3, the oligonucleotide of SEQ ID NO: 4, and a probe for detecting oligonucleotides derived from the oligonucleotide of SEQ ID NO: 5, from fish or environmental water through real-time PCR. It relates to a method for rapidly detecting numerical values with good specificity and sensitivity.

Kichulchoia brevifasciata is a fish belonging to the genus Cobitidae of the order Cypriniformes.

In general, the number one is very similar in morphology to the loach ( Kichulchoia multifasciata ), but the distribution area is different, and it can be distinguished by the difference in the vertebral marrow, bird wave number and horizontal pattern. It inhabits relatively small rivers with shallow water and fast-flowing gravel bottom, and its main food sources are aquatic insects and algae.

Habitat is decreasing due to river maintenance work and aggregate collection, and as a result, the number of individuals is rapidly decreasing. The Ministry of Environment designated it as an endangered wildlife class II in 2012, and in 2017, it was upgraded to class I endangered wildlife for legal protection. It is a paper in urgent need of protection. In the past, Jomsu number was distributed throughout the small rivers around Goheung Peninsula and Yeosu City, but now it has been investigated to inhabit only some sections, so the habitat range is very narrow. In particular, in recent years, the size of the remaining population has been reduced, making it very difficult to find the habitat.

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. 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 only environmental water. Therefore, it is possible to find out whether a target organism is inhabited without direct collection, 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.

Methods for analyzing environmental genes include metabarcoding, metagenome, and species-specific PCR. Metabarcoding and species-specific marker PCR are mainly used due to the nature of environmental genes that occur in very small amounts. Metabarcoding is mainly used to detect various species living in environmental waters, but it has disadvantages in that the analysis cost is high and it takes a long time. On the other hand, species-specific marker PCR takes time and cost to develop, but once developed, it has high sensitivity and can be analyzed in just a few hours. Therefore, there are many advantages of species-specific marker PCR in confirming and verifying the presence of endangered species populations. When using this method, if it is applied to river water that has the possibility of habitability, it is possible to confirm the potential habitat of the jomsu sugi, and if it is detected at a high concentration, the relative abundance can be derived.

In order to improve the specificity and sensitivity of the detection of small numbers in environmental water, the present invention developed a specific primer set and probe for detection of small numbers, and established a real-time PCR method using them.

Korean Patent Publication No. 10-2012-0136201

The present invention was derived to solve the problems of the above technology, and an object of the present invention is to provide a specific primer set and probe having excellent specificity and sensitivity capable of detecting a small number of genes from fish or environmental water.

In addition, it is possible to reduce the cumbersome and process of sequencing or electrophoresis, and to provide a specific number-specific real-time PCR method using primer sets and probes with high specificity and capable of quantitative analysis.

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

In order to achieve the above object, the present invention provides a primer set for detecting small numbers comprising the oligonucleotide of SEQ ID NO: 3 and the oligonucleotide of SEQ ID NO: 4.

In addition, the present invention provides a probe for detecting small numbers comprising the oligonucleotide of SEQ ID NO: 5.

In addition, the present invention provides a composition for detecting small numbers comprising the oligonucleotide of SEQ ID NO: 3, the oligonucleotide of SEQ ID NO: 4, and the probe.

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

the isolated DNA; and mixing the primer set or the composition and amplifying by PCR; and

It provides a method for detecting a small number, comprising the step of confirming the amplified product by electrophoresis.

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

the isolated DNA; and mixing the primer set or the composition and amplifying by PCR; and

It provides a method for detecting a small number, comprising the step of confirming a threshold cycle ( _CT ) value of the amplified product.

In one embodiment of the present invention, the step of confirming by electrophoresis is characterized in that when 353 ~ 355 bp is detected, it is determined as a small numerical value.

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 below the maximum number of cycles, it is determined as a small numerical value.

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 a small number.

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

In addition, if it is applied to river waters with potential habitat for Jomsusuchi, we can confirm the potential habitat for Jomsusumu, and if it is detected at a high concentration, it is possible to derive the relative abundance.

1 shows an electrophoresis picture showing representative results of PCR amplification of mitochondrial cytochrome b (COB) gene on 100 fish tissue samples of the present invention.
Figure 2 shows an image of a matrix suitable for decoding the DNA sequence information of the mitochondrial COB gene of freshwater fish and comparing the information.
3 shows a calibration curve ( r ² =0.999) of a real-time PCR amplification reaction for each concentration of numerical genomic DNA.
Figure 4 shows the results of verification of the specificity of molecular markers for real-time PCR amplification reactions specific to the number of freshwater fishes for 44 species of fish (the concentration of 1 × 10 ⁵ copies/μl of the plasmid of the number of mitochondrial COB gene region for the positive control of the number level) means).

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 in order to explain the present invention in more detail and help those of ordinary skill in the art 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.

The present invention relates to a primer set for detecting small numbers comprising the oligonucleotide of SEQ ID NO: 3 and the oligonucleotide of SEQ ID NO: 4.

In addition, the present invention relates to a probe for detecting small numbers comprising the oligonucleotide of SEQ ID NO: 5.

In addition, the present invention relates to a composition for detecting small numbers comprising the oligonucleotide of SEQ ID NO: 3, the oligonucleotide of SEQ ID NO: 4, and the probe.

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

the isolated DNA; and mixing the primer set or the composition and amplifying by PCR; and

It relates to a method for detecting a small number, comprising the step of confirming the amplified product by electrophoresis.

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

the isolated DNA; and mixing the primer set or the composition and amplifying by PCR; and

It relates to a method for detecting a small number, comprising the step of determining a C _T (threshold cycle) value of the amplified product.

The step of confirming by electrophoresis is characterized in that when 353 ~ 355 bp is detected, it is determined as a numerical value.

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 small numerical value.

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

A total of 100 fin samples of 4 types of fish were obtained.

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 Proteinase K solution (20 mg/ml) 6 μl was added and a genetic sample was obtained in the same manner as in the literature.

The results of checking the concentration and purity of the extracted genomic DNA using a spectrophotometer are shown in the table below (Table 1).

number scientific name country name taxa genomic DNA density
(ng/μl) A260/280 NIE-001 Misgurnus anguillicaudatus loach carp order loricaceae 434.7 1.82 NIE-002 Misgurnus anguillicaudatus loach carp order loricaceae 968.3 1.86 NIE-003 Misgurnus anguillicaudatus loach carp order loricaceae 313.9 1.86 NIE-004 Cyprinus carpio carp carp order carp family carp subfamily 316.5 1.84 NIE-005 Cyprinus carpio carp carp order carp family carp subfamily 275.1 1.77 NIE-006 Cyprinus carpio carp carp order carp family carp subfamily 201.5 1.75 NIE-007 Pseudogobio esocinus sand plain Carpidae Carpidae 558.2 1.82 NIE-008 Pseudogobio esocinus sand plain Carpidae Carpidae 165.1 1.90 NIE-009 Aceilognathus lanceolatus lead bag carpidae carpidae 130.8 1.84 NIE-010 Aceilognathus lanceolatus lead bag carpidae carpidae 86.1 2.18 NIE-011 Aceilognathus lanceolatus lead bag carpidae carpidae 391.7 1.87 NIE-012 Zacco platypus pyramid carpidae carpidae pyramidae 648.0 1.84 NIE-013 Zacco platypus pyramid carpidae carpidae pyramidae 1191.4 1.86 NIE-014 Zacco platypus pyramid carpidae carpidae pyramidae 93.5 2.04 NIE-015 Phoxinus oxycephalus willow carpidae carpidae 66.1 1.86 NIE-016 Phoxinus oxycephalus willow carpidae carpidae 1204.0 1.87 NIE-017 Channa argus gamulchi Perch and Gamul Dental Clinic 211.0 1.81 NIE-018 Channa argus gamulchi Perch and Gamul Dental Clinic 3813.9 1.87 NIE-019 Coreoperca herzi kkeokji perch 433.9 1.87 NIE-020 Coreoperca herzi kkeokji perch 465.9 1.88 NIE-021 Odontobutis interrupta speckled fish perch 2939.7 1.86 NIE-022 Odontobutis interrupta speckled fish perch 443.9 1.81 NIE-023 Pseudobagrus fulvidraco Dongjagae Catfish 81.3 2.13 NIE-024 Pseudobagrus fulvidraco Dongjagae Catfish 95.3 2.11 NIE-025 Silurus asotus catfish Catfish 78.4 2.05 NIE-026 Silurus asotus catfish Catfish 709.7 1.86 NIE-027 Silurus asotus catfish Catfish 270.9 1.83 NIE-028 Anguilla japonica eel eel family eel family 286.6 1.86 NIE-029 Anguilla japonica eel eel family eel family 528.3 1.88 NIE-030 Anguilla japonica eel eel family eel family 123.2 1.88 NIE-031 Arctoscopus japonicus patter sea smelt order sea smelt family 604.5 1.89 NIE-032 Arctoscopus japonicus patter sea smelt order sea smelt family 540.0 1.89 NIE-033 Arctoscopus japonicus patter sea smelt order sea smelt family 292.1 1.91 NIE-034 Mugil cephalus mullet mullet mullet 100.6 1.76 NIE-035 Mugil cephalus mullet mullet mullet 1338.9 1.90 NIE-036 Mugil cephalus mullet mullet mullet 799.2 1.91 NIE-037 Oryzias latipes killifish Donggalchidae Medaka 1204.8 2.00 NIE-038 Oryzias latipes killifish Donggalchidae Medaka 469.5 1.97 NIE-039 Oryzias latipes killifish Donggalchidae Medaka 304.9 2.00 NIE-040 Gasterosteus aculeatus aculeatus big fish Greater Spiny Species 264.4 1.99 NIE-041 Gasterosteus aculeatus aculeatus big fish Greater Spiny Species 1987.2 2.04 NIE-042 Gasterosteus aculeatus aculeatus big fish Greater Spiny Species 140.9 1.94 NIE-043 Monopterus albus dung waist Dreongheoridae 183.7 1.96 NIE-044 Monopterus albus dung waist Dreongheoridae 68.8 1.64 NIE-045 Cottus poecilopus poison brokerage Dept. of Poison Intervention 240.2 1.97 NIE-046 Cottus poecilopus poison brokerage Dept. of Poison Intervention 498.7 2.03 NIE-047 Cottus poecilopus poison brokerage Dept. of Poison Intervention 66.4 1.96 NIE-048 Microphysogobio jeoni ?瘟歷早? Carpidae Carpidae 570.8 1.90 NIE-049 Microphysogobio jeoni ?瘟歷早? Carpidae Carpidae 67.0 1.94 NIE-050 Microphysogobio jeoni ?瘟歷早? Carpidae Carpidae 468.1 1.94 NIE-051 Gobiobotia naktongensis white horse Carpidae Carpidae 1958.8 2.00 NIE-052 Gobiobotia naktongensis white horse Carpidae Carpidae 1851.6 2.07 NIE-053 Gobiobotia macrocephala kuguri Carpidae Carpidae 799.0 1.95 NIE-054 Gobiobotia macrocephala kuguri Carpidae Carpidae 338.0 1.99 NIE-055 Pseudopungtungia nigra persimmon Carpidae Carpidae 1319.5 1.88 NIE-056 Pseudopungtungia nigra persimmon Carpidae Carpidae 317.4 1.96 NIE-057 Gobiobotia brevibarba stone shark Carpidae Carpidae 88.5 1.72 NIE-058 Gobiobotia brevibarba stone shark Carpidae Carpidae 774.6 1.86 NIE-059 Microphysogobio koreensis sand shot Carpidae Carpidae 178.1 1.85 NIE-060 Microphysogobio koreensis sand shot Carpidae Carpidae 711.2 1.97 NIE-061 Microphysogobio koreensis sand shot Carpidae Carpidae 195.0 1.88 NIE-062 Pseudopungtungia tenuicorpa fine stone Carpidae Carpidae 522.4 1.98 NIE-063 Pseudopungtungia tenuicorpa fine stone Carpidae Carpidae 224.4 1.97 NIE-064 Pseudopungtungia tenuicorpa fine stone Carpidae Carpidae 140.3 1.77 NIE-065 Repomucenus olidus steel spatula mode perch sail mode 154.9 1.88 NIE-066 Repomucenus olidus steel spatula mode perch sail mode 737.4 1.98 NIE-067 Squalidus japonicus moron Carpidae Carpidae 646.0 1.96 NIE-068 Squalidus japonicus moron Carpidae Carpidae 574.5 2.02 NIE-069 Squalidus japonicus moron Carpidae Carpidae 531.6 1.98 NIE-070 Microphysogobio yaluensis as soon as you turn Carpidae Carpidae 266.7 1.95 NIE-071 Microphysogobio yaluensis as soon as you turn Carpidae Carpidae 448.9 1.96 NIE-072 Microphysogobio yaluensis as soon as you turn Carpidae Carpidae 1426.7 2.06 NIE-073 Coreoleuciscus aeruginos Chamsiri Carpidae Carpidae 1956.7 2.00 NIE-074 Coreoleuciscus aeruginos Chamsiri Carpidae Carpidae 501.3 1.95 NIE-075 Culter erythropterus Kang Jun-chi Carpoptera 1239.7 1.97 NIE-076 Culter erythropterus Kang Jun-chi Carpoptera 378.5 1.95 NIE-077 Aceilognathus yamatsutae sack carpidae carpidae 471.7 1.95 NIE-078 Aceilognathus yamatsutae sack carpidae carpidae 744.2 2.04 NIE-079 Tachysurus nitidus mother-of-pearl Catfish 3009.9 1.96 NIE-080 Tachysurus nitidus mother-of-pearl Catfish 1787.6 1.89 NIE-081 carassius carassius crucian carp carp order carp family carp subfamily 504.9 2.00 NIE-082 carassius carassius crucian carp carp order carp family carp subfamily 295.3 1.95 NIE-083 Siniperca scherzeri mandarin fish perch 260.7 1.88 NIE-084 Siniperca scherzeri mandarin fish perch 384.9 2.01 NIE-085 Pseudobagrus emarginatus mackerel Catfish 623.2 1.98 NIE-086 Pseudobagrus emarginatus mackerel Catfish 492.3 1.98 NIE-087 Sarcocheilichthys czerskii used Carpidae Carpidae 57.7 2.03 NIE-088 Sarcocheilichthys czerskii used Carpidae Carpidae 106.6 2.06 NIE-089 Aceilognathus rhombeus Napji-ri carpidae carpidae 53.9 2.04 NIE-090 Aceilognathus rhombeus Napji-ri carpidae carpidae 88.9 2.19 NIE-091 Nipponocypris koreanus Red seaweed carpidae carpidae pyramidae 1435.0 2.00 NIE-092 Nipponocypris koreanus Red seaweed carpidae carpidae pyramidae 696.6 1.97 NIE-093 Opsarichthys uncirostris amurensis off carpidae carpidae pyramidae 451.6 1.90 NIE-094 Iksookimia hugowolfeldi south pole koi family koi family loach family 442.5 1.98 NIE-095 Cobitis hankugensis oil pan koi family koi family loach family 523.1 1.85 NIE-096 Cobitis tetralineata line koi family koi family loach family 147.5 1.76 NIE-097 Cobitis pacifica northern dog koi family koi family loach family 249.3 2.01 NIE-098 Kichulchoia brevifasciata a little shame koi family koi family loach family 86.2 1.98 NIE-099 Kichulchoia brevifasciata a little shame koi family koi family loach family 94.4 1.77 NIE-100 Kichulchoia brevifasciata a little shame koi family koi family loach family 103.5 1.83

(Example 2) General PCR amplification and purification

The nucleotide sequence of the forward primer used to amplify the entire or specific region of the mitochondrial COB gene region of 100 fish using the designed PCR primer combinations was 5'-GATCCTRCAAAYTCYTAGTTAACAGCTA-3', and the nucleotide sequence of the reverse primer was 5 It was '-TGRAATCCTARTTGHGWGGG-3'. Including this, a PCR reaction solution was prepared as follows using AccuPower® PCR PreMix & Master Mix (Bioneer, Korea) (Table 2).

Reagent AccuPower® PCR MasterMix 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 3). At this time, when the PCR machine reaches the temperature (95℃) where the initial denaturation reaction occurs, put the PCR tube containing all the PCR reaction solutions into the PCR amplifier to start the PCR amplification reaction, that is, a 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, trim it appropriately, and create a contig proceeded.

The matrix including the nucleotide sequence obtained from the gene bank is as follows. A total of 1,140 bp of the COB gene sequence was successfully obtained in all subjects (FIG. 2).

By comparative analysis of the COB sequence matrix, primer sets (SEQ ID NOs: 3 and 4) and probes (SEQ ID NO: 5) for detecting small numbers that can be amplified only at small numbers were prepared as follows (Table 4).

name Base sequence (5'→3') note Kbr-0423f2 AGGRGCCACAGTGATC SEQ ID NO: 3 Kbr-0777r AGGTGTTACTAGGGGGTTA SEQ ID NO: 4 Kbr-0568fp Cy5-GCCGCAGCCACTATTTTACA-BHQ3 TaqMan probe
(SEQ ID NO: 5)

At both ends of the probe, a reporter and a fluorescent material of a quencher capable of quenching the reporter fluorescence may be bound.

The reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), NED, JOE, Cy3 and Cy5 It may be at least one selected from the group consisting of, and the quencher may be at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2, BHQ3, NFQ and Dabcyl, but is not limited thereto. .

For example, the probe (Kbr-0568fp) may be defined as Cy5-GCCGCAGCCACTATTTTACA-BHQ3.

The probe is a TaqMan probe, the 5' end of the reporter fluorescent labeling factor (FAM, Cy5, etc.), and the 3' end of the suppressor material (TAMRA, BHQ2, etc.), SEQ ID NO: 3 and It is preferably used together with the oligonucleotide of SEQ ID NO: 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 small numbers

DNA was isolated from the obtained fish or environmental water and PCR reaction was performed.

The present invention provides a set of PCR primers for detecting small numbers that can amplify specific target sequences of small numbers using the oligonucleotides of SEQ ID NO: 3 and SEQ ID NO: 4.

The present invention also provides a composition for detecting small numbers comprising the oligonucleotide of SEQ ID NO: 3, the oligonucleotide of SEQ ID NO: 4, and the probe.

In the case of electrophoresis after performing PCR using the PCR primer set or composition for detecting the number level, a band of 353-355 bp was detected in the case of the level number, but the band was not observed in other fish.

(Example 5) Securing environmental water samples

(Sample name: KBC, KBP samples)

Collect 1,000 ml of river water from the 3rd point of the Yeongsan River, which is known to inhabit the Jomsu, and filter it using disposable Celluose Nitrate filter paper (pore size 0.45㎛). It was stored by rapid freezing below.

(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.

In order to create a calibration curve of the real-time PCR amplification reaction, a primer set (SEQ ID NOs: 3 and 4) for detecting the number of species and a species-specific fluorescent probe (SEQ ID NO: 5) at the optimal binding temperature (64 ° C) were used A real-time PCR amplification reaction was performed for each numerical genomic DNA concentration. At this time, the plasmid of the mitochondrial COB gene region was diluted 10 times at a concentration of 1×10 ⁶ copies/μl and used for real-time PCR amplification.

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 5).

Reagent AccuPower® Plus DualStar™ qPCR PreMix & Master Mix 10 μl Genomic DNA or environmental DNA 2 μl Forward primer (5 pmoles) 1 μl Reverse primer (5 pmoles) 1 μl Probe (5 pmoles) 1 μl Nuclease-free water 5 μl Total 20 μl

PCR amplification was performed under the following conditions using QuantStudio5 (Life Technologies, USA) (Table 6).

Temperature Time No. cycles Initial denaturation 95℃ 2 min One Denaturation 95℃ 15 sec 45 Annealing/elongation 64℃ 1 min

As a result, it was confirmed that the C _T value confirmed through the real-time PCR amplification reaction increased in inverse proportion to the concentration of the numerical genomic DNA (Fig. 3, r ² =0.998). Therefore, it was determined that quantitative analysis was possible.

The present invention provides a C _T (critical threshold) which means a significant cycle when performing real-time PCR using the PCR primer set (SEQ ID NO: 3 and SEQ ID NO: 4) for detecting small numbers and a probe including the oligonucleotide of SEQ ID NO: 5 ) is detected in the maximum cycle or less, it is characterized in that it is judged as a numerical value.

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 small numbers and real-time PCR methods using the same are fast and accurate, and the sensitivity is excellent, so the efficiency and practicality are very high for the detection of small amounts of samples.

The results of verifying the specificity of the primer set and probe for detecting the level of Jomsu for 44 fishes (Table 7) including Jomsu level were as follows (FIG. 4). The real-time PCR amplification reaction was confirmed only in small numbers, and the specificity was verified, and the base line was stable up to a C _T value of 45. Therefore, it can be seen that the species specificity was very high, and there were no errors that could occur due to false-negative.

Sample name scientific name country name taxa NIE-001 Misgurnus anguillicaudatus loach carp order loricaceae NIE-004 Cyprinus carpio carp carp order carp family carp subfamily NIE-007 Pseudogobio esocinus sand plain Carpidae Carpidae NIE-009 Aceilognathus lanceolatus lead bag carpidae carpidae NIE-012 Zacco platypus pyramid carpidae carpidae pyramidae NIE-015 Moroco oxycephalus willow carpidae carpidae NIE-017 Channa argus gamulchi Perch and Gamul Dental Clinic NIE-019 Coreoperca herzi kkeokji perch NIE-021 Odontobutis interrupta speckled fish perch NIE-023 Pseudobagrus fulvidraco Dongjagae Catfish NIE-025 Silurus asotus catfish Catfish NIE-028 Anguilla japonica eel eel family eel family NIE-031 Arctoscopus japonicus patter sea smelt order sea smelt family NIE-034 Mugil cephalus mullet mullet mullet NIE-037 Oryzias latipes killifish Donggalchidae Medaka NIE-040 Gasterosteus aculeatus aculeatus big fish Greater Spiny Species NIE-043 Monopterus albus dung waist Dreongheoridae NIE-045 Cottus poecilopus poison brokerage Dept. of Poison Intervention NIE-048 Microphysogobio jeoni ?瘟歷早? Carpidae Carpidae NIE-051 Gobiobotia naktongensis white horse Carpidae Carpidae NIE-053 Gobiobotia macrocephala kuguri Carpidae Carpidae NIE-055 Pseudopungtungia nigra persimmon Carpidae Carpidae NIE-057 Gobiobotia brevibarba stone shark Carpidae Carpidae NIE-059 Microphysogobio koreensis sand shot Carpidae Carpidae NIE-062 Pseudopungtungia tenuicorpa fine stone Carpidae Carpidae NIE-065 Repomucenus olidus steel spatula mode perch sail mode NIE-067 Squalidus japonicus moron Carpidae Carpidae NIE-070 Microphysogobio yaluensis as soon as you turn Carpidae Carpidae NIE-073 Coreoleuciscus aeruginos Chamsiri Carpidae Carpidae NIE-075 Erythroculter erythropterus Kang Jun-chi Carpoptera NIE-077 Aceilognathus yamatsute sack carpidae carpidae NIE-079 Leiocassis nitidus mother-of-pearl Catfish NIE-081 carassius carassius crucian carp carp order carp family carp subfamily NIE-083 Siniperca scherzeri mandarin fish perch NIE-085 Pseudobagrus emarginatus mackerel Catfish NIE-087 Sarcochelichthys czerskii used Carpidae Carpidae NIE-089 Aceilognathus rhombeus Napji-ri carpidae carpidae NIE-091 Nipponocypris koreanus Red seaweed carpidae carpidae pyramidae NIE-093 Opsarichthys uncirostris amurensis off carpidae carpidae pyramidae NIE-094 Iksookimia hugowolfeldi south pole koi family koi family loach family NIE-095 Cobitis hankugensis oil pan koi family koi family loach family NIE-096 Cobitis tetralineata line koi family koi family loach family NIE-097 Cobitis pacifica northern dog koi family koi family loach family NIE-098 Kichulchoia brevifasciata a little shame koi family koi family loach family

As a result of performing a real-time PCR amplification reaction using the plasmid 1×10 ⁵ copies/μl of the Zomsui number mitochondrial COB gene region and the environmental DNA sample for the Zoms number test (eDNA test), the Zomsu number genomic DNA was confirmed at a C _T value of 19.99. , The environmental water sample (KBP8) for the test of Jomsu number was confirmed with a C _T value of 35.20. When converted by substituting it into the prepared calibration curve, the plasmid 1×10 ⁵ copies/μl sample of the mitochondrial COB gene region corresponds to 102,736 copies/μl at the plasmid concentration, and the environmental water sample KBP8 for the Zom number test corresponds to 3 copies/μl at the plasmid concentration (Table) 8).

number Sample name Ct value Plasmid equivalent (copies/ul) detection ^** One KBP8 35.20 3 ○ 2 positive control
(plasmid 1×10 ⁵ copies/μl) 19.99 102,736 ○ 3 negative control
(tertiary distilled water) ND ^* 0.000 -

^* ND: Not detected, ^** Criteria for detection (detection: ○, not detected: -)

<110> NATIONAL INSTITUTE OF ECOLOGY <120> PCR primer set and probe for environmental DNA detection of Kichulchoia brevifasciata and real-time PCR method using the same <130> PNC-2020-011 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Kbr-0423f2 <400> 1 aggrgccaca gtgatc 16 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Kbr-0777r <400> 2 aggtgttact aggggtta 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Kbr-0568fp <400> 3 gccgcagcca ctattttaca 20

Claims (7)

A primer set for detecting small numbers comprising the oligonucleotide of SEQ ID NO: 3 and the oligonucleotide of SEQ ID NO: 4.
A probe for detecting small numbers comprising the oligonucleotide of SEQ ID NO: 5.
A composition for detecting small numbers comprising the oligonucleotide of SEQ ID NO: 3, the oligonucleotide of SEQ ID NO: 4, and the probe of claim 2.
isolating DNA from fish or environmental water;
the isolated DNA; and mixing the primer set of claim 1 or the composition of claim 3 and amplifying by PCR; and
A method of detecting a small number, comprising the step of confirming the amplified product by electrophoresis.
isolating DNA from fish or environmental water;
the isolated DNA; and mixing the primer set of claim 1 or the composition of claim 3 and amplifying by PCR; and
A method of detecting a small number, comprising the step of checking a threshold cycle ( _CT ) value of the amplified product.
5. The method of claim 4,
In the step of confirming by electrophoresis, when 353 to 355 bp is detected.
6. The method of claim 5,
The step of determining the C _T value of the amplified product is a method for detecting a small number, characterized in that when _CT appears below the maximum number of cycles, the determination is made as a small number value.

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