KR101953574B1 - Identifying method of squid species using dual labeled probe and fluorescence melting curve analysis - Google Patents

Abstract

The present invention relates to a double-labeled probe and a squid species discrimination method using fluorescence melting curve analysis. More specifically, double-labeled probes and fluorescence-melting curve analysis for discrimination of domesticated squid ( Todarodes pacificus ), Sepia esculenta , Dosidicus gigas and Argentine short- tailed squid ( Illex argentinus ) (FMCA, fluorescence melting curve analysis).
It is possible to overcome the disadvantages of the conventional conventional squid species discrimination method in which it is difficult to distinguish the DNA base sequence of 1-2 bp effectively using the double-labeled probe and the fluorescence melting curve analysis (FMCA) according to the present invention, ( Todarodes pacificus ), Sepia esculenta (imported squid), and the American squid ( Dosidicus ), which are difficult to discriminate in the processed state based on the melting temperature (Tm) value obtained by the double- gigas , and Argentine short-tailed squid ( Illex argentinus ). In addition, using only one double-labeled LNA probe according to the present invention, it is possible to simultaneously discriminate between the two species of Todarodes pacificus , Dosidicus gigas , and Illex argentinus , However, there is an economic advantage that the inspection cost can be greatly reduced.

Description

[0002] Identification methods of squid species using dual labeled probes and fluorescence melting curve analysis [

The present invention relates to a double-labeled probe and a squid species discrimination method using fluorescence melting curve analysis. More specifically, double-labeled probes and fluorescence-melting curve analysis for discrimination of domesticated squid ( Todarodes pacificus ), Sepia esculenta , Dosidicus gigas and Argentine short- tailed squid ( Illex argentinus ) (FMCA, fluorescence melting curve analysis).

Squid is one of the cephalopods that are widely distributed worldwide and consumed worldwide as a food ingredient. Food and Agriculture Organization (FAO, http://www.fao.org) provides information on the geographical distribution, growth environment, economic importance, storage and processing methods of squid imported and distributed worldwide and distributed. The FAO species catalog also includes diagnostic characteristics, habitat and biological interest for each fish species of each squid species. Squids imported into the domestic aquaculture market can cause a musty odor depending on the storage period and handling method, which may lead to inedible food. Therefore, the authenticity of labels on species is important in the Consumer Rights Act.

In general, morphological features are used to distinguish squid species. However, because of the very similar morphological characteristics of some squid, it is difficult to differentiate squid species with the naked eye, and there are some detailed characteristics that can be identified only by the expert's eyes (Chen, X. et al. , Scientia Marina, 76 (3), 473-481, 2012). In addition, this morphological identification method can not be used on processed foods such as sliced, roasted, smoked, and fried foods. Thus, there is a need to develop a method for accurately and reliably displaying squid specimens and for identifying squid species to prevent counterfeiting. There are many analytical methods for species identification based on biomolecules such as proteins and nucleic acids. Protein-based assays to distinguish species have been developed. However, this method requires many complex steps and can be influenced by the type and status of the tissue (Hernandez-Lopez, JL et al. , Fishery Bulletin-National Oceanic and Atmospheric Administration, 99 (4) 684, 2001). Thus, DNA-based assays are widely applied because of the reliability of DNA analysis and the stability of DNA in the heating process (Rasmussen, RS et al. , Comprehensive Reviews in Food Science and Food Safety, 7 (3), 280-295, 2008).

The cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA in animals is usually evaluated for species-specific identification and is referred to as the DNA-barcode region (Waugh, J., Bioessays , 29 (2), 188 -197, 2007). Species identification studies are generally performed by probe-based real-time polymerase chain reaction (PCR) (Kubista, M. et al. , Molecular Aspects of Medicine, 27 (2- 3), 95-125, 2006). However, there are some obvious limitations to probe-based real-time PCR. For example, more probes would be needed because a single probe can only detect one specific target sequence if more species identification is needed (Zhang, T. et al. , Scientific Reports, 6 , 28494 , 2016). Furthermore, if hybridization occurs between the probe and another target sequence having a sequence difference of 1-2 bp, probe-based real-time PCR can be performed according to this mismatched base pair proportional to the length of the probe PCR) can result in false-positive results from mis-hybridization with annealing temperature (Gunson, RN et al. , Journal of Clinical Virology, 43 (4), 372 -375, 2008).

Thus, fluorescence melting curve analysis (FMCA) for species-specific sequence detection using two dual-labeled probes may be useful for species identification analysis. Probe-based FMCA is based on DNA base-pair mismatching (Tm) using the deduced melting temperature (Tm) due to the thermal denaturation of the hybrid between the double-labeled probe and the target strand DNA (Huang, Q. et al. , PLoS One, 6 (4), e19206, 2011).

Under these circumstances, the inventors of the present invention have made efforts to develop a method for quickly and accurately discriminating the species discrimination of squid, and as a result, they have found out that the domestic cuttlefish ( Todarodes pacificus ) The exact melting temperature values were measured using a double-labeled probe and a fluorescence melting curve analysis (FMCA) method to distinguish Sepia esculenta , Dosidicus gigas , Argentine short- lipped squid ( Illex argentinus ) The present invention has been accomplished by developing a probe-based fluorescence melting curve analysis (FMCA) method for discriminating squid species with higher accuracy.

Chen, X. et al., Scientia Marina, 76 (3), 473-481, 2012 Hernandez-Lopez, J. L. et al., Fishery Bulletin-National Oceanic and Atmospheric Administration, 99 (4), 679-684, 2001 Rasmussen, R. S. et al., Comprehensive Reviews in Food Science and Food Safety, 7 (3), 280-295, 2008 Waugh, J., Bioessays, 29 (2), 188-197, 2007 Kubista, M. et.al., Molecular Aspects of Medicine, 27 (2-3), 95-125, 2006 Zhang, T. et al., Scientific Reports, 6, 28494, 2016 Gunson, R. N. et al., Journal of Clinical Virology, 43 (4), 372-375, 2008 Huang, Q. et al., PLoS One, 6 (4), e19206, 2011

It is an object of the present invention to provide a method for the identification of squid species selected from the group consisting of domesticated squid ( Todarodes pacificus ), Sepia esculenta , Dosidicus gigas and Argentine short- tailed squid ( Illex argentinus ) To provide a set of DNA probes.

It is another object of the present invention to provide a method for determining the squid species by obtaining a melting temperature (Tm) value corresponding to each squid species using the double-labeled DNA probe set.

It is still another object of the present invention to provide the squid species discriminating kit comprising the double-labeled DNA probe set.

Another object of the present invention is to provide a double-labeled LNA (Locked Nucleic Acid) probe for simultaneous discrimination of domesticated domestic squid ( Todarodes pacificus ), imported American squid ( Dosidicus gigas ) and Argentine short-finned squid ( Illex argentinus species) .

It is still another object of the present invention to provide a method for simultaneously discriminating the three kinds of squid species by obtaining a melting temperature (Tm) value suitable for the three kinds of squid species using the double-labeled LNA probe.

It is still another object of the present invention to provide a kit for simultaneous discrimination of three kinds of squid species including the double-labeled LNA probe.

In order to achieve the above object, the present invention determines squid species selected from the group consisting of domestic squid (Todarodes pacificus), imported chamgap cuttlefish (Sepia esculenta), American giant squid (Dosidicus gigas) and Argentina short fin squid (Illex argentinus) A squid species discrimination method based on the fusion temperature (Tm) value for each squid species derived using the double-labeled DNA probe and the fluorescence melting curve analysis (FMCA) method, and And a kit for discriminating squid species comprising the double-labeled DNA probe set.

The present invention also relates to a double-labeled LNA (Locked Nucleic Acid) probe for simultaneous discrimination of domestic domesticated squid ( Todarodes pacificus ), Imported America Great Squid ( Dosidicus gigas ) and Argentine short fin squid ( Illex argentinus species) (Tm) value for the three kinds of squid species derived using the LNA probe and the fluorescence melting curve analysis (FMCA) method, and the method for simultaneous discrimination of the above three types of squid species, The present invention also provides a kit for simultaneous discrimination of three kinds of squid species including a probe.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention provides a set of squid species discriminating probes comprising a double-labeled DNA probe consisting of a nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6.

The term " probe " in the present invention is also referred to as a probe, and collectively refers to a substance that specifically detects a specific substance, site, state, and the like. "Dual-labeled DNA probes (dual-labeled deoxyribonucleic acid probe) " of the present invention domestic squid (Todarodes pacificus), imported chamgap cuttlefish (Sepia esculenta), American giant squid (Dosidicus gigas) and Argentina short fin squid (Illex argentinus ) Is a DNA probe for detecting or identifying a squid species gene selected from the group consisting of a 5 'end and a 3' end labeled with a quencher.

The squid species gene targeted by the probe is a gene group best suited for the genotype discrimination of squid, and is complementary to a nucleotide sequence of COI (Cytochrome c oxidase subunit Ⅰ) gene on the mitochondrial DNA.

Such fluorescent materials include, but are not limited to, 6-carboxyfluorescein, HEX (2 ', 4', 5 ', 7'-tetrachloro-6-carboxy-4,7dichlorofluorescein), fluorescein, Fluorescein chlorotriazinyl, rhodamine green, rhodamine red, TRITC, fluorescein isothiocyanate (FITC), Oregon green, (Alexa Fluor), JOE (6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein), ROX (6-carboxy-X- rhodamine), Texas Red, , 7'-dichloro-6-carboxy-4,7-dichlorofluorescein, TAMRA (N, N, N ', N'tetramethyl-6-carboxyrhodamine), cyanine dyes and thiadicarbocyanine. A dye, and a dye.

The quencher may include, but is not limited to, Dabcyl, TAMRA, Eclipse, Deep Dark Quencher, QSY, Blackberry Quencher, One or more selected from the group consisting of Black Hole Quencher, Iowa black FQ, Iowa black RQ and IRDye QC-1.

In the present invention, Domarid pacificus , Sepia esculenta , Dosidicus gigas and Argentinian short- tailed squid ( Illex argentinus ) species to be discriminated by the squid species are sea squirts , It can contain foods that are not seasoned, such as delicacy, powdered soup, etc., whose shape is unknown.

In another aspect, the present invention provides a squid species determination method comprising the following steps:

(a) isolating DNA from a squid sample selected from the group consisting of domesticated squid ( Todarodes pacificus ), Sepia esculenta , Dosidicus gigas and Argentine shortfin squid ( Illex argentinus );

(b) The DNA template isolated in step (a) is mixed with a primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4 and a double-labeled DNA probe consisting of the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6 Performing asymmetric PCR (Asymmetric PCR);

(c) melting the amplification product amplified by the asymmetric PCR of step (b) while changing the temperature to obtain a melting curve; And

(d) identifying the squid species by confirming the melting temperature of the melting curve obtained in the step (c).

Specifically, in the step (a), DNA is isolated from squid samples by extracting DNA from various tissues or various workpieces of the sample by various methods, and analyzing the extracted DNA to identify and identify the species Therefore, there is no particular limitation on which part of the sample the DNA is extracted or by which method it is extracted. In addition, the DNA extracted as described above can be amplified by PCR, which is intended to increase the number of test specimens. The method of obtaining the amplification product is not particularly limited. It is obvious that other DNA extraction methods and amplification methods known to those skilled in the art of the present invention belong to the scope of the present invention.

The primer set used in the step (b) is a DNA base sequence part commonly used in domestic squid squid, imported squid squid, American squid squid and Argentine short squid squid, and has a forward primer of SEQ ID NO: 3 and a forward primer of SEQ ID NO: Of reverse primers. The DNA base sequence portion in the forward and reverse primers is designed to include a species specific portion. This species-specific base sequence portion is applied as a complementary part to the double-labeled DNA probe.

Meanwhile, the double-labeled DNA probe used in the step (b) is as described above. Fluorescence Melting Curve Analysis (FMCA) technique is applied to the double-labeled DNA probe luminescence principle, Asymmetric PCR (asymmetric PCR) techniques can be used to increase the amount of single stranded PCR products that hybridize with DNA probes.

The term " Asymmetric PCR " in the present invention is a technique for producing DNA of a single strand, which can amplify more copies of the target single-stranded DNA hybridized with the double-labeled DNA probe . The double-labeled DNA probe binds to the fluorescence signal when a complementary base sequence exists in the mass-produced single stranded DNA.

The term " Fluorescence Melting Curve Analysis (FMCA) " in the present invention refers to monitoring and analyzing fluorescence intensity by gradually increasing the temperature after asymmetric PCR, and the inconsistency between the double-labeled DNA probe and the target single- The greater the number of mismatched base pairs, the more rapid the fluorescence intensity decreases at lower temperatures.

The temperature at which the fluorescence intensity sharply decreases in the melting curve analysis is referred to as the melting temperature (Tm), and Tm is a temperature at which the temperature of the reaction solution containing double-stranded DNA is gradually increased The temperature at which the double stranded structure suddenly begins to collapse and eventually breaks up into a single strand with a random structure means a temperature at which the degree of partial degeneration reaches an average of 50%.

In one embodiment of the present invention, when performing asymmetric PCR to produce a single strand to be annealed by a double-labeled DNA probe, the forward primer can be introduced 49 to 51 times, or 50 times more than the reverse primer . In one embodiment of the present invention, the asymmetric PCR can perform about 50 cycles because the amplification efficiency may be lower than the general PCR. This increases the number of single strands produced from the forward primer and a complementary base sequence portion can be formed in which double-labeled DNA probes can be annealed.

When the asymmetric PCR process is completed, a fluorescence melting curve analysis (FMCA) process can be performed using real-time PCR equipment.

In one embodiment of the invention, the FMCA was incubated at 4 ° C for about 10 minutes in the first step, in order to fully hybridize DNA with double-labeled DNA probes. Thereafter, the intensity of fluorescence was measured while increasing the temperature from 4 ° C to 70 ° C. The fluorescence intensity measurement was carried out after maintaining the temperature rise of 1 DEG C for 5 seconds so as to provide a hybridization time in which the double-labeled DNA probe hybridized.

When the FMCA process is terminated, the step of analyzing the fluorescence value with respect to temperature may be included. The fluorescence value can be analyzed by taking a differential value and then processing the values by a negative treatment, in order to analyze the melting temperature (Tm) value. The step of newly drawing the melting curve graph by setting the value obtained by subjecting the differential and negative values to the ordinate axis and the temperature value to the abscissa axis. Then, by analyzing the melting temperature (Tm) value, it is possible to identify what is the discriminated squid species. Since the species discrimination is performed by looking at the pattern of the melting temperature (Tm) value, This is possible.

In one embodiment of the present invention, a FMCA melting peak graph was plotted using a double-labeled DNA probe with two FAM fluorescence or HEX fluorescence to analyze the melting temperature (Tm) value of squid species.

Specifically, when the melting temperature derived from the probe having FAM fluorescence (SEQ ID NO: 5) is 52 캜 to 54 캜 or 53 캜, Todarodes pacificus ; When the melting temperature is 18 ° C to 20 ° C or 19 ° C, Sepia esculenta ; When the melting temperature is 27 캜 to 29 캜 or 28 캜, the American squid ( Dosidicus gigas ); And an Argentine short-finned squid ( Illex argentinus ) when the melting temperature was 40 캜 to 42 캜 or 41 캜. Also, when the melting temperature derived from the probe having HEX fluorescence (SEQ ID NO: 6) is 55 ° C to 57 ° C, or 56 ° C, Tetrarodes pacificus ; When the melting temperature is 48 캜 to 50 캜 or 49 캜, Sepia esculenta ; When the melting temperature is 41 캜 to 43 캜 or 42 캜, the American squid ( Dosidicus gigas ); And an Argentine short-finned squid ( Illex argentinus ) when the melting temperature was 43 ° C to 45 ° C or 44 ° C. The combination of the melting temperatures derived from the two double-labeled DNA probes can be used to clearly distinguish the four species of squid (Figs. 4 to 9).

In the squid species discrimination method according to an embodiment of the present invention, the PCR amplification product from the domestic Todarodes pacificus sample is 100% complementary to the double-labeled DNA probe having the FAM fluorescence without mismatching, The PCR amplification product from the above-mentioned Sepia esculenta and Dosidicus gigas samples has a complementary binding with a double-labeled DNA probe having the FAM fluorescence with mismatching of three bases, The PCR amplification product from Argentine Illex argentinus sample is characterized by a complementary binding with a double-labeled DNA probe with FAM fluorescence with mismatch of the two bases. Also, the PCR amplification product from the domestic Todarodes pacificus sample was 100% complementary to the double-labeled DNA probe having the HEX fluorescence without mismatching, and the PCR product from the Sepia esculenta sample The PCR amplification product has a complementary bond with a single base mismatch with a double-labeled DNA probe having the HEX fluorescence, and the PCR from the sample of the American Squid ( Dosidicus gigas ) and the Argentine Shortfin Squid ( Illex argentinus ) The amplification product is characterized in that it has a complementary binding with a double-labeled DNA probe having the HEX fluorescence with mismatching of two bases, respectively. On the other hand, the PCR amplification products from squid species samples complementarily binding with the double-labeled DNA probes having FAM fluorescence or HEX fluorescence, specifically, COI gene sites of mitochondrial DNA of squid species are amplified to a size of 226 bp (Figs. 2 and 3).

In another embodiment, the present invention relates to a method of treating a squid sample selected from the group consisting of domestic domesticated squid ( Todarodes pacificus ), Sepia esculenta (imported squid), Dosidicus gigas (American squid) and Illex argentinus A primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4 for amplifying DNA, and a double-labeled DNA probe consisting of a nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6.

The squid species discrimination kit of the present invention is characterized by comprising a primer set (SEQ ID NO: 3 and SEQ ID NO: 4) for amplifying a DNA of a squid sample selected from the group consisting of domestic squid, imported squid, American squid and Argentine short squid , And double-labeled DNA probes consisting of the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6, DNA polymerase, dNTPs, and PCR buffer solution. By applying the kit to the squid species discrimination method of the present invention, it is possible to easily and accurately discriminate the four kinds of squid species in the sample to be analyzed.

In yet another embodiment, the present invention provides a recombinant vector comprising the nucleotide sequence of SEQ ID NO: 9, wherein the 5 'end is labeled with a fluorescent substance, the 3' end is labeled with a quencher, And a double-labeled LNA probe in which the ninth base is modified to LNA.

The term " LNA (Locked Nucleic Acid) " in the present invention refers to a structure in which an oxygen atom (oxygen atom) and a carbon atom (carbon atom) Refers to one modified nucleic acid structure formed by a methylene linkage.

The "dual-labeled locked nucleic acid probe" of the present invention detects the genes of domesticated domestic squid ( Todarodes pacificus ), imported American squid ( Dosidicus gigas ) and Argentine short squid ( Illex argentinus ) species Or a LNA probe for simultaneous discrimination, wherein the 6th base and the 9th base from the 5'terminal are modified to LNA, and the 5'end is a fluorescent substance and the 3'end is labeled with a quencher.

The squid species gene targeted by the probe is a gene group best suited for the genotype discrimination of squid, and is complementary to a nucleotide sequence of COI (Cytochrome c oxidase subunit Ⅰ) gene on the mitochondrial DNA.

Such fluorescent materials include, but are not limited to, 6-carboxyfluorescein, HEX (2 ', 4', 5 ', 7'-tetrachloro-6-carboxy-4,7dichlorofluorescein), fluorescein, Fluorescein chlorotriazinyl, rhodamine green, rhodamine red, TRITC, fluorescein isothiocyanate (FITC), Oregon green, (Alexa Fluor), JOE (6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein), ROX (6-carboxy-X- rhodamine), Texas Red, , 7'-dichloro-6-carboxy-4,7-dichlorofluorescein, TAMRA (N, N, N ', N'tetramethyl-6-carboxyrhodamine), cyanine dyes and thiadicarbocyanine. A dye, and a dye.

The quencher may include, but is not limited to, Dabcyl, TAMRA, Eclipse, Deep Dark Quencher, QSY, Blackberry Quencher, One or more selected from the group consisting of Black Hole Quencher, Iowa black FQ, Iowa black RQ and IRDye QC-1.

In the present invention, the domesticated domestic squid ( Todarodes pacificus ), the imported American Great Squid ( Dosidicus gigas ) and the Argentine short-finned squid ( Illex argentinus ) are squid fish caught in the sea or seasoned squid And may include foods whose shape traits are unknown (for example, delicacy, powdered soup, etc.).

In another aspect, the present invention provides a method for simultaneous discrimination between domestic squid squid, imported squid squid, and Argentine short squid squid species comprising the steps of:

(a) isolating DNA from a squid sample selected from the group consisting of domestic domestic squid ( Todarodes pacificus ), imported American squid ( Dosidicus gigas ) and Argentine short-finned squid ( Illex argentinus );

(b) The DNA template isolated in step (a) is mixed with a primer set consisting of SEQ ID NO: 7 and SEQ ID NO: 8 and a double-labeled LNA probe consisting of the nucleotide sequence of SEQ ID NO: PCR);

(c) melting the amplification product amplified by the asymmetric PCR of step (b) while changing the temperature to obtain a melting curve; And

(d) identifying the squid species by confirming the melting temperature of the melting curve obtained in the step (c).

Specifically, in the step (a), DNA is isolated from squid samples by extracting DNA from various tissues or various workpieces of the sample by various methods, and analyzing the extracted DNA to identify and identify the species Therefore, there is no particular limitation on which part of the sample the DNA is extracted or by which method it is extracted. In addition, the DNA extracted as described above can be amplified by PCR, which is intended to increase the number of test specimens. The method of obtaining the amplification product is not particularly limited. It is obvious that other DNA extraction methods and amplification methods known to those skilled in the art of the present invention belong to the scope of the present invention.

The primer set used in the step (b) is a DNA base sequence part commonly used in domestic squid, imported American squid, and Argentine shortfin squid, and has a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: . ≪ / RTI > The DNA base sequence portion in the forward and reverse primers is designed to include a species specific portion. This species-specific base sequence portion is applied as a complementary part to a double-labeled LNA probe.

Meanwhile, the double-labeled LNA probe used in the step (b) is as described above. In order to apply the fluorescence melting curve analysis (FMCA) technique to the double-labeled LNA probe luminescence principle, asymmetric PCR Asymmetric PCR) technology can be used.

The term " Asymmetric PCR " in the present invention is a technique for producing DNA of a single strand, which can amplify more copies of the target single-stranded DNA hybridized with the double-labeled LNA probe . The double-labeled LNA probe binds to the fluorescence signal when a complementary base sequence exists in the mass-produced single stranded DNA.

The term " Fluorescence Melting Curve Analysis (FMCA) " in the present invention refers to monitoring and analyzing fluorescence intensity by gradually increasing the temperature after asymmetric PCR, and the inconsistency between the double-labeled LNA probe and the target single- The greater the number of mismatched base pairs, the more rapid the fluorescence intensity decreases at lower temperatures.

The temperature at which the fluorescence intensity sharply decreases in the melting curve analysis is referred to as the melting temperature (Tm), and Tm is a temperature at which the temperature of the reaction solution containing double-stranded DNA is gradually increased The temperature at which the double stranded structure suddenly begins to collapse and eventually breaks up into a single strand with a random structure means a temperature at which the degree of partial degeneration reaches an average of 50%.

In one embodiment of the present invention, when performing asymmetric PCR to produce a single strand to be annealed by a double-labeled LNA probe, the forward primer can be introduced 49 to 51 times, or 50 times more than the reverse primer . In one embodiment of the present invention, the asymmetric PCR can perform about 50 cycles because the amplification efficiency may be lower than the general PCR. This increases the number of single strands produced from the forward primer, and a complementary base sequence portion can be formed in which double-labeled LNA probes can be annealed.

When the asymmetric PCR process is completed, a fluorescence melting curve analysis (FMCA) process can be performed using real-time PCR equipment.

In one embodiment of the invention, the FMCA was incubated at 4 ° C for about 10 minutes in the first step, in order to completely adhere or hybridize DNA and double-labeled LNA probes. Thereafter, the intensity of fluorescence was measured while increasing the temperature from 4 ° C to 70 ° C. The fluorescence intensity measurement was carried out after the double-labeled LNA probe was maintained at a temperature rise of 1 DEG C for 5 seconds to provide a hybridization time margin.

When the FMCA process is terminated, the step of analyzing the fluorescence value with respect to temperature may be included. The fluorescence value can be analyzed by taking a differential value and then processing the values by a negative treatment, in order to analyze the melting temperature (Tm) value. The step of newly drawing the melting curve graph by setting the value obtained by subjecting the differential and negative values to the ordinate axis and the temperature value to the abscissa axis. Then, by analyzing the melting temperature (Tm) value, it is possible to identify what is the discriminated squid species. Since the species discrimination is performed by looking at the pattern of the melting temperature (Tm) value, This is possible.

In one embodiment of the present invention, the FMCA melting peak graph was plotted using one double-labeled LNA probe to analyze the melting temperature (Tm) value of squid species. Specifically, when the melting temperature is 47 캜 to 49 캜 or 48 캜, Todarodes pacificus ; When the melting temperature is 27 캜 to 29 캜 or 28 캜, the American squid ( Dosidicus gigas ); And when the melting temperature was 41 캜 to 43 캜 or 42 캜, it could be identified as Argentine short-tailed squid ( Illex argentinus ) (Fig. 12).

In the simultaneous discrimination method for domestic squid, imported squid, and squid species of Argentine short fin in an embodiment of the present invention, the PCR amplification product from the domestic Todarodes pacificus sample is the double-labeled LNA probe and mismatching And wherein the PCR amplification product from the sample of the Imported American Squid ( Dosidicus gigas ) has a complementary binding with the double-labeled LNA probe with a mismatch of the two bases, and the Argentine short fin The PCR amplification product from the squid ( Illex argentinus ) sample is characterized by the complementary binding with the double-labeled LNA probe with one base mismatch. Further, the PCR amplification product from the squid species sample complementarily binding to the double-labeled LNA probe, specifically, the COI gene region of the mitochondrial DNA of the squid species is amplified to a size of 227 bp (FIGS. 10 and 12) 11).

In the simultaneous discrimination method for domestic squid, imported North American squid and Argentine short fin squid species according to an embodiment of the present invention, the double-labeled LNA probe in which the 6th base and the 9th base from the 5 'end are transformed into LNA, The mismatched base and its surrounding base are transformed into LNA, whereby the difference in the melting temperature (Tm) value can be significantly increased.

In another embodiment, the present invention provides a method for amplifying the DNA of a squid sample selected from the group consisting of domestic domestic squid ( Todarodes pacificus ), imported American squid ( Dosidicus gigas ) and Argentine short fin squid ( Illex argentinus ) 7 and SEQ ID NO: 8, and a double-labeled LNA probe consisting of the nucleotide sequence of SEQ ID NO: 9, and a kit for simultaneous discrimination of argentina short fin squid species from imported Latin America squid.

The kit for simultaneous discrimination of domestic squid, imported squid, and short squid species of Argentinean squid according to the present invention comprises a sequence for amplifying the DNA of a squid sample selected from the group consisting of domestic squid, imported squid, and Argentine short squid A primer set consisting of SEQ ID NO: 7 and SEQ ID NO: 8, and a double-labeled LNA probe consisting of the nucleotide sequence of SEQ ID NO: 9, DNA polymerase, dNTPs, and PCR buffer solution. By applying the kit to the squid species determination method of the present invention, it is possible to simultaneously and easily determine the three kinds of squid species in the sample to be analyzed.

It is possible to overcome the disadvantages of the conventional conventional squid species discrimination method in which it is difficult to distinguish the DNA base sequence of 1-2 bp effectively using the double-labeled probe and the fluorescence melting curve analysis (FMCA) according to the present invention, ( Todarodes pacificus ), Sepia esculenta (imported squid), and the American squid ( Dosidicus ), which are difficult to discriminate in the processed state based on the melting temperature (Tm) value obtained by the double- gigas , and Argentine short-tailed squid ( Illex argentinus ). In addition, using only one double-labeled LNA probe according to the present invention, it is possible to simultaneously discriminate between the two species of Todarodes pacificus , Dosidicus gigas , and Illex argentinus , However, there is an economic advantage that the inspection cost can be greatly reduced.

1 is a schematic diagram schematically showing the principle of deriving a melting temperature (Tm) value using a double-labeled probe of the present invention and fluorescence melting curve analysis (FMCA).
Figure 2 shows the PCR amplification of the target sequence for four species of cuttlefish ( Todarodes pacificus ), Sepia esculenta , Dosidicus gigas , and Argentine shortfin squid ( Illex argentinus ) according to one embodiment of the present invention. The results are shown in FIG. Here, (-) Con. Is a control group in which the template DNA of squid species is not added.
FIG. 3 is a graph showing the results of two double-labeled probes in four species of Todarodes pacificus , Sepia esculenta , Dosidicus gigas and Argentine short- tailed squid ( Illex argentinus ) according to one embodiment of the present invention. And mismatched base number and position between the PCR amplification product and the PCR amplification product.
FIG. 4 is a graph showing the results of two double-labeled probes and Todarodes pacificus , Sepia esculenta , and Dosidicus gigas , which were derived using fluorescence melting curve analysis (FMCA) according to an embodiment of the present invention. ) And the melting temperature (Tm) of four Argentine short squid ( Illex argentinus ) species. Here, the blue line indicates the melting peak in the FAM fluorescence channel, and the green line indicates the melting peak in the HEX fluorescence channel.
FIG. 5 is a graph showing the effect of the two double-labeled probes according to one embodiment of the present invention on the FAM fluorescence channel for a control without added template DNA of squid species derived using Fusion Molecular Curve Analysis (FMCA) A melting peak graph and a melting peak graph in a HEX fluorescence channel.
FIG. 6 is a graph showing a melting peak in a FAM fluorescence channel for two squid-labeled probes and Todarodes pacificus derived using fluorescence microscopy (FMCA) according to an embodiment of the present invention. And a melting peak graph in a HEX fluorescence channel.
FIG. 7 is a graph showing the melting peak of a FAM fluorescent channel on a Sepia esculenta derived from two double-labeled probes and FMCA according to an embodiment of the present invention. And a melting peak graph in a HEX fluorescence channel.
Figure 8 shows a melting peak in the FAM fluorescence channel for two dominantly labeled probes and Dosidicus gigas derived using Fusion Molecular Curve Analysis (FMCA) according to one embodiment of the present invention. Graphs and melting peak graphs in HEX fluorescence channels.
Figure 9 is a graph showing the effect of melting peak (FAM) on the FAM fluorescence channel for Argentine shortfin squid ( Illex argentinus ) derived using two double-labeled probes and FMSA according to one embodiment of the present invention. ) Graph and a melting peak graph in a HEX fluorescence channel.
10 is a graph showing the results of a comparison between one double-labeled LNA probe and PCR amplification product in three species of Todarodes pacificus , Dosidicus gigas , and Argentine shortfin squid ( Illex argentinus ) according to one embodiment of the present invention. A schematic representation of relative mismatch patterns, i.e., mismatched number and locations of mismatches.
Fig. 11 is a graph showing the results of PCR amplification of target sequences for three species of Todarodes pacificus , Dosidicus gigas and Argentine short- tailed squid ( Illex argentinus ) according to one embodiment of the present invention. Fig. Here, (-) Con. Is a control group in which the template DNA of squid species is not added.
Figure 12 is a plot of one double-labeled LNA probe and Todarodes pacificus , Dosidicus gigas , and Argentine shortfin squid, derived using Fusion Molecular Curve Analysis (FMCA), according to one embodiment of the present invention. (Tm) for three species of Illex argentinus . Herein, the blue line represents the melting peak in the FAM fluorescence channel, and the NTC (no template control) is a control group in which no template DNA of squid species is added.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1: Preparation of experimental materials and experimental methods

Example  1-1: Preparation of squid sample

Four species of labeled and morphologically confirmed squid species, specifically, Todarodes pacificus , Sepia esculenta , the American Squid ( Dosidicus gigas ) and Argentine shortfin squid ( Illex argentinus ) Samples were transferred to the laboratory and raw fillets were cut and frozen at -20 ° C. After freezing, the squid samples were prepared for subsequent experiments.

Example  1-2: Genomic DNA ( genomic  DNA) extraction and quantification

Genomic DNA was extracted from each sample prepared in Example 1-1 using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's manual. After genomic DNA extraction, the concentration and purity of genomic DNA extracted using a NanoDrop-2000 UV-vis Spectrophotometer (Thermo Fisher Scientific, Waltham, Mass., USA) were measured. The DNA samples were then diluted with distilled water (DW) to a final concentration suitable for the experiment.

Example  1-3: For Squid Species Universal primer  Production and Sequence Identification

The COI gene of mitochondrial DNA was selected as the target amplification region. COI sequence information for the four squid species prepared in Example 1-1 was obtained from the National Center for Biotechnology Information (NCBI) GenBank database (http://www.ncbi.nlm.nih.gov/genbank) The accession numbers of the COI genes of Todarodes pacificus , Sepia esculenta , Dosidicus gigas and Argentine shortfin squid ( Illex argentinus ) were NC_006354.1, NC_009690.1, NC_009734 .1 and NC_026908.1. Then, using ClustalW (http://www.mbio.ncsu.edu/bioedit/bioedit.html) provided by BioEdit, a universal primer pair for the four squid species for sequencing The sequence information was sorted and analyzed. The universal primer pairs designed for the four squid species are as shown in Table 1 below and commonly amplify a part of COI (Cytochrome c oxidase subunit I) among the mitochondrial DNAs of the four squid species.

primer Base sequence SEQ ID NO: Forward primer
(forward primer) 5'-GCGATGACTATTTTCTACAAACCAT-3 '(25 mer) One Reverse primer
(reverse primer) 5'-TGTGAAATAATACCAAAGGCAGGTA-3 '(25 mer) 2

The primer pair was synthesized by Bioneer (Daejeon, Korea) and PCR was performed by conventional PCR. The well-amplified PCR products were then sequenced using a 3730x1 DNA analyzer (Applied Biosystems, Foster City, CA, USA) to the Marcrogen Sequencing Service (Macrogen, Seoul, Korea). The sequenced data was entered into the identification request engine of the BOLD (Barcode Of Life Database system; http://www.barcodeinglife.org) for species identification. After confirming the similarity of barcode index numbers (BINs) (Ratnasingham & Hebert, 2013) listed in Table 2 below, 22 additional squid ( Todarodes pacificus ) samples, 5 squid esculenta A total of 43 samples were selected, including 8 samples of the American Squid ( Dosidicus gigas ) and eight Argentine shortfin squid ( Illex argentinus ).

Squid species Bar code index number
(barcode index numbers; BINs) Squid
( Todarodes pacificus ) AAD5750 Cuttlefish squid
( Sepia esculenta ) AAE9622 Great American squid
( Dosidicus gigas ) AAB8140 Argentine short fin squid
( Illex argentinus ) ABA9189

Example  1-4: for discrimination of squid species For FMCA primer  And DNA Probe  design

The sequences of the samples selected in Examples 1-3 were sorted and analyzed using ClustalW. Based on the results, pairs of primers for fluorescence melting curve analysis (FMCA) and double-labeled DNA probes dual-labeled deoxyribonucleic acid probe). The designed primer pairs were named S-FMCA_F and S-FMCA_R, respectively, and the DNA probes were named FAMsig_P (first probe) and HEXsig_P (second probe), respectively (Table 3). The primer pairs were synthesized by Bioneer, and the two double-labeled DNA probes were synthesized with reference to Integrated DNA Technologies (IDT, Coralville, IA, USA). FAM-labeled probes were used to identify the sequences of Sepia esculenta, S. esculenta , Dosidicus gigas, D. gigas , and Argentine short squid ( Illex argentinus, I. argentinus ) It has a mismatch of 3 bp, 3 bp and 2 bp, respectively, and is designed to be perfectly matched to the sequence of the cuttlefish ( Todarodes pacificus, T. pacificus ). In addition, HEX-labeled probes were identified as 1 bp, 2 bp and 2 bp for the sequences of S. esculenta , D. gigas and Argentine I. argentinus , respectively It has mismatches and is designed to be perfectly matched to the sequence of T. pacificus . The two probes were labeled with an Iowa Black ^® FQ quencher regardless of the fluorophores used. The mismatch pattern for the target strand DNA and the dual-labeled DNA probe is shown in FIG.

A primer pair sequence for fluorescence melting curve analysis (FMCA) for discrimination of squid species, and a DNA probe sequence in which a fluorescent substance capable of complementarily binding with the amplified nucleic acid molecule and a small mineral substance are labeled designation Base sequence SEQ ID NO: S-FMCA_F
(forward primer) 5'-GCG ATG ACT ATT TTC TAC AAA CCA T-3 '(25 mer) 3 S-FMCA_R
(reverse primer) 5'-ACC AAA TCC TCC AAT TAT AAT AGG-3 '(24mer) 4 FAMsig_P
(First probe) 5'-6FAM-CAG ATA CCA AAG ATA AAA TA-Iowa Black ^® FQ-3 ' 5 HEXsig_P
(Second probe) 5'-HEX-ATT ATA ATG AAT CCG TGC GC-Iowa Black ^® FQ-3 ' 6

Example  1-5: PCR  analysis

Prior to fluorescence melting curve analysis (FMCA), PCR was first performed to confirm the presence or absence of target DNA amplification. The PCR contained 10 μl of Premix Ex Taq (TaKaRa, Shiga, Japan), 10 pmol of the forward primer S-FMCA_F designed in Example 1-4, 10 pmol of the reverse primer S-FMCA_R and 2 ng template DNA The reaction was carried out in a final volume of 20 [mu] l volume, at which time the volume was adjusted using distilled water (DW). On the other hand, the PCR was pre-denaturated at 95 ° C for 2 minutes, followed by denaturation at 95 ° C for 5 seconds and annealing and extension at 35 ° C for 30 seconds at 58 ° C (cycle).

Example  1-6: Asymmetry PCR  Analysis and fluorescence melting curve analysis

Asymmetric PCR was performed for further amplification of target DNA strands hybridized with complementary dual-labeled DNA probes.

Asymmetric PCR can amplify more copies of the target single-stranded DNA to be hybridized with two dual-labeled DNA probes. The double-labeled DNA probe is labeled with a fluorophore at the 5 'end and a quencher at the 3' end (Marras, Kramer, & Tyagi, 2002). In the hybridization status, the fluorescent material is an absorption energy from a light source and can strongly emit its wavelength to return to the ground state. As temperature increases, fluorescence signals in the denatured state are quenched by quenchers due to the proximity between the fluorophore and the quencher, called the quenching effect (Didenko, 2001). The greater the number of mismatched base pairs between the double-labeled DNA probe and the target strand DNA, the lower the melting temperature of the probe due to the lower number of hydrogen bonds (Tsuboi, 1964).

Asymmetric PCR and fluorescence melting curve analysis (FMCA) were performed using 10 μl Premix Ex Taq (TaKaRa, Shiga, Japan), 50 pmol of the forward primer S-FMCA_F designed in Example 1-4, 1 pmol of the reverse primer S-FMCA_R, 0.5 pmol of FAMsig_P (first probe) designed in Example 1-4, 0.5 pmol of HEXsig_P (second probe) designed in Example 1-4 as a HEX probe, 2 ng template DNA (CFX96 real-time PCR instrument; Bio-Rad, Hercules, Calif., USA) with a final volume of 20 [mu] l. At this time, the volume was adjusted using distilled water (DW). PCR was performed by pre-denaturation at 95 ° C for 2 minutes followed by denaturation at 95 ° C for 5 seconds and annealing and extension at 50 ° C for 30 seconds at 58 ° C. ). After the amplification phase, the samples were denatured at 95 ° C for 30 seconds and then cooled for 10 minutes at 4 ° C for hybridization between the target strand and the probe. Fluorescence melting curve analysis (FMCA) was then performed by increasing the temperature from 4 ° C to 70 ° C in order to detect fluorescent intensity. At every 1 ° C, fluorescence intensity was detected for 5 seconds at a specific wavelength emitted by FAM and HEX.

Asymmetric PCR composition for FMCA after confirmation of target sequence amplification Ex Taq premix 10 μl Forward primer 50 pmole Reverse primer 1 pmole FAMsig_P (first probe) 0.5 pmole HEXsig_P (second probe) 0.5 pmole Samples (Squid ' s genomic DNA) 2 ng Total volume 20 μl

After asymmetric PCR, conditions for FMCA process Temperature time Cycles Pre-denaturation < RTI ID = 0.0 > 95 ℃ 2 minutes Denaturation 95 ℃ 5 seconds 50 times Annealing and extension 58 ℃ 30 seconds Hybridization and incubation < RTI ID = 0.0 > 4 ℃ 10 minutes * Melting curve: Fluorescent probe every 1 ° C from 4 ° C to 70 ° C

Example  2: Double labeled DNA Probe  And squid species discrimination using fluorescence melting curve analysis

Example  2-1: PCR  analysis

The primer pair designed in Example 1-4 successfully amplified a part of the COI gene of two double-labeled DNA probes and hybridized mitochondrial DNA. The length of the amplicon was 226 bp regardless of the template DNA of the other squid species (Fig. 2). PCR amplification products were separated on 1.5% agarose gel and visualized with EtBr (ethidium bromide) staining.

Example  2-2: Fluorescence Melting Curve Analysis (FMCA)

As a result of the fluorescence melting curve analysis, it was confirmed that double-labeled DNA probes of the hybridized Example 1-4 can accurately discriminate squid species as shown in FIG. As the temperature increased by 1 ° C, the fluorescence intensity emitted from the fluorescent material was detected. Melt peak graphs showed a negative regression of Relative Fluorescence Units (RFU) at each temperature.

In the FAM channel, the melting peak graph shows the fusing temperature (Tg) for T. pacificus , S. esculenta , D. gigas and Argentine short squid ( argentinus ) Tm) values were 53 캜, 19 캜, 28 캜 and 41 캜, respectively. The melting temperature (Tm) values for the squid ( T. pacificus ), S. esculenta , D. gigas and Argentinian argentinus in the HEX channel were 56 C, 49 C, 42 C, and 44 C, respectively.

On the other hand, a no template control (NTC) without a template confirmed that there was no fusion peak in both FAM and HEX fluorescence channels, as shown in Fig. 5 below.

Thus, using a combination of the two fusion temperatures with each fluorescence channel, we found that T. pacificus , S. esculenta , D. gigas , and Argentine short squid ( I. argentinus) ), It was found that the four kinds of squid species can be clearly discriminated.

The melting temperature of each squid species, The first probe (FAM fluorescence) Second probe (HEX fluorescence) Squid
( Todarodes pacificus ) 53 ℃ 56 ℃ Cuttlefish squid
( Sepia esculenta ) 19 ℃ 49 ℃ Great American squid
( Dosidicus gigas ) 28 ℃ 42 ° C Argentine short fin squid
( Illex argentinus ) 41 C 44 ° C

In a recent study, a DNA-based anlaytical method such as restriction fragment length polymorphism (RFLP) (McKeown, Robin, & Shaw, 2015) and probe-based real- Were applied to species-level identification. RFLP requires much time for technically complex conditions, labor-intensive and restriction enzyme digestion and gel electrophoresis. In addition, probe-based real-time PCR is sometimes associated with false-positive results when there is a sequence difference of 1 bp or 2 bp. Moreover, because of the different annealing temperatures of species-specific probes, the multiplexing competency is poor (Kutyavin et al. , 2000).

Therefore, it can be seen from the above results that the probe-based fluorescence melting curve analysis (FMCA) can be applied to identification of squid species. That is, it was confirmed that squid species can be identified by confirming the melting temperature (Tm) value using the above two double-labeled DNA probes.

Example  3: Double labeled LNA Probe  And simultaneous discrimination of squid species using fluorescence melting curve analysis

Example  3-1: For simultaneous discrimination of squid species For FMCA primer  And Locked Nucleic Acid (LNA) Probe  design

The sequences of the samples selected in Examples 1-3 were sorted and analyzed using ClustalW. Based on the results, a pair of primers for fluorescence melting curve analysis (FMCA) for the simultaneous discrimination of squid species and a pair of LNA modified double-labeled probes (locked nucleic acid-modified dual-labeled probe). The designed primer pairs were named S'L-FMCA_F and S'L-FMCA_R, respectively, and the LNA probe was named LNA-FAMsig_P (third probe) (Table 7). The primer pairs were synthesized by Bioneer, and the double-labeled LNA probes were synthesized with reference to Integrated DNA Technologies (IDT, Coralville, IA, USA). FAM-labeled probes have mismatches of 2 bp and 1 bp for the sequences of the Americas Great Squid ( Dosidicus gigas, D. gigas ) and the Argentine Shortfin Squid ( Illex argentinus, I. argentinus ) , And the squid ( Todarodes pacificus, T. pacificus ). The probe was labeled with an Iowa Black ^® FQ quencher regardless of the fluorophores used. The mismatch pattern for the target strand DNA and the double-labeled LNA probe is shown in FIG.

A primer pair sequence for fluorescence melting curve analysis (FMCA) for simultaneous discrimination of squid species, and a fluorescent substance capable of complementarily binding with the amplified nucleic acid molecule and a LNA probe sequence designation Base sequence SEQ ID NO: S 'L-FMCA_F
(forward primer) 5'-ATG CGA TGA CTA TTT TCT ACA AAC C-3 '(25 mer) 7 S 'L-FMCA_R
(reverse primer) 5'-CCA AAT CCT CCA ATT ATA ATA GGT ATA AC-3 '(29mer) 8 LNA-FAMsig_P
(Third probe) 5'-6FAM-ATA CC + A AA + G ATA AAA TA-Iowa Black ^® FQ-3 ' 9

* Means a nucleotide modified by (+) LNA, in which the 6th base and the 9th base from the 5 'end are transformed into LNA.

Example  3-2: PCR  analysis

Prior to fluorescence melting curve analysis (FMCA), PCR was first performed to confirm the presence or absence of target DNA amplification. 10 μl of Premix Ex Taq (TaKaRa, Shiga, Japan), 10 pmol of the forward primer S'L-FMCA_F designed in Example 3-1, 10 pmol of the reverse primer S'L-FMCA_R, 2 ng of template ) DNA was subjected to reaction at a final volume of 20 [mu] l, at which time the volume was adjusted using distilled water (DW). On the other hand, the PCR was pre-denaturated at 95 ° C for 2 minutes, followed by denaturation at 95 ° C for 5 seconds and annealing and extension at 35 ° C for 30 seconds at 58 ° C (cycle).

The primer pair designed in Example 3-1 successfully amplified a part of the COI gene of the mitochondrial DNA hybridized with the double-labeled LNA probe. The length of the amplicon was 227 bp, regardless of the template DNA of the other squid species (Fig. 11). PCR amplification products were separated on 1.5% agarose gel and visualized with EtBr (ethidium bromide) staining.

Example  3-3: Asymmetry PCR  Analysis and fluorescence melting curve analysis

Asymmetric PCR was performed for further amplification of the target DNA strand hybridized with a complementary LNA modified dual-labeled probe.

Asymmetric PCR can amplify more copies of target single-stranded DNA hybridized with double-labeled LNA probes. The double-labeled LNA probe is labeled with a fluorophore at the 5 'end and a quencher at the 3' end (Marras, Kramer, & Tyagi, 2002). On the other hand, the 6 < th > base and the 9 < th > base from the 5 '

LNA (Locked Nucleic Acid) is based on the structural form of RNA (Ribose Nucleic Acid), which is a methylene linkage between the oxygen atom (Oxygen atom) and the 4th carbon atom (Carbon atom) (Nucleic Acid) structure. The purpose of modification of DNA to LNA is that when the probe binds to a complementary sequence of a probe sequence composed of nucleic acid molecules, the binding force becomes relatively higher than that of DNA. The melting temperature (Tm) of the probe can be increased based on the increased binding force. In addition, when the positions of the LNA modifications are located in the mismatched sequence, the difference in melting temperature can be made larger depending on the inconsistency, and the difference in melting temperature can be more clearly shown.

In the hybridization status, the fluorescent material is an absorption energy from a light source and can strongly emit its wavelength to return to the ground state.

Asymmetric PCR and fluorescence melting curve analysis (FMCA) were performed using 10 μl of Premix Ex Taq (TaKaRa, Shiga, Japan), 50 pmol of the forward primer S'L-FMCA_F designed in Example 3-1 and the reverse primer S'L-FMCA_R 1 pmol, 0.5 pmol of the LNA-FAMsig_P (third probe) designed in Example 3-1, 2 ng template DNA as the FAM probe, and the final 20 μl volume of the CFX96 real-time PCR instrument CFX96 real-time PCR instrument; Bio-Rad, Hercules, Calif., USA). At this time, the volume was adjusted using distilled water (DW). PCR was performed by pre-denaturation at 95 ° C for 2 minutes followed by denaturation at 95 ° C for 5 seconds and annealing and extension at 50 ° C for 30 seconds at 61 ° C ). After the amplification phase, the samples were denatured at 95 ° C for 30 seconds and then cooled for 10 minutes at 4 ° C for hybridization between the target strand and the probe. Fluorescence melting curve analysis (FMCA) was then performed by increasing the temperature from 4 ° C to 70 ° C in order to detect fluorescent intensity. Every 1 ° C, fluorescence intensity was detected for 5 seconds at a specific wavelength emitted by FAM.

Asymmetric PCR composition for FMCA after confirmation of target sequence amplification Ex Taq premix 10 μl Forward primer 50 pmole Reverse primer 1 pmole LNA-FAMsig_P (third probe) 0.5 pmole Samples (Squid ' s genomic DNA) 2 ng Total volume 20 μl

After asymmetric PCR, conditions for FMCA process Temperature time Cycles Pre-denaturation < RTI ID = 0.0 > 95 ℃ 2 minutes Denaturation 95 ℃ 5 seconds 50 times Annealing and extension 61 ℃ 30 seconds Hybridization and incubation < RTI ID = 0.0 > 4 ℃ 10 minutes * Melting curve: Fluorescent probe every 1 ° C from 4 ° C to 70 ° C

Example  3-4: Derivation of Fluorescence Melting Curve Analysis (FMCA) and melting temperature (Tm) values

As a result of the fluorescence melting curve analysis, the hybridized double-labeled LNA probe of Example 3-1 hybridized to T. pacificus , D. gigas and Argentine short fin squid I. argentinus ) were identified at the same time. As the temperature increased by 1 ° C, the fluorescence intensity emitted from the fluorescent material was detected. Melt peak graphs showed a negative regression of Relative Fluorescence Units (RFU) at each temperature.

In the FAM channel, the melting peak graph shows the values of melting temperature (Tm) values for T. pacificus , D. gigas and Argentinian argentinus at 48 ° C, 28 ° C and 42 ° C, respectively.

On the other hand, a no template control (NTC) without a template confirmed that there was no fusion peak in the FAM fluorescence channel, as shown in FIG.

The melting temperature of the LNA-modified dual-labeled probe, which is specific for each squid species, LNA-FAMsig_P (third probe) Cuttlefish ( Todarodes pacificus ) 48 ° C The Great American Squid ( Dosidicus gigas ) 28 ℃ Argentine short-finned squid ( Illex argentinus ) 42 ° C

Thus, the dual labeled LNA probes with a probe-based fluorescent melting curve analysis to determine the melting temperature (Tm) values derived from the (Fluorescence Melting Curve Analysis, FMCA), squid (T. pacificus), American giant squid (D gigas , and Argentine I. argentinus , the three species of squid can be clearly identified at the same time, and the double-labeled LNA probe and probe-based fluorescence melting curve analysis can be usefully applied to identify squid species .

<110> Industry-University Cooperation Foundation Hanyang University ERICA Campus <120> Identifying method of squid species using dual labeled probe and          분광 이 <130> P17U22C0048 <160> 9 <170> KoPatentin 3.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> squid universal primer_F <400> 1 gcgatgacta ttttctacaa accat 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> squid universal primer_R <400> 2 tgtgaaataa taccaaaggc aggta 25 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> S-FMCA_F <400> 3 gcgatgacta ttttctacaa accat 25 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> S-FMCA_R <400> 4 accaaatcct ccaattataa tagg 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FAMsig_P <400> 5 cagataccaa agataaaata 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEXsig_P <400> 6 attataatga atccgtgcgc 20 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> S 'L-FMCA_F <400> 7 atgcgatgac tattttctac aaacc 25 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> S 'L-FMCA_R <400> 8 ccaaatcctc caattataat aggtataac 29 <210> 9 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> LNA-FAMsig_P <400> 9 ataccaaaga taaaata 17

Claims (18)

delete delete delete delete delete delete delete delete delete delete delete delete (a) isolating DNA from a squid sample selected from the group consisting of domestic domestic squid ( Todarodes pacificus ), imported American squid ( Dosidicus gigas ) and Argentine short-finned squid ( Illex argentinus );
(b) a DNA primer separated from the step (a) by a forward primer consisting of SEQ ID NO: 7, a reverse primer consisting of SEQ ID NO: 8, and a reverse primer having a 5 'end labeled with a fluorescent substance and a 3' quencher and a double labeled LNA probe consisting of the nucleotide sequence of SEQ ID NO: 9 in which the 6th base and the 9th base from the 5 'end are mutated to LNA, is added in an amount of 1 pmole Asymmetric PCR (Asymmetric PCR) by mixing in an amount within the range of not more than 0.5 pmole and an amount in the range of not more than 0.5 pmole;
(c) melting the amplification product amplified by the asymmetric PCR of step (b) while changing the temperature to obtain a melting curve; And
(d) confirming the melting temperature of the melting curve obtained in the step (c), and when the melting temperature is 47 to 49 ° C, Todarodes pacificus ; When the melting temperature is 27 ° C to 29 ° C, the American squid ( Dosidicus gigas ); Identifying each squid species with Argentine shortfin squid ( Illex argentinus ) when the melting temperature is between 41 ° C and 43 ° C. Method for simultaneous identification of domestic squid, imported squid, and Argentine shortfin squid species.
14. The method according to claim 13, wherein the asymmetric PCR is carried out by further adding a forward primer to the reverse primer at a ratio of 49 to 51 times as much as that of the reverse primer to discriminate between domestic squid, imported squid, and argentina short squid.
14. The method according to claim 13, wherein the step of obtaining the melting curve is performed by measuring the fluorescence intensity with increasing temperature while analyzing the fluorescent value with respect to the temperature, thereby determining the simultaneous discrimination between domestic squid, imported squid, and Argentine shortfin squid species .
The method according to claim 15, wherein the fluorescence intensity is measured by increasing the temperature stepwise from 4 ° C to 70 ° C while maintaining the 1 ° C increase for 5 seconds and then measuring the fluorescence intensity, And Argentine short-fin squid species.
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