TW201814053A - Genetic marker for detecting yellow head virus genotype 1 and method for detecting yellow head virus genotype 1 using the same - Google Patents

Abstract

The present invention relates to a genetic marker for detecting yellow head virus genotype 1 (YHV1) and a method for detecting yellow head virus genotype 1 using the same, and more particularly, to a method for amplifying a specific nucleic acid of yellow head virus genotype 1 (YHV1), hybridizing peptide nucleic acid (PNA) specifically recognizing the amplification product, controlling a temperature of the hybridization product to obtain a melting curve depending on temperature, and then detecting yellow head virus genotype 1 or identifying whether or not shrimp is infected with the virus by analyzing the melting curve and confirming a melting temperature. According to the present invention, infection of the yellow head virus genotype 1 (YHV1) recognized as the largest threat to shrimp aquaculture, and spread of yellow head disease caused by this virus can be blocked at an early stage by simply, rapidly, and accurately detecting the yellow head virus genotype 1 (YHV1).

Description

Genetic marker for detecting yellowhead virus genotype 1 and method for detecting yellowhead virus gene type

The invention relates to a method for detecting genetic markers of yellow head virus genotype 1 (YHV1) and detecting a type of yellowhead virus gene, and more particularly to an amplification of yellowhead virus gene A specific nucleic acid method, a peptide nucleic acid (PNA) specifically recognizes an amplification product, obtains a melting curve according to temperature control of the temperature of the hybrid product, and then detects a yellow head virus gene. Type 1 either identifies the shrimp as a virus by analyzing the melting curve and confirming the melting temperature.

Shrimp accounts for 17% of global trade, and 75% of shrimp are produced by aquaculture (FAO, 2009). Although the global shrimp farming industry has grown substantially, the loss of shrimp has continued to increase due to disease outbreaks. In fact, since 1994, the loss of shrimp caused by viral diseases is estimated to be more than $3 billion per year.

Especially in recent years, damage caused by new diseases and viral diseases has not only increased in Asia, but also in all parts of the world, leading to serious problems such as a sharp decline in shrimp aquaculture production (Walker and Mohan, 2009; Tran et al . 2013), the quarantine and disinfection of aquaculture and aquatic ecosystems in various countries is increasing.

Yellow head virus genotype 1, YHV1, is one of the viruses that cause yellow head disease (YHD), a shrimp disease regulated by the World Organisation for Animal Health and Aquatic Animal Disease Control Act. Is an infectious disease that produces a chain reaction.

The yellowhead virus genotype 1 and other genotypes contained in the viral yellow head syndrome are classified by the International Committee on taxonomy viruses as belonging to the genus okavirus. (family roniviridae) is a single species of the wild-type genus (鳃-associated virus). The yellow-head virus gene type is known as one of the eight genotypes of the yellow-headed syndrome and is a unique pathogen known to cause yellow head disease. .

The yellow-headed virus genotype 1 is highly contagious in shrimp species known as aquaculture shrimp (Penaeus Japonicus), and it is reported that Penaeus monodon (black tiger shrimp) and white shrimp (Peneus setiferus) ) (a type of aquaculture shrimp) is naturally infected with YHV1.

The symptoms of shrimp infected with YHV1 are as follows: i) The consumption of shrimp feed increased significantly during the scheduled time, but suddenly, the shrimp no longer eat food, resulting in increased mortality, ii) found to be drowsy Or dead shrimp on the surface of the water, and shrimp that slowly and irregularly swim on the edge, iii) bright yellow under the chest of the shrimp, making the cephalosporin yellow and the liver and pancreas extremely soft , iv) The overall color of the shrimp is unusually bright or discolored.

At present, in the diagnosis method of the yellow head virus gene type 1 (YHV1), a diagnostic method and a kit for using molecular diagnosis, and a method of performing a general polymerase chain reaction (PCR) method are developed, and then By using a fluorescent probe or SYBR Green, it is confirmed that the amplification product of the reaction using electrophoresis or the method of confirming amplification by a method such as real-time polymerase chain reaction (RT-PCR) product.

In particular, in order to diagnose the yellow head virus gene type Y (YHV1), after the general conventional PCR or nested real-time RT-PCR (Nest RT-PCR) method, and then according to the World Organisation for Animal Health (OIE) Aquatic Animal Health Code The PCR amplification product is confirmed by electrophoresis, and a method of cloning the amplification product and confirming the base sequence of the purified plasmid DNA by using Sanger sequencing method or the like is performed again. Therefore, this procedure has the disadvantage of analyzing and diagnosing the disease for a long time due to complexity.

In addition, when electrophoresis is used to confirm PCR amplification products according to the OIE standard, it is impossible to perform objective analysis of PCR amplification products (grey regions) or ambiguous PCR amplification products using cloning vectors and reconfirm the bases of amplification products. Cloning validation steps for sequences, etc. This verification step is a step of confirming and verifying a sequence of non-specific PCR bands, the analysis process of which is complicated and time consuming. Particularly in the case where the amplification product is confirmed to be positive by a conventional PCR method (before sequence determination), this case corresponds to the case where there is a risk of infecting an aquatic animal infectious disease, and an epidemic is prevented; when the amplification product is subsequently subjected to sequencing When determined to be a false positive, the reliability of the inspection agency may deteriorate and the fishermen may suffer losses. Therefore, an improved method for the rapid and accurate diagnosis of a designated legal infectious disease is urgently needed.

Against this technical background, the inventors have found that the yellowhead virus gene type I (YHV1) can be identified simply, quickly and accurately by using a genetic marker for identifying and/or detecting the yellow head virus genotype 1 (YHV1). The present invention was confirmed by amplification using a genetic marker-specific peptide nucleic acid and a primer to obtain a melting curve.

The object of the present invention is to provide a genetic marker and a peptide nucleic acid (PNA) probe for detecting yellowhead virus gene type 1 (YHV1).

Another object of the present invention is to provide a composition and kit for detecting yellow-head virus gene type Y (YHV1) comprising an primer for detecting yellow-head virus gene type Y (YHV1) and the above-described PNA probe.

Another object of the present invention is to provide a method for detecting whether a yellow-head virus gene type Y (YHV1) is infected, which is obtained by generating a single-stranded genetic marker sequence fragment using the above primer to hybridize the PNA probe to the generated genetic marker sequence fragment. And the melting temperature (T _m ) value is obtained according to the hybridization of the PNA probe.

According to an aspect of the present invention, a genetic marker for detecting a type 1 of a yellowhead virus gene is provided, which is represented by a nucleotide sequence of SEQ ID NO: 1.

According to another aspect of the present invention, there is provided a peptide nucleic acid (PNA) probe for detecting yellowhead virus genotype 1 (YHV1), wherein the PNA probe is complementary to the above-described genetic marker by a nucleotide sequence of SEQ ID NO: Ground combination.

According to another aspect of the present invention, there is provided a composition and kit for detecting a yellowhead virus gene type Y (YHV1) comprising a primer represented by a nucleotide sequence of SEQ ID NO: 2 and the aforementioned PNA probe .

According to another aspect of the present invention, there is provided a method for detecting a yellow head virus genotype 1 (YHV1) comprising: (a) extracting a target nucleic acid from a test sample; (b) using a serial number of 2 A primer sequence represented by a base sequence and a gene sequence of a yellowhead virus gene type 1 using the target nucleic acid as a template to generate a single strand genetic maker sequence fragement; (c) will be numbered 3 The PNA probe represented by the base sequence is hybridized with the generated single-stranded genetic marker sequence fragment; (d) while increasing the temperature of the product heterozygous with the PNA probe of step (c), Obtaining a melting curve depending on temperature; and (e) detecting the yellowhead virus gene type by analyzing the melting curve obtained in the step (d) and confirming a melting temperature.

In the present invention, a genetic marker for detecting yellowhead virus genotype 1 (YHV1) is selected, and a virus can be detected simply, quickly, and accurately using a PNA probe and an primer for genetic marker. Thus, the present invention has the ability to effectively block the infection and spread of viruses believed to be the greatest threat to farmed shrimp at an early stage.

Unless otherwise defined herein, all technical and scientific terms used in the specification have the same meaning meaning Generally, the terms used in this specification are well known and commonly employed in the art.

The present inventors have attempted to develop a method for detecting the yellow-head virus gene type Y (YHV1) of an infectious virus as described in the Aquatic Animal Code of the World Organisation for Animal Health (OIE), without a sequencing step or before the sequencing step, by It is determined whether a PCR amplification product for a virus-specific genetic marker will specifically/non-specifically amplify, and an individual infected with the virus (for example, shrimp) is detected. As a result, using a marker for a nucleotide sequence specific for the yellow head virus genotype 1 (YHV1), and a primer and peptide nucleic acid probe virus corresponding to the marker, the yellow head virus gene type 1 (YHV1) can be used. Tested in a simple, fast and accurate way.

More specifically, a composition or kit (including a melting array) comprising the following components (1) and (2) which detects yellowhead virus gene type 1 (YHV1) is used:

(1) An oligomer mixture comprising a PNA probe for detection (SEQ ID NO: 3) and a primer for detection (SEQ ID NO: 2), which is specific for the yellow head virus genotype 1 (YHV1) ;as well as

(2) A single-strand generation buffer (SSG buffer) for generating a single-stranded DNA using a PCR amplification product produced by the primer as a template, and heterozygous the PNA probe with single-stranded DNA .

Thus, according to one aspect, the invention relates to a genetic marker for detecting a type 1 of a yellow-headed virus gene, which is represented by a base sequence of SEQ ID NO: 1.

In another aspect, the invention relates to an primer for detecting a yellow-headed virus gene type Y (YHV1), which is represented by a base sequence of SEQ ID NO: 2.

According to another aspect, the present invention relates to a peptide nucleic acid (PNA) probe for detecting yellow-head virus gene type Y (YHV1), which is represented by a nucleotide sequence of SEQ ID NO: 3.

In the present invention, a fluorescent reporter and a fluorescent quencher capable of quenching the fluorescent light of the reporter can be connected to both ends of the PNA probe. The reporter conductor may be selected from the group consisting of 6-carboxyfluorescein (FAM), Texas Red, 2', 4', 5', 7', '- tetrachloro-6-carboxy- One or more of the group consisting of 4,7-dichloroluciferin (hexadecyl (HEX)), JOE, Cy3, and Cy5; and the quencher may be selected from 6-carboxytetramethyl - one or more of the group consisting of rhodamine (TAMRA), BHQ1, BHQ2, and Dabcyl, but is not limited thereto. Preferably, FAM-labeled Dabcyl can be used.

Peptide nucleic acids (PNA) are DNA analogs with nucleic acid nucleotides joined by peptide bonds rather than phosphate bonds, which were first synthesized in 1991 by Nielsen et al. PNA is chemically synthesized and found in natural systems.

PNA is one of the substances that recognize genes, such as locked nucleic acid (LNA) or morpholino nucleic acid (MNA). It has a skeleton composed of polyamine. PNA has the advantages of excellent affinity and selectivity, and has high stability to nucleases, and thus is not cleaved by existing restriction enzymes. Furthermore, it is advantageous that the PNA is highly thermally and chemically stable, making it easy to store and not easily degradable.

PNA forms a duplex by hybridizing a natural nucleic acid having a nucleotide sequence complementary thereto. PNA/DNA duplexes are more stable than DNA/DNA duplexes when they are of equal length, and PNA/RNA duplexes are more stable than DNA/RNA duplexes. Furthermore, PNAs are superior to natural nucleic acids in their ability to detect single nucleotide polymorphisms (SNPs) when PNAs have single base mismatches that render double strands unstable.

Furthermore, the binding affinity of PNA-DNA is much higher than the binding affinity of DNA-DNA, so there is a melting point difference of about 10 ° C to 15 ° C even in the presence of a nucleotide mismatch. Using this difference in binding affinity, SNP and In/Del nucleotide changes can be detected.

Although the length of the PNA nucleotide sequence according to the present invention is not particularly limited, a length of 12 to 18 mers may be constructed so as to include a specific nucleotide sequence depending on the kind of the virus (for example, nucleotide variation) (nucleotide variation) or single nucleotide polymorphism (SNP)). In the present invention, by adjusting the length of the PNA probe, the PNA probe can be designed to a desired T _m value, and even in the case of a PNA probe having the same length, the T can be adjusted by changing the nucleotide sequence. _m value. Furthermore, since the PNA probe has a higher binding affinity than the DNA probe, it has a higher T _m value. Thus, a PNA probe can be designed to have a shorter length than a DNA probe, such that it can even detect adjacent nucleotide variations or SNPs. In the conventional high resolution melt (HRM) method, the difference in the T _m value of the target nucleic acid is as low as about 0.5 ° C, so an additional analysis procedure or minor changes or corrections in temperature are required. When two or more nucleotide variations or SNPs occur, analysis is difficult. However, the PNA probe according to the present invention will not be affected by the PNA probe sequence and SNP, and thus can be analyzed in a simple and convenient manner.

As described in the present invention, when the PNA probe comprises 17 nucleotides, preferably, the PNA probe comprises one or more nucleotides corresponding to nucleotide variations or SNP positions of the virus in the sequence. . Furthermore, the PNA probe may have a structural modification in the middle portion of the nucleotide sequence comprising a sequence corresponding to a nucleotide variation or a SNP site of the virus, thereby further increasing the melting temperature of the target nucleic acid to which it is perfectly matched (T _m ) difference.

According to another aspect, the present invention relates to a composition and kit for detecting yellow-head virus gene type 1 (YHV1), comprising a primer and a PNA probe.

The kit of the present invention may optionally include reagents required for performing a nucleic acid amplification reaction (e.g., a PCR reaction), such as a buffer, a DNA polymerase cofactor, and deoxyribonucleotide. -5-triphosphate). Alternatively, the kit of the present invention may also include various polynucleotide molecules, reverse transcriptase, various buffers and reagents, and antibodies that inhibit DNA polymerase activity. Moreover, in the kit, one skilled in the art can readily determine the optimal amount of reagent for use in a particular reaction by obtaining the disclosure described herein. In general, the kits of the invention can be made into separate packages or compartments containing the above ingredients.

When a kit is used, it is possible to efficiently detect a single nucleotide mutation and a mutation caused by a nucleotide deletion or insertion in a target nucleic acid by analyzing a melting curve obtained by using PNA, thereby detecting yellow. Head virus gene type 1 (YHV1).

The inventors can detect the yellow head virus gene type Y (YHV1) and perform a comparative analysis of the base sequence of the gene corresponding to the PCR product according to the OIE standard for detecting the yellow head virus gene type 1 (YHV1) to confirm whether the virus infection occurs. And by using the detection primer represented by the nucleotide sequence of SEQ ID NO: 2 and the PNA probe represented by the nucleotide sequence of SEQ ID NO: 3, the melting curve confirmed by artificial hybrid single-strand PCR amplification product confirms the melting temperature. (T _m ).

Thus, according to another aspect, the invention relates to a method for detecting a yellow head virus gene type Y (YHV1) comprising the steps of: (a) extracting a target nucleic acid from a test sample; (b) using a sequence number of two A primer expressed by a base sequence and a gene sequence of a yellowhead virus gene type 1 using the target nucleic acid as a template generate a single strand genetic maker sequence fragement; (c) a base having a sequence number of 3 The PNA probe represented by the base sequence is hybridized with the generated single-stranded genetic marker sequence fragment; (d) while increasing the temperature of the product heterozygous with the PNA probe of step (c), A melting curve of temperature; and (e) a melting temperature obtained by analyzing step (d) confirms a melting temperature to detect the yellowhead virus gene type.

The method of the present invention may further comprise adding a single strand generation buffer (SSG buffer) in step (b) to produce the single stranded genetic marker sequence fragment.

In the present invention, the single-strand production buffer may comprise a DNA polymerase, deoxynucleotides (dNTPs), and a stabilizer, wherein the DNA polymerase may be Taq polymerase, but is not limited thereto.

In the present invention, step (b) may comprise amplifying a nucleotide sequence having a genetic marker using a conventional primer pair, and then using the amplification product as a template to generate a single-stranded genetic marker sequence fragment. A TaqMan probe can be added to obtain an amplification curve.

In the present invention, amplification can be carried out by an instant PCR (polymerase chain reaction) method.

As used herein, the term "sample" is intended to include a variety of samples. Preferably, the biological sample is analyzed using the methods of the invention. More preferably, the sample may be a sample mixed with the viral material described herein or a sample from an individual infected with the virus (eg, shrimp, etc.). It can be analyzed from plants. Biological samples of animals, humans, fungi, bacteria and viruses. When analyzing a sample of mammalian or human origin, it can be derived from a particular tissue or organ. Representative examples of tissue include connective tissue, skin, muscle or neural tissue. Representative examples of organs include the eye, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, small intestine, testis, ovary, uterus, rectum, nervous system , glands and internal blood vessels. The biological sample to be analyzed includes any cells, tissues or liquids derived from a biological source, or any other medium that can be well analyzed by the present invention. Biological samples also include samples obtained from food prepared for food for humans and/or animals. In addition, biological samples to be analyzed include body fluid samples including, but not limited to, blood, serum, plasma, lymph, breast milk, urine, feces, eye fluid, saliva, semen, brain extracts (eg, ground brain), spinal fluid Extracts from the appendix, spleen and tonsil, but are not limited thereto.

As used herein, the term "target nucleic acid", "synthetic DNA" or "synthetic oligonucleotide" refers to a nucleic acid sequence (containing nucleotide variations or SNPs) to be detected. The target nucleic acid comprises a specific region of a nucleic acid sequence encoding a "target gene" of a protein having both physiological and biochemical functions, and annealed or hybridized to a primer or probe under annealing, hybridization, or amplification conditions.

As used herein, the term "heterozygous" refers to the formation of a double stranded nucleic acid by a complementary single stranded nucleic acid. Hybridization can occur when the complementarity between two nucleic acid strands is perfect (complete match) or when there are some mismatched residues. The degree of complementarity required for heterozygosity may vary depending on the heterozygous conditions and is particularly likely to be temperature controlled.

In the present invention, the melting curve analysis can be carried out by a fluorescence melting curve analysis (FMCA) method.

According to the present invention, a PNA probe comprising a reporter conductor and a quencher is hybridized with a target nucleic acid to produce a fluorescent signal. As the temperature increases, the PNA probe and the target nucleic acid rapidly melt at a suitable melting temperature, and the fluorescent signal is quenched. The presence or absence of nucleotide modifications (including nucleotide variations or SNPs) can be detected by analysis of high-resolution rate melting curves obtained from fluorescent signals of temperature changes. If the PNA probe exactly matches the nucleotide sequence of the target nucleic acid, the expected melting temperature (T _m ) value is displayed, but if the PNA probe is mismatched with the target nucleic acid in which the nucleotide mutation is present, the melting temperature is displayed. The (T _m ) value is lower than expected.

As used herein, the term "nucleotide variation" refers to a change in the nucleotide sequence of a target nucleic acid relative to a reference sequence (eg, substitutions, deletions or insertions of one or more nucleotides, and single nucleotide polymorphisms) Sex (SNP)). The PNA probe of the present invention can analyze changes in the nucleotide sequence of a target nucleic acid by melting curve analysis, such as a SNP of a target nucleic acid or a substitution, deletion or insertion of a nucleotide of a target nucleic acid.

The PNA probe according to the present invention may have a reporter conductor and a fluorescent quenching body connected to both ends. The fluorescent quencher is capable of quenching the fluorescent light of the conductor. The reporter conductor may be selected from the group consisting of 6-carboxyfluorescein (FAM), Texas Red, 2', 4', 5', 7', '- tetrachloro-6-carboxy- One or more of the group consisting of 4,7-dichloroluciferin (hexadecyl), JOE, Cy3, and Cy5. The quenching body may be selected from one or more of the group consisting of 6-carboxytetramethyl-rhodamine (TAMRA), BHQ1, BHQ2, and Dabcyl, but is not limited thereto, and preferably, FAM-labeled Dabcyl.

The T _m value also varies depending on the difference between the nucleotide sequence of the PNA probe and the nucleotide sequence of the DNA complementary thereto, and thus the application based on the change is easy to implement. The PNA probe is analyzed using a hybrid method different from the hydrolysis method for the TaqMan probe, and the probe having a function similar to the PNA probe includes molecular beacon probes and scorpion probes. ).

Use a forward/reverse primer set for PCR (according to the International Epidemiological Office Standard (OIE) for conventional primer pairs) and a probe containing nucleotides identifying a specific nucleotide sequence, and use of primers The use of the panel as a template to amplify a nucleotide sequence of a genetic marker to generate a single-stranded genetic marker sequence fragment can be fully achieved using a specific nucleotide sequence (e.g., nucleotide variation or SNP) analysis of the PNA probe. PCR can be carried out using conventional methods, and after the completion of the PCR, a melting process is required. When the melting temperature was increased by 0.5 ° C, the fluorescence intensity was measured to obtain a T _m value. In particular, general real-time PCR systems are well known and have the advantage that no additional procedures such as HRM (High Resolution Melting Curve) programs or minute temperature changes need to be purchased.

The melting curve analysis according to the present invention is a method of analyzing double-stranded nucleic acids and probes formed from a target nucleic acid DNA or RNA. This method is called "melting curve analysis" because it is performed by, for example, T _m analysis or analysis of a melting curve of double-stranded nucleic acid. A hybrid (double-stranded DNA) of the target single-stranded DNA and the probe is formed using a probe complementary to a specific nucleotide sequence (including a nucleotide variation or a SNP) of the target to be detected. Subsequently, the formed hybrid is heated, and the dissociation (melting) of the hybrid caused by the temperature rise is detected based on the change of the signal such as the absorbance. The T _m value is determined based on the result of the detection, so that the presence or absence of the specific nucleotide sequence can be determined. The T _m value increases as the homology of the formed hybrid increases, and the T _m value decreases as the homology decreases. To this end the target, the value of T _m T _m value of the hybrid to be formed is a target specific nucleotide sequence and its complementary detection probe determined in advance (reference value for evaluation), and measuring the sample to be detected The T _m value of the hybrid formed by the single-stranded DNA and the T _m value (measured value) of the probe. If the measured value is approximately equal to the reference value, it can be determined that the probe matches, ie, a specific nucleotide sequence is present in the target DNA. If the measured value is lower than the reference value, the probe does not match, ie there is no mutation in the target DNA.

The fluorescence melting curve analysis of the present invention is a method of analyzing a melting curve using a fluorescent material, and more specifically, a melting curve can be analyzed by using a probe containing a fluorescent material. The fluorescent material may be a reporter conductor or a quenching body, preferably an intercalating fluorescent material.

In the instant polymerase chain reaction (PCR) method according to the present invention, a fluorescent substance is inserted into a double-stranded DNA duplex during PCR, and the temperature is increased as the amplification increases to melt the double-stranded DNA, thereby reducing the presence The amount of fluorescent material between the DNA duplexes. Melting curve obtained pattern may be analyzed, in particular at a temperature (T _m) DNA is melted (denatured (denatured)), whereby the presence or absence of a particular nucleotide sequence-based (or comprising nucleotide changes SNP) to detect and / or Differentiate the type of virus.

1 is a conceptual diagram showing the technical features of each step of the conventional melting array method and each step of the melting array according to the present invention; and FIG. 2 is a view showing the effect of the melting array method of the present invention compared with the conventional melting array method. Schematic diagram. As shown, the PNA probe can hybridize to the target nucleic acid and then produce a fluorescent signal. As the temperature increases, PNA probe from the target nucleic acid rapid melting in a suitable melting temperature (T _m), the fluorescent signal is thus quenched. According to the present invention, the presence or absence of a target nucleic acid and a difference in nucleotide sequence can be detected by high-resolution fluorescence melting curve analysis (FMCA) obtained from a fluorescent signal as a result of the temperature change. If the PNA probe according to the present invention completely matches the nucleotide sequence of the target nucleic acid, the expected melting temperature (T _m ) value is displayed, but if the PNA probe is mismatched with the target nucleic acid, wherein the nucleotide exhibits a mutation, A melting temperature (T _m ) value lower than the expected value is shown. If there is no target nucleic acid, the PNA probe shows no melting temperature (T _m ) value.

The invention will be described in detail below by way of examples. However, the examples are only intended to illustrate the invention, and those skilled in the art will understand that these embodiments should not be construed as limiting the scope of the invention.

Example 1: Genetic marker for detection of yellowhead virus genotype 1 (YHV1) and preparation of primers and PNA probes against viruses

In order to obtain the genetic marker specific for the yellow head virus genotype 1 (YHV1), the six viral bases shown in Table 1 below were analyzed in the nucleotide database (DB) of the National Center for Biotechnology (NCBI, US). Sequence, and attempt to determine a consensus sequence containing a genetic marker specific for the yellow head virus genotype 1 (YHV1) based on the results of the analysis. Here, the names of the IUB codes used in the consensus sequence are summarized in Table 2 below.

[Table 1]

[Table 2]

The base sequence represented by 5'-TCAGTGAGAGAACAGGA-3' (SEQ ID NO: 1) was selected as the detection of the yellow head virus gene type Y (YHV1) from the results of the consensus analysis of the yellow head virus genotype 1 (YHV1). Genetic marker.

Further, a primer represented by a nucleotide sequence of SEQ ID NO: 2 was prepared as a primer for generating a single-stranded DNA of a genetic marker, and a PNA probe represented by a nucleotide sequence of SEQ ID NO: 3 was prepared as a probe for hybrid genetic markers. needle.

Figure 3 shows the base sequence of the yellow head virus genotype 1 (YHV1), including genetic markers, primers, and PNA probes derived from genetic markers. In Figure 3, the genetic marker regions heterozygous for the PNA probe are indicated in blue.

[table 3]

The PNA probe was constructed using the nucleotide sequence, reporter conductor and quencher shown in Table 3 above. The PNA probe was synthesized using the HPLC purification method of Panagene (Korea). The purity of the synthetic probe was analyzed by mass spectrometry. The probe avoids unnecessary secondary structures for efficient binding to a target nucleic acid.

Example 2: Optimization of a melting array kit for detecting yellow head virus gene type 1 (YHV1).

The amplification curve and the melting curve were obtained from the nucleic acid sample of the yellow head virus gene type 1 (YHV1) using the PNA probe and primer prepared in Experimental Example 1. The obtained curve was analyzed to verify the PCR product to optimize the detection of the yellow head virus gene type 1 (YHV1). Here, a PCR product is produced according to a primer set of the International Office of Episodes (OIE) standard (in accordance with the introduction of the related art, Table 4).

[Table 4]

The melting array reaction was carried out using a CFX96 real-time system (BIO-RAD, USA). To generate a single-stranded target nucleic acid from a PCR product, a single-stranded production buffer (SSG buffer) and a single primer complementary to the binding strand of the probe are used. The composition of SSG buffer contains 2X nTaq-HOT (0.2 units/μl), nTaq-HOT buffer (containing 4 mM MgCl ₂ ), dNTP mixture (A, T, G and C; each 0.4 mM) and one stabilizer.

The reactant compositions used in the melt array reaction are shown in Table 5 below. After the main mixing in the molten array kit, 1 to 3 μl of the PCR product was added for analysis.

[table 5]

Table 6 shows the conditions for the reactant heterozygous reaction. Specifically, Table 6 shows the steps of producing single-stranded DNA from the PCR product, a denaturation step, a process of hybridizing the PNA probe, and increasing the temperature of the hybrid product.

[Table 6]

As a result, as shown in Fig. 5, by analyzing the melting curve, it was confirmed that the yellow head virus gene type 1 (YHV1) can be detected using the PNA probe at a T _m value of 58 ° C to 64 ° C .

Example 3: Sensitivity confirmation of a melting array kit for detecting yellow head virus gene type 1 (YHV1)

Using a primer set for virus detection according to the OIE standard, 1 μl, 0.5 μl, 0.25 μl, and 0.125 μl of PCR products were obtained, each loaded onto a 2% agarose gel, and passed through a melting array kit. Analyze.

As a result, as shown in Fig. 6, when the PCR product was loaded onto the agar colloid, a clear melting peak of up to 0.25 μl appeared in the melting curve obtained using the PNA probe, in which the band could be detected, indicating The melt array kit of the present invention is very sensitive. Furthermore, the concentration of the PCR product on the strip of agar colloid can be sufficiently detected by the melting set of kits according to the present invention.

Although the present invention has been described in detail based on the specific features thereof, it is obvious to those skilled in the art that these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited to the embodiments. Therefore, the scope of the invention is defined by the scope of the appended claims and their equivalents.

no

1 is a conceptual diagram showing the technical features of each step of the conventional melting array method and each step of the melting array method according to the present invention. Fig. 2 is a view showing the effect of the melting array method of the present invention compared with the conventional melting array method. Figure 3 shows a consensus sequence for the detection of a yellow head virus genotype 1 (YHV1) according to the invention, comprising a genetic marker, a primer and a peptide nucleic acid. Figure 4 shows a PCR method for detecting yellowhead virus gene type 1 (YHV1) according to the OIE standard, and the electrophoresis results of the amplified product were obtained. Figure 5 shows a method for detecting the yellow head virus gene type Y (YHV1), which obtains an amplification curve and a melting curve according to the use of an primer and a peptide nucleic acid for the virus. Figure 6 shows the results of the detection of the yellow-head virus gene type 1 (YHV1) by PCR according to the OIE standard, and the sensitivity of the detection of the yellow-head virus gene type Y (YHV1) using the primer and the peptide nucleic acid of the present invention. the result of.

<110> REPUBLIC OF KOREA (National Institute of Fisheries Science) <120> The genetic marker for detecting genotype 1 of the yellow head virus and the method for detecting the type of yellowhead virus gene <130> PF-B1946 <140 > TW 106134725 <141> 2017-10-11 <150> 10-2016-0132025 <151> 2016-10-12 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 17 <212> DNA < 213> Artificial sequence <220> <223> Genetic Marker for YHV1 <400> 1 tcagtgagag aacagga 17 <210> 2 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 2 aaggtgttat Gtcgaggaag t 21 <210> 3 <211> 17 <212> DNA <213> Artificial sequence <220> <223> PNA probe <400> 3 tcctgttctc tcactga 17 <210> 4 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Conventional Primer(Forward) <400> 4 ccgctaattt caaaaactac g 21 <210> 5 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Conventi Onal Primer(Reverse) <400> 5 aaggtgttat gtcgaggaag t 21

Claims (8)

A genetic marker for detecting yellow head virus genotype 1, YHV1, represented by a base sequence of SEQ ID NO: 1. A Peptide Nucleic Acid (PNA) probe for detecting a yellowhead virus gene type, represented by a single base sequence of SEQ ID NO: 3, wherein the PNA probe is complementary to the inheritance described in claim 1 mark. The PNA probe of claim 2, wherein a reporter or a quencher is attached to the PNA probe. A composition for detecting a type 1 of a yellow head virus gene comprising a one base sequence of SEQ ID NO: 2 and a primer represented by the PNA probe of claim 2. A kit for detecting a type 1 of a yellow head virus gene, comprising a one base sequence of SEQ ID NO: 2 and an primer represented by the PNA probe of claim 2. A method for detecting a type of yellowhead virus gene comprises the steps of: (a) extracting a target nucleic acid from a test sample; (b) using a primer represented by a base sequence of SEQ ID NO: 2 and using the target nucleic acid As a template, a gene sequence of the yellow head virus gene type 1 generates a single strand genetic maker sequence fragement; (c) a PNA probe represented by a single base sequence of SEQ ID NO: The needle is hybridized with the generated single-stranded genetic marker sequence fragment; (d) obtaining a melting curve depending on temperature while increasing the temperature of the product heterozygous with the PNA probe of step (c) (melting curve); and (e) determining the yellowhead virus gene type by confirming a melting temperature by analyzing the melting curve obtained in the analysis step (d). The method of claim 6, wherein the single-stranded genetic marker sequence fragment is produced by adding a single strand generation buffer (SSG buffer) in step (b). The method of claim 7, wherein the single-strand production buffer comprises a DNA polymerase, a deoxynucleotide (dNTP), and a stabilizer.