KR100983873B1 - Miniarray for differential diagnosis between the porcine circovirus type 1 and 2 - Google Patents

Abstract

The present invention relates to a method for differentially diagnosing pig circoviruses 1 and 2 from swine degenerative neoplastic consumption syndrome or swine dermatitis syndrome, and more specifically, nylon consisting of DNA and common probes of pig circovirus types 1 and 2 The present invention relates to a method for accurately diagnosing pig circoviruses 1 and 2 from pig post-neoplastic consumable syndrome or swine dermatitis syndrome by introducing a mini-array method using a membrane.

Miniarray method, differential diagnosis of pig circovirus

Description

Miniarray for differential diagnosis between the porcine circovirus type 1 and 2}

The present invention relates to a method for differentially diagnosing pig circoviruses 1 and 2 from swine degenerative neoplastic consumption syndrome or swine dermatitis syndrome, and more specifically, nylon consisting of DNA and common probes of pig circovirus types 1 and 2 The present invention relates to a method for accurately diagnosing pig circoviruses 1 and 2 from pig post-neoplastic consumable syndrome or swine dermatitis syndrome by introducing a mini-array method using a membrane.

Swine circoviruses are divided into types 1 and 2. Swine circovirus type 1 has been isolated from pig kidney cells in 1974 and has no clinical symptoms in pigs. The disease pattern has been separated from the piglets and piglets spinal cord, and controversial pathogenicity. In addition, circovirus type 1 is persistent infection in porcine kidney cells, and thus may be present as a nephroplasmic renal virus.

Swine circovirus type 2 emerged around the end of 1990 and is closely related to swine degenerative consumable syndrome or swine dermatitis syndrome and has been mixed with other pathogens to show complex diseases.

Until now, the type 1 and 2 of swine circoviruses have been distinguished by gene amplification and tissue PCR. However, gene amplification (PCR) methods generally have the potential to amplify nonspecific products, and in situ hybridization (PCR) methods can be used only in infected tissues, and in cases where the virus is in liquid or non-tissue suspensions. There was a problem that can not be detected only in virus infected pig organ tissues.

Therefore, an accurate and reliable differential diagnosis method has been made for them, and thus, it is necessary to identify a virgin virus in pig kidney cells used for vaccine production and differential diagnosis in pigs infected with swine wasting disease.

DNA microarray is a high density arrangement of DNA molecules with known sequence on a small substrate. DNA microarrays, also known as DNA chips, are collections of genes planted on a small piece of glass, nylon, silicon, or silica. When a DNA labeled with fluorescent material is extracted from a tissue such as blood and reacted with a chip, fluorescence is generated when there is DNA of a tissue that matches the DNA on the chip. Using the program of the computer, the gene located at the point is identified, and the function is identified to analyze the meaning of the pattern given by the fluorescent points.

The advantage of microarrays is that they provide a comprehensive view of the state of gene expression. DNA microarray technology is a fusion of various fields such as chemistry, molecular biology, mechanical engineering, and electronic engineering, and contributes greatly to genome-level research related to life phenomena. Because DNA microarray technology can put tens of thousands of fragments into a single microarray, information about the entire gene can be obtained in a single experiment. Unlike the existing technology using Southern or Northern method, which only targets a few genes, it has a characteristic that can get quite a lot of information. There are various types of microarrays, such as DNA chips, protein chips, cell chips, and nerve cell chips, depending on the type of material currently loaded on the chip. Microarrays have a wide range of applications, including gene discovery, cancer classification, disease risk assessment, disease prognosis, disease diagnosis, new drug development, toxicology research, food safety assessment, and genetic disease research.

Microarrays are used to find genes that are expressed differently in different tissues or conditions and to discover new genes based on known gene expression patterns. It is also widely used to study how one gene interacts with other genes.

However, the microarray method is widely used in the human body field because it can be tested at a high cost because it is expensive. However, in the veterinary field, a minimum test cost is required and the results of analyzing the microarray result are very high. Since it is impossible to equip all the expensive equipment such as a reader, it is difficult to introduce the microarray method.

Therefore, the present inventors have tried to find a more accurate and reliable differential diagnosis of swine circovirus types 1 and 2, and as a result, by developing a mini-array method using an expensive microarray method, the identification of a fresh virus in pig kidney cells used for vaccine production and The present invention has been found to facilitate the differential diagnosis of circovirus types 1 and 2 in pigs infected with post-swine epidemic consumable syndrome.

Accordingly, it is an object of the present invention to provide a method for accurately diagnosing swine circoviruses 1 and 2 from swine degenerative neoplastic syndrome or swine dermatitis syndrome.

In order to achieve the above object, in the present invention

1) dropping swine circovirus type 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2) onto the nylon membrane and the common probe (SEQ ID NO: 3), respectively;

2) amplifying the pig circovirus DNA isolated from the field sample by PCR;

3) hybridizing the nylon membrane of step 1) and the PCR amplification product of step 2);

4) dyeing the hybridized product of step 3) with nitrobrotetrazolium and bromochloroindolin phosphate; And

5) determining the negative or positive based on the color development result of step 4);

Provides differential diagnosis method of pig circovirus 1 and 2 comprising a.

The miniarray method according to the present invention is economical than the conventional microarray method because it can be read by the naked eye without the need of a reader for the final reading, and the pig circovirus 1 and Could distinguish two correctly.

Hereinafter, the present invention will be described in detail.

The term "mini-array method" used in the present invention refers to a series of processes in which a probe is dropped on a nylon membrane, and a gene amplified by PCR is hybridized with the nylon membrane and visually read.

The miniarray method is a modified version of the microarray method of chip development technology using DNA, RNA and protein, and is much cheaper than the microarray method and is economical because it does not require a reader or the like. In addition, by applying the mini-array method according to the present invention will be able to create a variety of merchandise value.

The miniarray method according to the present invention is carried out through a series of processes shown in FIG. 1 and includes the following steps:

1) dropping swine circovirus type 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2) onto the nylon membrane and the common probe (SEQ ID NO: 3), respectively;

2) amplifying the pig circovirus DNA isolated from the field sample by PCR;

3) hybridizing the nylon membrane of step 1) and the PCR amplification product of step 2);

4) dyeing the hybridized product of step 3) with nitrobrotetrazolium and bromochloroindolin phosphate; And

5) determining negative or positive through the color development result of step 4).

The miniarray method according to the present invention is composed of three probes (SEQ ID NOs: 1 to 3), a pair of primers (SEQ ID NOs: 4 and 5), nylon membrane, and hybridization reagents described in Table 1, and mini The action of the array is that regardless of the pig circovirus type, a pair of primers are located within the OFR1 gene region, which is a site with little mutation of the circovirus, and the amplified gene is bound to the corresponding type of probes loaded on the nylon membrane. It is determined by.

division order SEQ ID NO: Probe Swine Circovirus 1 5'-GCGGAACCAGGGGAAGT ₄₄ -3 ' One Porcine Circovirus 2 5'-AGCGGGAGTCTGGTGACCT ₄₂ -3 ' 2 Common probe 5'-GACCTGTCTACTGCTGTGAGTACCT ₃₆ -3 ' 3 primer Sense Primer 5'-CCGCTGCCACATCGAGAAAG-3 ' 4 Antisense Primer 5'-CCCGCTCACTTTCAAAAGTTCAGC-3 ' 5

The preparation of porcine circovirus types 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2) and common probe (SEQ ID NO: 3) used in the present invention is a sequence of nucleotide sequences for 17 strains of porcine circoviruses in a gene bank. After mutation, the ORF1 gene region was selected, and the primer for this region was amplified by one. That is, the three probes were all prepared inside the ORF1 primer preparation site, and the total probe length was 60 bp.

Looking at the differential diagnosis process of porcine circovirus types 1 and 2 through the mini-array method according to the present invention in detail, first, after cutting the nylon membrane into 1.5cm equilateral triangle, the common probe of SEQ ID NO: 3 on the upper side of the equilateral triangle nylon membrane, left In Fig. 1, pig circovirus type 1 of SEQ ID NO: 1 and swine circovirus type 2 of SEQ ID NO: 2 were added dropwise at 10 pM per μm of the nylon membrane, and then allowed to stand at 70 ° C. for 30 minutes.

PCR is performed by mixing primer pairs of SEQ ID NOs: 4 and 5 and biotin to PCR the DNA for each pig circovirus type isolated from an open field sample as a target gene. The PCR reaction was performed by mixing 5 μl of DNA, 1 × PCR buffer, 1.5 mM MgCl ₂ , 0.2 mM dNTPs, 0.3 mM Biotin-16-dUTP, 1 U Taq DNA polymerase, and 10 pM of primers of SEQ ID NOs: 4 and 5. To 20 μl, wherein the reaction conditions were pre-modified at 94 ° C. for 5 minutes, followed by 35 cycles of denaturation at 95 ° C. for 30 seconds, annealing at 65 ° C. for 40 seconds, and expansion at 72 ° C. for 50 seconds, followed by 72 ° C. The reaction proceeds for 5 minutes at.

The PCR amplification products were dropped into three probes and placed in a 1.5 ml Eppendorf tube along with a nylon membrane prepared in advance to perform a series of hybridization processes. In other words, the nylon membrane loaded with three probes was placed in an eppendorf tube and reacted for 10 minutes at 45 ° C. using prehybridization buffer for 10 minutes, and then the PCR amplification products were mixed to add a hybridization buffer. React for minutes. After the hybridization is completed, two washes with washing buffer 2 at room temperature for two minutes and one wash for 10 minutes and the last wash with washing buffer 1 for three minutes. The prehybridization buffer used in the above process may be composed of 5ⅹSSC (Roche), 0.5% SDS and 1% blocking solution (Roche), the hybridization buffer may be composed of 5ⅹSSC and 0.5% SDS, wash buffer 1 May be composed of 1 μs PBS and 0.3% Tween 20 (v / v), and wash buffer 2 may be composed of 1 μs SSC and 0.5% SDS, but is not limited thereto.

The washed reaction was reacted with alkaline phosphatase containing streptoavidin for 30 minutes and then treated with washing buffer 2 for 5 minutes and then stained with nitrobrotetrazolium and bromochloroindoline phosphate. After reacting until the color appears, it is judged positive when it is navy blue, and negative when it is colorless. Usually, the reaction time is completed within 10 minutes, and the eye can be directly read without a reader.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are intended to illustrate the present invention in detail, and the scope of the present invention is not limited by the following examples.

Example 1 Miniarray Method for Differential Diagnosis of Swine Circovirus

The nylon membrane cut into 1.5 cm equilateral triangles was doped with a common probe of SEQ ID NO: 3 on the top, a pig circovirus type 1 of SEQ ID NO: 1 on the left, and a pig circovirus type 2 of SEQ ID NO: 2 on the right, 10 pM each. dotting) and left at 70 ° C. for 30 minutes.

5 μl of pig circovirus DNA isolated from the field sample, 1 × PCR buffer, 1.5 mM MgCl ₂ , 0.2 mM dNTPs, 0.3 mM Biotin-16-dUTP, 1 U Taq DNA polymerase, and 10 pM primers of SEQ ID NOs: 4 and 5 After mixing and adjusting to a total of 20 μl, pre-modifying at 94 ° C. for 5 minutes, and then repeating 35 cycles of denaturation at 95 ° C. for 30 seconds, annealing at 65 ° C. for 40 seconds, and expanding at 72 ° C. for 50 seconds, and then reacting at 72 ° C. for 5 minutes. PCR amplification products were prepared.

The nylon membrane prepared above was placed in a 1.5 ml eppendorf tube and reacted for 10 minutes at 45 ° C. using prehybridized buffer (5ⅹSSC (Roche), 0.5% SDS, and 1% blocking solution (Roche)) for 10 minutes. PCR amplification products were mixed, hybridization buffers (5 μS SSC and 0.5% SDS) were added and reacted at 45 ° C. for 60 minutes. After hybridization is complete, perform two washes for 5 minutes at room temperature twice with Wash Buffer 2 (1 및 SSC and 0.5% SDS), 1 wash for 10 minutes, and the last wash for 3 minutes Wash buffer 1 (1ⅹPBS and 0.3% Tween 20). (v / v)) and then reacted with alkaline phosphatase containing streptoavidin for 30 minutes, followed by treatment with washing buffer 2 (1ⅹSSC and 0.5% SDS) for 5 minutes, followed by nitrobrotrazolium and bromochloro. Stained with indorin phosphate. After the reaction until the color appeared, the result was visually judged as negative or positive, the same experiment was repeated twice and the results are shown in Figures 2a and 2b, respectively.

In the results of Figures 2a and 2b, Depending on whether the sample is PCV1 or PCV2, it reacts with the probes planted on the left and right and commonly on the top and then hybridizes. About 10 minutes after staining with nitrobrotetrazolium and bromochloroindorine phosphate, the ultramarine blue color was clearly differentiated into PCV1 (FIG. 2A) and PCV2 (FIG. 2B) according to the infection patterns of the samples. In addition, it was confirmed that repeat reproducibility exists by obtaining the same result from the results of the repetition performed twice in FIGS. 2A and 2B (each a and b).

The mini-array method according to the present invention can measure porcine circovirus infection in porcine kidney cells used in the production of porcine vaccines in animal drug vaccine industry, and porcine circovirus infection according to the type when the quality of the incoming vaccine against the produced vaccine is tested. Differential diagnosis can be made. In addition, porcine circovirus causative agents for outdoor pig post-neoplastic consumable syndrome and swine dermatitis syndrome can be accurately discriminated from other pathogens.

In addition, the mini-array method according to the present invention is more useful in the field of diagnosis for other animal diseases and pathogens by hybridizing genes amplified by PCR method after loading dozens of disease gene probes to nylon membrane in accordance with future conditions. It is expected to be used for the prevention of livestock epidemic, which is a big obstacle for domestic livestock industry due to its high efficiency in differential diagnosis.

1 is a schematic diagram of a mini-array method for differential diagnosis of pig circovirus according to the present invention.

2a and 2b shows the results of differential diagnosis of porcine circovirus PCV1 and PCV2 through the miniarray method according to the present invention, respectively.

<110> REPUBLIC OF KOREA (Management: Ministry of Agriculture and Forestry, National Veterinary Research) <120> Miniarray for differential diagnosis between the porcine          circovirus type 1 and 2 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 60 <212> DNA <213> porcine circovirus type 1 <400> 1 gcggaaccag gggaagtttt tttttttttt tttttttttt tttttttttt tttttttttt 60                                                                           60 <210> 2 <211> 60 <212> DNA <213> porcine circovirus type 2 <400> 2 agcgggagtc tggtgacctt tttttttttt tttttttttt tttttttttt tttttttttt 60                                                                           60 <210> 3 <211> 60 <212> DNA <213> common probe of porcine circovirus type 1 and 2 <400> 3 gacctgtcta ctgctgtgag tacctttttt tttttttttt tttttttttt tttttttttt 60                                                                           60 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer <400> 4 ccgctgccac atcgagaaag 20 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> antisense primer <400> 5 cccgctcact ttcaaaagtt cagc 24  

Claims (2)

1) dropping swine circovirus type 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2) onto the nylon membrane and the common probe (SEQ ID NO: 3), respectively; 2) amplifying the pig circovirus DNA isolated from the field sample by PCR; 3) hybridizing the nylon membrane of step 1) and the PCR amplification product of step 2); 4) dyeing the hybridized product of step 3) with nitrobrotetrazolium and bromochloroindolin phosphate; And 5) determining the negative or positive based on the color development result of step 4); Differential diagnosis method of pig circovirus 1 and 2 comprising a. The PCR amplification reaction according to claim 1, wherein the PCR amplification reaction of step 2) is repeated 35 cycles of pre-modification at 94 ° C for 5 minutes, denaturation at 95 ° C for 30 seconds, annealing at 65 ° C for 40 seconds, and expansion at 72 ° C for 50 seconds. And then differential diagnosis of pig circovirus 1 and 2, characterized in that for 5 minutes to proceed the reaction.

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