KR20040074264A - Nucleocapsid gene of infectious bronchitis virus - Google Patents

Abstract

PURPOSE: A nucleocapsid gene of infectious bronchitis virus is provided, which gene is useful as a molecular marker for determining and classifying the type of infectious bronchitis virus. CONSTITUTION: The nucleocapsid gene of infectious bronchitis virus has the nucleotide sequence set forth in SEQ ID NO:1, wherein the nucleocapsid gene is selected from the nucleotide sequences set forth in SEQ ID NO:2 to SEQ ID NO:39. The method for determining the type of infectious bronchitis virus comprises the steps of: (1) PCR amplifying a testing DNA using primers having the nucleotide sequences set forth in SEQ ID NO:40 and SEQ ID NO:41; (2) sequencing the PCR amplified product; and (3) comparing the sequenced nucleotide sequence with the nucleocapsid gene of SEQ ID NO:1.

Description

Nucleocapsid gene of infectious bronchitis virus

The present invention relates to a nucleocapsid gene of infectious bronchitis virus, and more particularly to a nucleocapsid gene of an infectious bronchitis virus having a nucleotide sequence set forth in SEQ ID NO: 2 to SEQ ID NO: 39 , and It relates to a method of determining the type of infectious bronchitis virus using the gene sequence.

Of Infectious Bronchitis Virus (Infectious bronchitis virus: IBV) belong to a single chain (single-stranded) positive corona irregularities for (Coronaviridae) and (family) coronavirus (Coronavirus) genus (genus) into (positive-sense) RNA virus Although there is a difference in degree according to the isolates, it causes respiratory symptoms such as runny nose, cough and bronchitis, mortality caused by nephritis, spawning deterioration and poor quality due to scattering organ damage (Holmes, Virology 2nd ed, Raven press, New York). : 841-856; King and Cavanagh, Diseases of poultry, 9th ed.Iowa State University Press, Ames, Iowa: 471-494; Van Roekel et al ., Am J Vet Res , 1951, 12: 140-146).

Infectious bronchitis viruses are known to be prevalent in various strains due to genetic recombination with other strains, as well as substitution, deletion and addition of sequences (Lai et al ., J. Virology , 1985, 56, 449-456). Keck et al ., J. Virol., 1988, 62, 1810-1813; Cavanagh et al ., Avian Path ., 1992, 21, 401-408. In addition, the virus has been reported worldwide since it was first reported in 1931 (Schalk and Hawn, J. Am Vet. Med. Assoc. , 1931, 78, 413-422). More than 13 serotypes and multiple variants have been reported, including the Massachusetts type (abbreviated as 'Mass'), and serotypes and variants other than Mass have also been reported in Europe, Australia and Japan. (Purchase et al ., Avian Dis ., 1965, 9, 111-120; Hopkins, Avian Dis ., 1978, 22, 71-81; Gelb et al ., Avian Dis ., 1991, 35, 82-87; Karaca et al ., Avian Dis ., 1990, 34, 899-904; Kwon et al ., Avian Dis ., 1993, 37, 194-202; Cook, Avian Path . 1984, 13, 711-741; Picault et al . , Avian Path ., 1986, 15, 367-383; Kusters et al ., J Gen. Virol ., 1987, 68, 343-352; Doi et al ., Avian Dis ., 1982, 26, 946-956).

In Korea, antibody-positive strains for the infectious bronchitis virus were reported in 1968 (Yu et al., Journal of Korean Veterinary Medicine, 1968, 8, 24-29) and 1980 (Kim et al., Korean Journal of Veterinary Medicine, 1980, 20, 59-64). In 1986 (Lee et al., Korean Journal of Veterinary Medicine, 1986, 26, 277-282), the virus was isolated from spawning breeders and reported the first disease outbreak.

Existing diagnosis of infectious bronchitis requires much time and effort because it is required to inoculate SPF (specific pathogenic free) eggs and observe the lesions appearing after 3-5 blind passages and neutralize the proliferated virus with positive serum. If different or mutated, false negatives may appear in identification by neutralization testing (Lukert, Infectious bronchitis in Isolation and identification of avian pathogens, 2nd ed., 1980, 70-72). In addition, another method for diagnosing infectious bronchitis is to detect infectious bronchitis virus antigens directly by fluorescent antibody method or histochemical staining method using monoclonal antibodies against common antigens, or infecting bronchitis virus primary culture with nitrocellulose membrane. Immunoblotting methods for adsorption on nitrocellulose membranes have been reported (Mocket et al ., J. Gen. Virol ., 1984, 65, 2281-2286; Naqi, Avian Dis ., 1990, 34 , 893-898; Wainright et al ., Avian Dis ., 1989, 33, 482-490; Song et al ., Avian Dis ., 1998, 42, 92-100).

Recently, RT-PCR method has been developed to confirm the presence of virus by converting RNA from infectious bronchitis virus to complementary DNA by the development of molecular biological techniques and accumulation of genetic information, and then amplifying a specific site. -PCR-RFLP method that detects products amplified by PCR using probes, or PCR-RFLP method that separates the amplified products by restriction enzymes to distinguish between virus strains. Developed and applied (Andreasen et al ., Avian Dis ., 1991, 35, 216-220; Zwaagstra et al ., J. Clin.Microbiology, 1992, 30, 79-84; Falcone et al ., J. Virol Methods , 1997, 64, 125-130; Kwon et al ., Avian Dis ., 1993, 37, 194-202).

The isolation of infectious bronchitis virus was first performed in Korea in 1986, but it was reported by RT-PCR and sequencing method that infectious bronchitis virus existed in 1980 before the first isolation. (Kwon et al., Korean Journal of Veterinary Medicine, 2001, 41, 59-65). Since the infectious bronchitis virus isolated in Korea is different from foreign strains and its sequencing, it is difficult to make an accurate diagnosis by existing foreign-developed methods such as RT-PCR or PCR-RFLP. There is an urgent need to develop an accurate diagnostic method.

Accordingly, the present inventors have found that infectious bronchitis isolated from Korea, which can be useful for the development of a diagnosis and vaccine suitable for virus strains existing only in Korea in order to reduce the damage of infectious bronchitis, which is underestimated due to the lack of rapid and accurate diagnosis. The present invention has been completed by providing a novel nucleocapsid gene of a viral strain.

It is an object of the present invention to provide a nucleocapsid gene of an infectious bronchitis virus and a method of determining the type of infectious bronchitis virus using said gene.

1 is an electrophoretic photograph confirming the presence or absence of infectious bronchitis virus by PCR with primers described in SEQ ID NO: 40 and SEQ ID NO: 41 ,

M; DNA ladder, 1: H120, 2: Ma5, 3: IBV-SNU80108, 4: IBV-SNU869,

5: IBV-SNU8615, 6: IBV-SNU8622, 7: IBV-SNU8628, 8: IBV-SNU8644,

9: IBV-SNU8650, 10: IBV-SNU8910A, 11: IBV-SNU90110, 12: IBV-SNU90129C,

13: IBV-SNU9993, 14: IBV-SNU99114, 15: IBV-SNU99122, 16: IBV-SNU99133,

17: IBV-SNU99143, 18: IBV-SNU99169, 19: IBV-SNU99192, 20: IBV-SNU99193,

21: IBV-SNU99199, 22: IBV-SNU99204

2 compares nucleotide (152-396) similarity of nucleocapsid proteins between infectious bronchitis virus strains,

3 plots phylogenetic tree based on nucleotide (152-396) similarity of nucleocapsid protein between infectious bronchitis virus strains.

Figure 4 is an electrophoresis picture of whether the infectious bronchitis virus is an outdoor strain or a vaccine strain by performing PCR-RFLP with the primer and restriction enzyme Fnu4H I of the present invention.

M; DNA ladder, 1: H120, 2: Ma5, 3: IBV-SNU80108, 4: IBV-SNU869,

5: IBV-SNU8615, 6: IBV-SNU8622, 7: IBV-SNU8628, 8: IBV-SNU8644,

9: IBV-SNU8650, 10: IBV-SNU8910A, 11: IBV-SNU90110, 12: IBV-SNU90129C,

13: IBV-SNU9993, 14: IBV-SNU99114, 15: IBV-SNU99122, 16: IBV-SNU99133,

17: IBV-SNU99143, 18: IBV-SNU99169, 19: IBV-SNU99192, 20: IBV-SNU99193,

21: IBV-SNU99199, 22: IBV-SNU99204

In order to achieve the above object, the present invention provides a nucleocapsid gene of infectious bronchitis virus.

The present invention also provides a method for determining the type of infectious bronchitis virus using the gene.

Hereinafter, the present invention will be described in detail.

The present invention provides a nucleocapsid gene of infectious bronchitis virus as set forth in SEQ ID NO: 1 .

Infectious bronchitis virus is a virus in which genetic recombination occurs frequently, and among them, the spike gene distributed on the surface of the virus has a wide variety of bases by selection by an immune response, and thus, a primer that can detect all infectious bronchitis viruses is selected. There are a lot of difficulties. In comparison, nucleocapsid proteins are diverse enough to classify viruses, and are present in the virus so that their nucleotide sequences are relatively conserved, making it easy to select primers for detecting all viruses.

Nucleocapsid gene of the present invention is a gene characterized by having a nucleotide sequence described in SEQ ID NO: 1 having a base sequence different from the previously known nucleocapsid gene, it is described in SEQ ID NO: 2 to SEQ ID NO: 39 It is characterized in that it is selected from the group consisting of genes. The genes set forth in SEQ ID NO: 2 to SEQ ID NO: 39 of the present invention include IBV-SNU80108 ( SEQ ID NO: 2 ), IBV-SNU8615 ( SEQ ID NO: 3 ), IBV-SNU8622 ( SEQ ID NO: 4 ), IBV-SNU8628 ( SEQ ID NO: 5 ) , IBV-SNU8644 ( SEQ ID NO: 6 ), IBV-SNU8650 ( SEQ ID NO: 7 ), IBV-SNU869 ( SEQ ID NO: 8 ), IBV-SNU90110 ( SEQ ID NO: 9 ), IBV-SNU90129C ( SEQ ID NO: 10 ), IBV-SNU9993 ( SEQ ID NO: 11 ), IBV-SNU99133 ( SEQ ID NO: 12 ), IBV-SNU99143 ( SEQ ID NO: 13 ), IBV-SNU99169 ( SEQ ID NO: 14 ), IBV-SNU99186 ( SEQ ID NO: 15 ), IBV-SNU99193 ( SEQ ID NO: 16 ), IBV-SNU99199 ( SEQ ID NO: 17 ), IBV-SNU99204 ( SEQ ID NO: 18 ), IBV-SNU0049 ( SEQ ID NO: 19 ), IBV-SNU011005 ( SEQ ID NO: 20 ), iIBV011018 ( SEQ ID NO: 21 ), iIBV01935 ( SEQ ID NO: 22 ) iIBV02 -01 ( SEQ ID NO: 23 ), iIBV02-09 ( SEQ ID NO: 24 ), iIBV02-10 ( SEQ ID NO: 25 ), iIBV02-11 ( SEQ ID NO: 26 ), iIBV02-23 ( SEQ ID NO: 27 ), iIBV02-26 ( SEQ ID NO: 28 ), iIBV02-31 ( SEQ ID NO: 29 ), iIBV02-62 ( SEQ ID NO: 30 ), iIBV02 -68 ( SEQ ID NO : 31 ), iIBV02-72 ( SEQ ID NO: 32 ), iIBV02-73 ( SEQ ID NO: 33 ), iIBV02-74 ( SEQ ID NO: 34 ), IB96213 ( SEQ ID NO: 35 ), IB96266 ( SEQ ID NO: 36 ), Nucleocapsid genes present in IB97041 ( SEQ ID NO: 37 ), IBEY95 ( SEQ ID NO: 38 ), and IBKC90 ( SEQ ID NO: 39 ) virus strains, all of which are found in nucleotides 152 to 396 of the gene encoding the nucleocapsid protein The partial base sequence that contains the corresponding moiety.

The present inventors refer to an infectious bronchitis virus having a nucleocapsid comprising the nucleotide sequence set forth in SEQ ID NO: 23 as "iIBV0201 (chicken coronavirus)", which was assigned to the Korea Biotechnology Research Institute Gene Bank on May 30, 2002. It was deposited (Accession Number: KCTC 10266BP).

Comparing the novel nucleotide sequence of the infectious bronchitis virus nucleocapsid of the present invention described in SEQ ID NO: 2 to SEQ ID NO: 39 with foreign infectious bronchitis virus strains, the nucleotide sequence of the nucleocapsid described in SEQ ID NO: 5 is a vaccine subject It showed a high homology of 99.5% with H120 strain but only 89.6% homology with M41, another vaccine strain, and was isolated after H120 vaccination. However, SEQ ID NO: 2 to SEQ ID NO: 4 , SEQ ID NO: 6 to SEQ ID NO: 8 , SEQ ID NO: 9 to SEQ ID NO: 39 show homology between the vaccine strains H120 and M41 89.1% to 94.0%, 87.4% to 94.0%, respectively. Therefore, it is an outdoor strain different from these attenuated vaccine strains. SEQ ID NO: 2 to SEQ ID NO: 4 , SEQ ID NO: 6 to SEQ ID NO: 8 , SEQ ID NO: 9 to SEQ ID NO: 39 show very low homology with N188 strains isolated in Australia, but 86.3% to 96.2% homology, and 88.5% to 100% homology with each other (see FIG. 2 ). In the above, the vaccine is attenuated by subcultured several times in chick embryos to attenuate the outdoor strain, and in general, the outdoor strain is more pathogenic than the vaccine strain.

As a result of producing a phylogenetic tree based on the nucleotide sequence of the infectious bronchitis virus nucleocapsid gene of the present invention, the pathogenic infectious bronchitis virus strain isolated from Korea between 1980 and 2001 was isolated from foreign virus strains. And clusters distinguished from the vaccine strain (see FIG. 3 ).

Therefore, the infectious bronchitis genes of SEQ ID NO: 2 to SEQ ID NO: 39 of the present invention has a base sequence different from the known virus strains and forms infectious bronchitis isolated from Korea because it forms a cluster different from the general vaccine strains. It can accurately determine the type and pathogenicity of the virus, and can be usefully used to develop a vaccine for treating the infectious bronchitis virus existing only in Korea.

The present invention also provides a method for determining the type of infectious bronchitis virus using the gene.

The virus type determination method of the present invention

1) performing PCR using DNA of the sample and primers set forth in SEQ ID NO: 40 and SEQ ID NO: 41 ;

2) analyzing the nucleotide sequence of the PCR product of step 1; And

3) comparing the base sequence analyzed in step 2 with the base sequence described in SEQ ID NO: 1 .

In step 1, the primers described in SEQ ID NO: 40 and SEQ ID NO: 41 is a sequence common to the infectious bronchitis virus gene, when the PCR is performed using the DNA and the primer of the sample infectious bronchitis virus in the sample If present, amplification may occur to easily confirm the presence or absence of infectious bronchitis virus (see FIG. 1 ). In addition, the DNA of the sample may be extracted from tissue or blood of an animal suspected of being infected with infectious bronchitis virus, but is not necessarily limited thereto.

In step 2, the sequencing of the PCR product of step 1 may be performed by any method that is obvious to those skilled in the art.

In the step 3, the base sequence described in SEQ ID NO: 1, because it is specific for SEQ ID NO: infectious bronchitis virus gene present in the Republic of Korea to diagnosis by comparing the nucleotide sequence amplified by the base sequence as in Step 1 described in the SEQ ID NO: 1 Can accurately diagnose what type of virus it is. The base sequence described in SEQ ID NO: 1 is preferably selected from the group consisting of the base sequence described in SEQ ID NO: 2 to SEQ ID NO: 39 .

In addition to the above diagnostic method, another method of determining the type of infectious bronchitis virus strain using the genes set forth in SEQ ID NOs: 2 to 39 of the present invention may be performed by using RT-PCR. PCR-RFLP (restriction fragment length polymorphism) or hybridization such as Southern blotting using the base sequence as a probe.

In addition, by performing PCR using the infectious bronchitis virus diagnostic primer and cutting it with restriction enzymes, genotype can be analyzed by distinguishing whether the infectious bronchitis virus is a vaccine strain or an outdoor strain. In a preferred embodiment of the present invention, after performing RFLP using Fnu4H I which recognizes a GCNGC site as a restriction enzyme, the vaccine product was distinguished from an outdoor strain by checking whether the PCR product was cleaved by the restriction enzyme (see FIG. 4 ). ). In other words, the vaccine strains M41 and H120 were isolated from the field, but the H120 vaccine was isolated from one group, and similar to the genotype H120, only IBV-SNU 8628 and IBV-SNU 8910A, which are considered vaccines , were not cleaved by Fnu4H I. It is distinguished from outdoor owners.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation of Infectious Bronchitis Virus Strain

The present inventors isolated 52 infectious bronchitis viruses from chickens of various classes in order to isolate the infectious bronchitis virus specific to Korea ( Table 1 and Table 2 ). Specifically, the infectious bronchitis virus was used as a material for the chicken trachea, the proventricle, the small intestine, the cecal tonsil, and the kidney. 5% carbon dioxide, 37% by inoculating fetal embryonic liver or kidney cells grown in DMEM (GIBCO / BRL, Grand Island, NY) medium with 10% BCS (bovine calf serum, GIBCO / BRL, Grand Island, NY) After wet culture, the inoculum was inoculated into the allantoic cavity of the embryo (Sunrise Co., NY) or directly inoculated to recover the allantoic fluid. The recovered ureter was stored at −20 ° C. and used for the experiment.

virus Separation Material ^* Year Breed ^** Week Clinical symptoms IBV-SNU80108 CT 1980 ? ? ? IBV-SNU869 CT 1986 L 42.0 Spawning IBV-SNU8615 CT 1986 LB 14.3 Respiratory signs IBV-SNU8622 T 1986 L 17.5 Respiratory signs IBV-SNU8628 TS 1986 LB 34.0 Respiratory signs, reduced scattering IBV-SNU8644 T, L 1986 BB 5.0 Respiratory signs IBV-SNU8650 T, L 1986 L 23.2 Respiratory signs, reduced scattering IBV-SNU8910A T 1989 BB 1.3 Respiratory signs IBV-SNU90110 T 1990 BB 3.1 Respiratory signs IBV-SNU90129C K 1990 B 3.3 Respiratory signs IBV-SNU9993 T 1999 B 2.6 Respiratory signs IBV-SNU99114 T, P 1999 B 3.3 Respiratory signs IBV-SNU99122 L, S 1999 B 3.3 Respiratory signs IBV-SNU99133 S, P 1999 B 7.5 Respiratory signs IBV-SNU99143 T, S 1999 B 5.2 Respiratory signs, diarrhea IBV-SNU99169 T 1999 BB 9.2 Respiratory signs IBV-SNU99186 K 1999 SPF ? Respiratory signs IBV-SNU99192 K 1999 LB 14.3 Respiratory signs IBV-SNU99193 T 1999 B 14.4 Respiratory signs IBV-SNU99199 K 1999 L 6.6 Respiratory signs IBV-SNU99204 K 1999 L 12.7 Respiratory signs IBV-SNU0049 T 2000 B ? Respiratory signs

Caecum tonsil (CT), trachea (T), lung (L), dislocation (P), small intestine (S), kidney (K)

** Laying hens (L), Laying hens (LB), Broiler (B), Broiler breeders (BB)

virus Separation Material ^* Year Breed ^** Week Clinical symptoms IBV-SNU0081 T 2000 B ? Respiratory signs IBV-SNU0091 T 2000 L ? Respiratory signs iIBV01935 T 2001 L ? Respiratory signs iIBV011005 T 2001 B ? Respiratory signs iIBV011018 T 2001 B ? Respiratory signs iIBV02-01 T / CT 2002 BB ? Respiratory signs, kidney tumors iIBDV02-09 T / CT 2002 Indigenous 11.0 ? iIBDV02-10 T / CT 2002 B 3.3 ? iIBDV02-11 T / CT / L 2002 BB 34.0 Bad and steady personality iIBDV02-16 L 2002 BB 18.0 ? iIBDV02-23 T / CT / L / K 2002 ? ? ? iIBV02-26 T / CT 2002 BB 38.0 Respiratory signs iIBV02-31 T 2002 B 4.5 Respiratory signs iIBV02-34 T / CT / K 2002 ? ? Respiratory signs iIBV02-36 T / CT 2002 B 3.0 Respiratory signs iIBV02-40 T 2002 L 12.1 Respiratory signs, decreased spawning iIBV02-41 T / S / K 2002 B 2.6 Respiratory signs iIBV02-52 T / CT / K 2002 ? 2.5 Respiratory signs iIBV02-62 T 2002 Indigenous 8.0 Respiratory signs, Kidneys / Diarrhea iIBV02-64 T / CT / K 2002 Indigenous 2.4 Respiratory signs, kidney tumors iIBV02-68 T 2002 L 15.0 Respiratory signs iIBV02-69 T / CT 2002 BB ? Respiratory signs, kidney tumors and uric acid deposition iIBV02-72 T / K 2002 ? ? Respiratory signs, death iIBV02-73 T / K 2002 ? ? Respiratory signs, death iIBV02-74 T / CT 2002 Indigenous ? Respiratory signs IB96213 ? 1996 ? ? ? IB96266 ? 1996 ? ? ? IB97041 ? 1997 ? ? ? IBEY95 ? 1995 ? ? ? IBKC90 T / K 1990 ? ? Respiratory signs, kidney tumors and uric acid deposition, mortality

Caecum tonsil (CT), trachea (T), lung (L), dislocation (P), small intestine (S), kidney (K)

** Laying hens (L), Laying hens (LB), Broiler (B), Broiler breeders (BB)

Example 2 Diagnosis of Infectious Bronchitis Virus

In order to confirm the presence or absence of infectious bronchitis virus, the present inventors performed PCR with primers described in SEQ ID NO: 40 and SEQ ID NO: 41 , which are commonly present in infectious bronchitis virus genes. Specifically, viral genomic RNA was extracted from the ureter fluid by the acid-guanidinium-phenol method (Chomzinski, P., and Sacci, N. Anal. Biochem. 1987, 162, 156) . . Specifically, in order to separate RNA, 1 ml of easyBlue (iNtRON co. Ltd., Seoul, Republic of Korea) was added to 100 μl of the embryonic egg culture ureum solution, and the RNA was isolated according to the method provided by the manufacturer. It was dissolved in 10 μl of sterile tertiary distilled water treated with diethyl-pyrocarbonate (Sigma Chemical Co, St. Louis, Mo., USA). 4 μl of 1st strand buffer (iNtRON Co.), 1 μl of 0.1 M DTT (iNtRON Co.), 1 μl of 2.5 mM dNTP (iNtRON Co.), random hexamer, 50 for cDNA synthesis ng / μl) 1 μl and 10 μl RNA were added and warmed at 70 ° C. for 15 minutes and then quenched in ice. After standing at room temperature for 10 minutes, 1 μl of reverse transcriptase (iNtRON Co.) was added and reacted at 42 ° C. for 1 hour, and the reverse transcriptase was inactivated at 95 ° C. for 5 minutes. PCR was performed using 1 μl of 10 × PCR buffer, 0.2 μl of 2.5 mM dNTP, 0.2 μl of primers described in SEQ ID NO: 40 and SEQ ID NO: 41 , respectively, and 0.2 μl of Taq polymerase (iNtRON Co., 1 U / μl), respectively. After mixing ㎕, sterile distilled water 7.2 ℓ, cDNA 1 ℓ and denaturing at 94 ° C. for 4 minutes, the reaction was performed 94 times to 30 seconds, 48 ° C.-15 seconds, and 72 ° C.-20 seconds for 35 minutes, followed by 7 minutes at 72 ° C. (Thermocycler 9600; Perkin-Elmer Co, Foster City, CA, USA). The amplified product was observed by electrophoresis on 1.5% agarose gel and stained with EtBr.

As a result, amplification products of 263 bp size, which were predicted as a result of PCR using the primers, were observed, and as a result of nucleotide sequence analysis, it was confirmed that they were specific bands ( FIG. 1 ). From the above results, PCR was performed using the primer pair of the present invention, and it was confirmed that the presence of the infectious bronchitis virus was accurately known.

Example 3 Sequence Analysis of Nucleocapsid Gene

The present inventors analyzed the nucleotide sequence of the nucleocapsid gene obtained in Example 2 by using an ABI377 DNA automatic analyzer and a die terminator kit (dye terminator kit, Perkin Elmer, Foster, CA). , The Korean Journal of Veterinary Medicine, 41, 1, 59-65, 2001). Specifically, 12.5 μl of 95% ethanol and 1 μl of 3 M sodium acetate (pH 5.2) were added to 5 μl of the amplification product, which was allowed to stand at -20 ° C for 10 minutes, followed by centrifugation at 14,000 rpm at 4 ° C for 15 minutes to remove ethanol. Then, 100 μl of 70% ethanol was added and mixed, followed by centrifugation under the same conditions for 5 minutes. Ethanol was removed, dried and dissolved in 10 μl of 1 × PCR buffer. 1 µl of the amplified product precipitated for sequencing, 1 µl of PCR primer (3 pmol / µl), 1 µl of die terminator (Perkin-Elmer Co), and 2 µl of 1x PCR buffer were added at 96 ° C. After denaturation for 10 seconds, a series of reactions of 96 ° C.-10 seconds, 52 ° C.-5 seconds, and 60 ° C.-30 seconds was repeated 25 times. The reactants were precipitated with ethanol in the same manner, and after drying, 2 μl of blue dextran / EDTA loading buffer (1 volume of dextran / EDTA + 5 volumes of formamide) was dissolved, and the resultant was heated at 95 ° C. for 5 minutes and placed in ice. It was. ABI Prism 377 (Perkin-Elmer Co) and sequencing program version 1.01 (Perkin-Elmer Co) were used for electrophoresis and sequencing.

As a result, it was confirmed that the nucleocapsid gene analyzed in the strain has a nucleotide sequence described in SEQ ID NO: 2 to SEQ ID NO: 39 .

The strain used in the present invention and the novel infectious bronchitis virus nucleocapsid gene analyzed in the strain were IBV-SNU80108 ( SEQ ID NO: 2 ), IBV-SNU8615 ( SEQ ID NO: 3 ), IBV-SNU8622 ( SEQ ID NO: 4 ), and IBV. -SNU8628 ( SEQ ID NO: 5 ), IBV-SNU8644 ( SEQ ID NO: 6 ), IBV-SNU8650 ( SEQ ID NO: 7 ), IBV-SNU869 ( SEQ ID NO: 8 ), IBV-SNU90110 ( SEQ ID NO: 9 ), IBV-SNU90129C ( SEQ ID NO: 10 ), IBV-SNU9993 ( SEQ ID NO: 11 ), IBV-SNU99133 ( SEQ ID NO: 12 ), IBV-SNU99143 ( SEQ ID NO: 13 ), IBV-SNU99169 ( SEQ ID NO: 14 ), IBV-SNU99186 ( SEQ ID NO: 15 ), IBV- SNU99193 ( SEQ ID NO: 16 ), IBV-SNU99199 ( SEQ ID NO: 17 ), IBV-SNU99204 ( SEQ ID NO: 18 ), IBV-SNU0049 ( SEQ ID NO: 19 ), IBV-SNU011005 ( SEQ ID NO: 20 ), iIBV-011018 ( SEQ ID NO: 21 ) Or iIBV-01935 ( SEQ ID NO: 22 ), iIBV02-01 ( SEQ ID NO: 23 ), iIBV02-09 ( SEQ ID NO: 24 ), iIBV02-10 ( SEQ ID NO: 25 ), iIBV02-11 ( SEQ ID NO: 26 ), iIBV02-23 (SEQ ID NO: 27), iIBV02-26 (standing SEQ ID NO : 28 ), iIBV02-31 ( SEQ ID NO: 29 ), iIBV02-62 ( SEQ ID NO: 30 ), iIBV02-68 ( SEQ ID NO: 31 ), iIBV02-72 ( SEQ ID NO: 32 ), iIBV02-73 ( SEQ ID NO: 33 ), iIBV02-74 ( SEQ ID NO: 34 ), IB96213 ( SEQ ID NO: 35 ), IB96266 ( SEQ ID NO: 36 ), IB97041 ( SEQ ID NO: 37 ), IBEY95 ( SEQ ID NO: 38 ), IBKC90 ( SEQ ID NO: 39 ), all of which are nucleocapsids The partial nucleotide sequence corresponding to nucleotides 152 to 396 of the gene encoding the.

The present inventors refer to an infectious bronchitis virus having a nucleocapsid comprising the nucleotide sequence set forth in SEQ ID NO: 23 as "iIBV0201 (chicken coronavirus)," which was assigned to the Korea Biotechnology Research Institute Gene Bank on May 30, 2002. It was deposited (Accession Number: KCTC 10266BP).

Example 4 Comparative Analysis of Sequences with Other Strains

The present inventors compared the base sequences analyzed in Example 3 with existing base sequences using the cluster method of MegAlign package (Windows version 3.12e; DNASTAR, Madison, Wis, USA). . Comparative infectious bronchitis virus strains were obtained from GenBank and their accession numbers were as follows: Beaudette (M28565), KB8523 (M21515), A-ga 99 (M85244), CU-T2 (U04805), D1466U (X58002), M41 (M28566), Gray (S48137), M85246, N1 / 62 (U52596), N1 / 88 (U52599), HT120 (AY028296).

As a result of analyzing the new nucleotide sequence of the isolated infectious bronchitis virus of the present invention with the existing infectious bronchitis virus strain as described above, the nucleotide sequence of the nucleocapsid protein as set forth in SEQ ID NO: 5 was 99.5% The virus was isolated after the H120 vaccination with only 89.6% homology with M41, another vaccine strain. However, SEQ ID NO: 2 to SEQ ID NO: 4 , SEQ ID NO: 6 to SEQ ID NO: 8 , SEQ ID NO: 9 to SEQ ID NO: 39 show homology between vaccine strains H120 and M41, 89.1% to 94.0%, 87.4% to 94.0%, respectively. Vaccines and other outdoor strains. SEQ ID NO: 2 to SEQ ID NO: 4 , SEQ ID NO: 6 to SEQ ID NO: 8 , SEQ ID NO: 9 to SEQ ID NO: 39 showed very low homology with N188 strains isolated in Australia, but 86.3% to other foreign isolates 96.2% homology and 88.5% to 100% homology with each other ( FIG. 2 ).

Based on the nucleocapsid sequences of the infectious bronchitis virus, the phylogenetic tree was isolated from Korea. Between 1980 and 2002, pathogenic infectious bronchitis virus strains isolated from Korea form different clusters from foreign virus strains except for SEQ ID NO: 3 . In addition, it was easy to distinguish by forming a cluster also distinguished from the vaccine strain ( Fig. 3 ).

Example 5 Analysis of Vaccines and Fields by PCR-RFLP

The inventors performed PCR using the primers set forth in SEQ ID NO: 40 and SEQ ID NO: 41 of the present invention to determine whether the infectious bronchitis virus isolated from the vaccine strain or the field strain, and then 5 μl of the amplified PCR product. Into the restriction enzyme Fnu4H I 5 unit 1 ㎕ 10 × buffer and put the rest to a total of 10 ㎖ with distilled water and reacted for 1 hour at 37 ℃ to cut the PCR product. 10 μl of the cleaved reaction product was observed by electrophoresis on a 2% agarose gel.

As a result, it was found that the PCR product was cut by the restriction enzyme Fnu4H I and the vaccine strain, and the outdoor strain was not cut ( Fig. 4 ). The vaccine is a virus that eliminates the pathogenicity by passing several times in the fetuses, there is a difference in pathogenicity and genotype with the outdoor strain. Therefore, if PCR is performed using the primer pair of the present invention and RFLP is performed using Fnu4H I, it is easy to distinguish whether the infectious bronchitis virus is a vaccine strain or an outdoor strain, and thus it can be easily used for genotyping. It can be easily used for differentiation from municipal vaccine strains.

As described above, the gene encoding the infectious bronchitis virus nucleocapsid protein of the present invention contains a sequence specific to the infectious bronchitis virus isolated in Korea, and can be used as a marker for determining the infectious bronchitis virus type. It can be useful for developing vaccines.

<110> iNtRON Biotechnology Co., Ltd. <120> Nucleocapsid gene of infectious bronchitis virus <130> 1p-11-12 <160> 41 <170> KopatentIn 1.71 <210> 1 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 1 ccnaangtng gnnnnncngg anannnntnn tggttncann cnntaaangn naanaannnn 60 annnnnnnnn nncnnanntt nnnaggnagn ggngtncctg ataannnnaa nntnaannna 120 nnnnnncanc anggntantg gannngncan nnnanntnnn anncnnnnaa aggnngnaga 180 nn 182 <210> 2 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 2 cctaaagttg gttctgctgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aatacacctc cacctaagtt tgaaggtagc ggtgttcctg ataatgaaaa tcttaaaaca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaagtaa aggcgggaga 180 aa 182 <210> 3 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 3 cctaaagttg gttcttctgg aaatgtatct tggtttcaag caataaaagc caagaagtta 60 aattcacctc cgcctaagtt tgaaggtagc ggtgttcctg ataatgaaaa tctaaaacca 120 agtcagcagc atggatattg gagacgccaa gctaggttta agccaggtaa aggtggaaga 180 aa 182 <210> 4 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 4 cctaaagttg gttcttctgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aattcacctg ctcctaagtt tcaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 5 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 5 cctaaagttg gttcttctgg aaatgcatct tggtttcaag cactaaaagc caagaagtta 60 aattcacctc ctcctaagtt tgaaggtagc ggcgttcctg ataatgaaaa tcttaaatta 120 agccagcaac atgggtactg gagacgtcaa gccaggtaca agccaggtaa aggcggaaga 180 aa 182 <210> 6 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 6 cctaaagttg gttcttctgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aatacacctc cacctaagtt tgaaggtagt ggtgtgcctg ataatgaaaa tcttaaaaca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 7 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 7 cctaaagttg gttcttctgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aatacacctc cacctaagtt tgaaggtagt ggtgtgcctg ataatgaaaa tcttaaaaca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 8 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 8 ccaaaagttg gttcttctgg aaatgcattt tggtttcaag ccctaaaagc caagaagcta 60 aatacacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 gg 182 <210> 9 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 9 ccaaaagttg gttcttctgg aaatgcatct tggtttcagg cactaaaagc caagaagcta 60 agttcacctg ctcctaagtt tgaaggtagt ggtgtgcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gaggcgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 10 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 10 ccaaaagttg gttcttctgg aaatgcgtct tggtttcagg cactaaaagc caagaagcta 60 ccaaaagttg gttcttctgg aaatgcgtct tggtttcagg cactaaaagc caagaagcta 120 agccagcaac atggatactg gagacgtcaa gccaggtata agccaggtaa aggcggaaga 180 aa 182 <210> 11 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 11 ccaaaagttg gttcttctgg aaatgcatct tggtttcaag ccctaaaagc caagaagcta 60 aatacacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 12 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 12 ccaaargtag gttcttctgg aaatgcatct tggtttcaag ccctaaaagc caagaagcta 60 aattcacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaayca 120 agccarcaac atggatactg gacacgccaa kyyaggttta agccaggtaa aggcgggaga 180 aa 182 <210> 13 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 13 cctaaagttg gttcttctgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aattcacctg ctcctaagtt tcaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgtcaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 14 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 14 cctaaagttg gttctgctgg aaatgcattt tggtttcaag ccataaaagc caagaagcta 60 aatacacctc cacctaagtt tgaaggtagc ggtgttcctg ataatgaaaa tcttaaaaca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccacgtaa aggcgggaga 180 ga 182 <210> 15 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 15 ccaaaagttg gttcttctgg aaatgcatct tggtttcaag ctttaaaagc caagaagcta 60 aatacacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 16 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 16 ccaaaagttg gttcttctgg aaatgcatct tggtttcaag ctctaaaagc caagaagcta 60 aatacacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 ag 182 <210> 17 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 17 cctaaagttg gttcttctgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aagtcacccg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcgggaga 180 aa 182 <210> 18 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 18 cctaaagttg gttcttctgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aagtcacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 ga 182 <210> 19 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 19 ccaaaagttg gttcttctgg aaagtcatct tggtttcagg caataaaagc caagaagtta 60 aattcacctg ctcctaggtt tgaaggcagc ggtgttcctg ataatgaaaa tcttaaacca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggcaa aggcggaaga 180 aa 182 <210> 20 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 20 ccaaaagttg gttcttctgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aattcacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaagtca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 21 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 21 ccaaaagttg gttcttctgg aaatgcatct tggtttcaag ccctaaaagc caagaagcta 60 aatacacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcgggaga 180 aa 182 <210> 22 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 22 cctaaagttg gttcttctgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aagtcacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 23 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 23 ccaaaagttg gctcttctgg aaatgcatct tggtttcagg caataaaagc caagaagcta 60 aatacacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaacca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 24 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 24 ccaaaagttg gttcttctgg aaatgcatct tggtttcaac ccataaaagc caagaagcta 60 aatacacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccatgtaa aggcggaaga 180 aa 182 <210> 25 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 25 ccaaaagttg gttcttctgg aaatgcatct tggtttcaag ccctaaaagc caagaagcta 60 aatacacctg ctcctaagtt tgaaggcagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 26 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 26 ccaaaagttg gttcttctgg aaatgcatct tggttccaag ccctaaaagc caagaagcta 60 aatacacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcgggaga 180 aa 182 <210> 27 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 27 cctaaagttg gttcttctgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aagtcacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaagtca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 28 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 28 ccaaaagttg gttcttccgg aaatgcatct tggtttcagg cactaaaagc caagaagcta 60 aattcaactg ctcctaagtt tggaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 29 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 29 cctaaagttg gtccttctgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aagtcacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 30 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 30 cctaaagttg gttcttccgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aagtcacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 31 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 31 ccaaaagttg gttcttctgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aatacacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaacca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 32 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 32 cctaaagttg gttcttccgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aaktcacctk ctcctaaatt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 33 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 33 ccaaaggtag ggtcttccgg aaatgcatct tggtttcaag ccataaaagc caagaagcta 60 aagtcacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 34 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 34 ccaaaagttg gttcttctgg aaatgtatct tggtttcaag ctctaaaagc caagaagcta 60 aatacacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 35 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 35 ccaaaagttg gttcttccgg aaatgcatct tggtttcagg cactaaaagc caagaagtta 60 aattcacctg ctcctaaatt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaacca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 36 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 36 ccaaaagttg gttcttccgg aaatgcatct tggtttcagg cactaaaagc caagaagtta 60 aattcacctg ctcctaaatt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaaaca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 37 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 37 ccaaaagttg gttcttccgg aaatgcatct tggtttcagg cactaaaagc caagaagcta 60 aattcaactg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tctcaaatca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 38 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 38 cctaaagttg gttcttctgg aaatgcatct tggtttcaag cactaaaagc caagaagtta 60 aattcacctg ctcctaagtt tgaaggtagt ggtgttcctg ataatgaaaa tcttaaatca 120 agccagcaac atggatactg gagacgccaa gctaggttta agccaagtaa aggcggaaga 180 aa 182 <210> 39 <211> 182 <212> DNA <213> Infectious bronchitis virus <400> 39 ccaaaagttg gttcttctgg aaatgcatct tggtttcagg caataaaagc caagaagcta 60 aattcacctg ctcctaggtt tgaaggcagc ggtgttcctg ataatgaaaa tcttaaacca 120 agccagcaac atggatactg gagacgccaa gccaggttta agccaggtaa aggcggaaga 180 aa 182 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IBNF <400> 40 ccagtyatya aactaggagg 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IBNR <400> 41 gcggcwggtc ctgttccagt 20

Claims (3)

Nucleocapsid gene of infectious bronchitis virus set forth in SEQ ID NO: 1 . The gene of claim 1, wherein the gene is selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 39. 3 . 1) performing PCR using DNA of the sample and primers set forth in SEQ ID NO: 40 and SEQ ID NO: 41 ; 2) analyzing the nucleotide sequence of the PCR product of step 1; And 3) A method of determining the type of infectious bronchitis virus consisting of comparing the nucleotide sequence analyzed in step 2 with the gene of claim 1.