KR102170821B1 - Primers for LAMP based detection of Marek's disease virus in poultry and its use - Google Patents

Abstract

The present application discloses a primer set capable of detecting viruses that cause various diseases by suppressing immunity in poultry by an isothermal amplification method, and a method for detecting the virus using the same. The primer according to the present application can be widely used in the field of veterinary medicine, food industry, environment and human body as it can quickly check whether the pathogen is infected or contaminated at the source of infection or contamination, as well as high sensitivity and specificity, and terminate the reaction if necessary. Convenience is increased as it can be detected immediately without any separate treatment such as post electrophoresis.

Description

Primers for LAMP based detection of Marek's disease virus in poultry and its use

The present application relates to a primer for accurately and quickly detecting Marek's disease virus, a kind of poultry immunosuppressive virus using the LAMP method, and its use.

Chicken infectious anemia (CIA) is a disease characterized by aplastic anemia and atrophy of lymph nodes as well as immunosuppression. When infected, the disease defense ability decreases, causing secondary diseases such as viruses, bacteria, and fungi. Young chickens infected with the disease slow growth and mortality rates generally reach 10-20%, sometimes reaching 60%. The virus that causes CIA (CIAV) is a DNA virus belonging to the genus Gyrovirus of Circoviridae. Methods for diagnosing chicken infectious anemia include virus isolation and identification, and serological testing. Viruses are mainly isolated from liver, spleen, and thymus tissue, but inoculating cells and separating them is time-consuming, so gene amplification methods using PCR are widely used. Chicken infectious anemia has similar symptoms to avian leukemia virus, chicken infectious F. cyst disease, and Marek's disease. The damage caused by chicken infectious anemia is mainly caused by egg-line transmission or early infection, so it is best to prevent it by searching for infected individuals and vaccination against the breeder.

Marek's Disease (MD) is a neoplastic disease commonly found in chickens, characterized by the invasion of monocytes in peripheral blood vessels and various tissues such as eyes and skin. In particular, it causes cytolytic infections in lymphocytes such as thymus, F sac, and spleen. It can kill up to 60% in laying hens affected by this disease, and in broilers, it can lead to a death rate of up to 10%. Marek's disease virus (MDV) is a cell-binding Herpes virus, a DNA virus that has affinity for lymphocytes. Marek's disease can be diagnosed by isolation and identification of the virus or by serological testing. Lymphocytes, blood, spleen, and tumor cells in the blood are used for virus isolation, and viruses can be isolated by inoculating chicken kidney cells or embryonic cells. It can be diagnosed using histopathology and genetic tests using PCR. Because of similar symptoms during infection, it is important to differentiate them from avian leukemia, chicken infectious anemia, reticuloendothelial disease, and chicken infectious cyst F. There is no effective treatment for Marek's disease so far, and early detection and prevention are the best measures.

Reticuloendotheliosis (RE) is a disease in which immunity decreases and tumors appear in internal organs such as liver, spleen, and kidney. Infection can cause dwarfism or chronic tumors, leading to serious economic losses, and immunosuppression, reducing the effectiveness of the vaccine. The virus that causes RE (REV) is a retrovirus belonging to the retroviridae family and is a DNA virus. The diagnosis of reticuloendothelial disease includes virus isolation and identification, and serological testing. For the isolation of viruses, various tissues such as blood, liver, glandular, thymus, and F can be used as samples, and after inoculation using fetal fibrous cells, a fluorescent antibody method using a specific antibody or a gene amplification method such as PCR Use It is important to discriminate against diseases that cause atrophy of the thymus and F cyst. The damage caused by REV infection is mainly caused by the infection of the egg zone or early infection, so preventive measures are important. However, since no vaccine has been developed to prevent retinoendothelial virus, it should be managed hygienically to prevent REV from being infected in the breeder, and the infected flock should not be searched for and used as a breeder. In addition, since REV can be contaminated and transmitted by Marek's disease and poultry vaccines, it must be managed to prevent contamination of REV when manufacturing these vaccines.

Chicken Infectious Bursal Disease (IBD) is an acute infectious disease of chickens and is characterized by causing severe damage to the F cyst, the lymphatic tissue of young chickens, causing immunosuppression. This can increase the chance infection of secondary pathogens and cause a decrease in immunity against other vaccines, causing great economic damage to the poultry industry worldwide. When infected with this disease, the mortality rate is up to 60% in chickens over 3 weeks of age, depending on the pathogenicity of the virus, and in young chickens, it causes severe immune deterioration, resulting in economic loss. The virus that causes chicken infectious cyst F (IBDV) is a double helix RNA virus consisting of two segments. Diagnosis includes isolation and identification of viruses and serological testing. F sac tissue is used as a sample for virus isolation and inoculated into chicken embryonic intestinal urinary membrane (CAM). In addition, gene amplification methods such as RT-PCR are widely used by using the VP protein portion of the virus as a target gene. Although the lesion is remarkable, such as atrophy of the F cyst, it is important to distinguish it from other immunosuppressive diseases that cause similar symptoms. Chicken infectious cyst F is an acutely progressing disease, so treatment after onset is of little significance, and early detection and prevention are important.

The viral immunosuppressive disease is infected alone or in combination in the poultry industry, resulting in damage not only from the viral infection itself, but also from the secondary disease infection to immunosuppression. Preventive measures against these immunosuppressive diseases overcome the limitations of gene amplification methods such as isolation and identification and PCR methods to prepare effective countermeasures through early detection and differential diagnosis, and can be effectively applied in the field. Diagnostics need to be developed.

US Patent 5,605,792 US Patent Publication 2006-0188871

The present application is intended to provide a method capable of detecting infection of Marek's disease virus quickly and easily while exhibiting high sensitivity and specificity.

In one aspect, the present application is Chicken anemia virus selected from the group consisting of the following, Marek's disease virus and Reticuloendotheliosis virus It provides a primer set for LAMP analysis for detection: Chicken anemia virus-specific primer set comprising primers represented by SEQ ID NOs: 1 to 4, SEQ ID NOs: 7 to 10, or SEQ ID NOs: 13 to 16; Marek's disease virus-specific primer sets and SEQ ID NOs: 37 to 40, SEQ ID NOs: 43 to 46, and SEQ ID NOs: 49 to 52 including primers represented by SEQ ID NOs: 19 to 22, SEQ ID NOs: 25 to 28 or SEQ ID NOs: 31 to 34 or Reticuloendotheliosis virus comprising primers represented by SEQ ID NOs: 55 to 58 Specific primer set.

In addition, a primer set for RT-LAMP analysis for detecting infectious bursal disease virus is provided: a primer set represented by SEQ ID NOs: 61 to 64, SEQ ID NOs: 67 to 70 or SEQ ID NOs: 73 to 76.

The primer set according to the present application may further include an LF primer and an LB primer for rapid reaction, and the Chicken anemia virus-specific primer set is SEQ ID NO: 5 and 6, SEQ ID NO: 11 and 12, or SEQ ID NO: 17 and 18. Indicated primers; The Marek's disease virus-specific primer set includes primers represented by SEQ ID NOs: 23 and 24, SEQ ID NOs: 29 and 30, or SEQ ID NOs: 35 and 36; The Reticuloendotheliosis virus The specific primer set includes primers represented by SEQ ID NOs: 41 and 42, SEQ ID NOs: 47 and 48, SEQ ID NOs: 53 and 54, or SEQ ID NOs: 59 and 60; The infectious bursal disease virus specific primer set is selected from primers represented by SEQ ID NOs: 65 and 66, SEQ ID NOs: 71 and 72, or SEQ ID NOs: 77 and 78.

When the primer set according to the present application is required for detection of an amplification product in real time or at the end of the reaction, one or more primers included in each set may be labeled with a detectable labeling material.

In another aspect, the present application also provides Chicken anemia virus in vitro using the primer set disclosed herein, Marek's disease virus, Reticuloendotheliosis virus And As a method of detecting infectious bursal disease virus, the method is Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And Providing a sample of a subject suspected of being infected with an infectious bursal disease virus; Providing any one of the primer sets according to the present application; Performing a LAMP or RT-LAMP reaction using the sample and primer set; And if there is a difference by analyzing the result of the LAMP or RT-LAMP reaction and comparing it with the control sample, this is Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And Infectious bursal disease virus infection or contamination.

The primer set according to the present application is Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And Various samples requiring detection of infectious bursal disease virus, such as thymus, spleen, and F sac, may be used, but are not limited thereto.

In the method according to the present application, analysis of the LAMP or RT-LAMP reaction result can be performed in real time or after the reaction is terminated, and whether amplification is determined through, for example, measurement using one or more of color change measurement or turbidity, and control If the color changes or turbidity is increased compared to the chicken anemia virus, it is amplified. Marek's disease virus, Reticuloendotheliosis virus And It can be judged as positive for infectious bursal disease virus.

The primer set capable of specifically detecting Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus, and Infectious bursal disease virus by isothermal amplification method according to the present application, and the detection method of the viruses using the same, as well as high sensitivity and specificity, various samples Can quickly detect the contamination of pathogens. This can be widely used in pathogenicity test samples in the veterinary field and disease monitoring in poultry farms, and if necessary, it can be detected immediately without additional treatment such as electrophoresis after the reaction is terminated, increasing convenience.

1 is a result of detecting the specificity and sensitivity of the Chicken anemia virus-specific LAMP primer set for the combination of the primer set in Table 1 according to an embodiment of the present application by color change and agarose gel electrophoresis.
Figure 2 is Marek's disease virus for the primer set of the combination described in Table 2 according to another embodiment of the present application This is the result of detecting the specificity and sensitivity of the specific LAMP primer set by color change and agarose gel electrophoresis.
3 is a Reticuloendotheliosis virus for the primer set of the combination described in Table 3 according to another embodiment of the present application This is the result of detecting the specificity and sensitivity of the specific LAMP primer set by color change and agarose gel electrophoresis.
4 is an Infectious bursal disease virus for the primer set of the combination described in Table 4 according to another embodiment of the present application This is the result of detecting the specificity and sensitivity of a specific RT-LAMP primer set by color change and agarose gel electrophoresis.
In each of the above figures, the sensitivity is indicated in red. The reactions were performed in a total of 7 different concentrations and negative controls for each set, and each concentration was as follows: DNA concentration of Chicken anemia virus was 1: 6.25 x 10 ³ copies, 2: 1.25 x 10 ³ copies, 3: 2.5 x 10 ² copies, 4: 50 copies, 5: 10 copies, 6: ² copies, 7: 0.4 copies and 8: negative control; The DNA concentration of Marek's disease virus is 1: 1.25 x 10 ⁴ copies, 2: 2.5 x 10 ³ copies, 3: 5.0 x 10 ² copies, 4: 1.0 x 10 ² copies, 5: 20copies, 6: 4copies, 7: 0.8 copy and 8: negative control; The DNA concentration of the reticuloendotheliosis virus is 1: 1.0 x 10 ⁴ copies, 2: 2.0 x 10 ³ copies, 3: 4.0 x 10 ² copies, 4: 80 copies, 5: 16 copies, 6: 3.2 copies, 7: 0.64 copies and 8: The DNA concentration of negative control and infectious bursal disease virus was 1: 6.25 x 10 ³ copies, 2: 1.25 x 10 ³ copies, 3: 2.50 x 10 ² copies, 4: 50copies, 5: 10copies, 6: 2copies, 7: 0.4 copy and 8: negative control.

This application uses the LAMP and RT-LAMP method to use Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And Infectious bursal disease virus It is based on the discovery that it can be easily detected with high accuracy and sensitivity at the nucleic acid level.

Viral immunosuppressive diseases are very important in the poultry industry and in the industry that produces SPF (Specific Pathogen Free) chickens and eggs. This is because these immunosuppressive diseases cause serious economic damage by acting as a major cause of secondary disease outbreak for immunosuppression as well as damage caused by viral infection itself due to single or multiple infection. Effective countermeasures against these immunosuppressive diseases are to quickly identify the presence of the causative virus through early detection and elimination of the causative agents of these immunosuppressive diseases and differential diagnosis from similar diseases, and to prepare preventive measures such as vaccination. Therefore, we attempted to develop a detection method that overcomes the limitations of the existing isolation and identification and gene amplification methods such as PCR, can be effectively applied in the field, and can simultaneously detect major immunosuppressive diseases of poultry.

The LAMP-CIAV:MDV:REV:IBDV method according to the present application creates specific primers for chicken infectious anemia virus, Marek's disease virus, reticuloendothelial virus, and chicken infectious cyst F virus, and the Loop-Mediated Isothermal Amplification (LAMP) method. By using, the above four types of viruses can be detected simultaneously.

Therefore, this application uses the LAMP method, Chicken anemia virus, Marek's disease virus and Reticuloendotheliosis virus, RT-LAMP method Primer set capable of specifically detecting infectious bursal disease virus and Chicken anemia virus using the same, Marek's disease virus, Reticuloendotheliosis virus And Infectious bursal disease virus A detection method is disclosed.

In one aspect, the present application is Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And It relates to a primer set for LAMP analysis that can specifically detect each infectious bursal disease virus.

The term “primer” as used herein refers to a nucleic acid molecule that is a single-stranded oligonucleotide in which a nucleotide is added to the 3′ end by covalent bonds in a nucleic acid amplification or synthesis reaction using a polymerase. The primer set according to the present application is used for amplification of nucleic acids, that is, LAMP or RT-LAMP analysis.

As used herein, the term “nucleic acid” refers to a single-stranded or double-stranded oligonucleotide or polynucleotide, and refers to an RNA or DNA molecule containing one or more nucleotides, or analogs thereof, and derivatives thereof. Chicken anemia virus in which the primer set according to the present application is used, Marek's disease virus, Reticuloendotheliosis virus And Infectious bursal disease virus nucleic acids include all or part of the genome, and in one embodiment, DNA or RNA.

As used herein, the terms “oligonucleotide” and “polynucleotide” are used interchangeably, and any one includes both. The term “primer” may also be used interchangeably with “oligonucleotide” and “polynucleotide”, and includes nucleic acid (RNA or DNA), aptamer, and the like.

As used herein, the term “detection” refers to Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And Infectious bursal disease virus contamination or infection is analyzed for the presence or amount of nucleic acid, and is based on LAMP and RT-LAMP analysis.

The term “target” or “target nucleic acid” as used herein refers to a nucleic acid sequence to which the primer set of the present application is bound and specifically amplified by the primer set according to the present application, and includes RNA or DNA. Chicken anemia virus, Marek's disease virus, and reticuloendothelial virus are detected by the LAMP method, and the infectious F cyst virus is detected by the RT-LAMP method.

The primers according to the present application specifically bind to the target nucleic acid through complementarity and amplify the target nucleic acid in the LAMP manner. Herein, in particular, only genes specific for each strain/virus described in Tables 1 to 4 can be specifically detected.

LAMP is a nucleic acid amplification method capable of amplifying a target nucleic acid with high sensitivity and specificity under isothermal conditions (Notomi, T. et al. 2000. Loop-Mediated Isothermal Amplification of DNA. Nucleic Acids Res 28, E63). displacement) active DNA polymerase and six sites of the target nucleic acid, i.e., in the direction of 5′→3′, B3 region, B2 region, B1 region, F1c region, F2c region and F3c region (region complementary to the region is B3c , B2c, B1c, F1, F2 and F3) specifically designed primer sets of at least 4 to 6 are used (Korean Patent No. 612551; Nagamine, K. et al. 2002. Accelerated reaction by Loop mediated isothermal amplification using loop primers.See Mol Cell Probes 16, 223-9, etc.). The standard LAMP reaction includes at least four types of two inner primers FIP (forward inner primer), BIP (backward inner primer), and two outer primers F3 (forward outer primer) and B3 (backward outer primer). For rapid reaction (Accelerated LAMP), a total of 6 primers can be used, including additionally LF (Loop F) and LB (Loop B).

The LAMP reaction largely includes an initial structure production step, a cycling amplification step, and an extension and recycling step. In the initiation step, the F2 moiety constituting the inner primer FIP binds to the F2c region of the target nucleic acid to initiate the reaction. Subsequently, the outer primer F3 primer binds to the F3c region of the target nucleic acid to initiate strand-replacement DNA synthesis, thereby generating a single-stranded nucleic acid. This single strand forms a ring at the 5'end through the F1/F1c linkage, and the second inner primer BIP and the outer primer B3 bind to this, resulting in DNA synthesis and strand replacement DNA synthesis. As a result, a single strand with a stem-loop formed at both ends of the dumbbell shape is formed, and DNA synthesis starts from the 3'end of the F1 region. Then, in the cycling amplification step, only two inner primers are used, and at first, one inner primer binds to the ring, resulting in the synthesis of strand-replacement DNA, forming the original ring, forming a single strand and a new ring that doubled the stem site in the ring structure. Formation single strands are generated, and the amplification products are amplified up to about 10 ⁹ within 1 hour by continuing to synthesize strand-replacement DNA using this as a template. For more detailed principles and characteristics and functions of each F3 primer, B3 primer, FIP primer, BIP primer, Loop F primer (LF), and Loop B (LB) primer, the previously mentioned documents Notomi et al., 2000 and Nagamine et al. al., 2002 may be referred to. RT (reverse transcription) LAMP is a template that first synthesizes RNA into DNA and then performs LAMP, and a method of synthesizing RNA into DNA is known in the art.

As used herein, the term "complementary" or "complementary" refers to a state in which a primer or an oligonucleotide can sequence-specifically bind to a target nucleic acid through pairing of a Watson-Crick base under predetermined hybridization (hybridization), binding or annealing conditions. As a term, it includes all of partial complementarity, substantially complementary, and perfectly complementary. Substantially complementarity means that the two strands of nucleic acid sequences are not completely complementary to each other, but the effect according to the present application by binding to the target nucleic acid, that is, the degree of complementarity to amplify the target sequence through the LAMP method.

As used herein, the term “hybridization” or “hybridization” refers to a reaction in which two strands of complementary nucleic acid molecules form a sequence-specific complex through hydrogen bonding. Hybridization may constitute a step in which the primers bind to the target nucleic acid or template in the LAMP reaction in which the primers according to the present invention are used. The degree of hybridization is influenced by various factors such as the degree of complementarity of any two nucleic acid molecules involved in it, their melting temperature (Tm), or the stringency of the hybridization. Hybridization reaction conditions can also be determined in consideration of this. The stringency of the hybridization reaction is a condition that determines how easily any two nucleic acid molecules to be hybridized with each other can be combined.Complementarity, reaction temperature, ionic strength, and/or concentration of a general substance included in the hybridization reaction solution And it may be determined according to the type, for example J. Sambrook and DW Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Ed., 2012; and F. M. Ausubel, Ed., Short Protocols in Molecular Biology, Wiley; You can refer to those described in 5th Ed, 2002.

Specific binding or hybridization herein means that a specific nucleic acid molecule or primer does not substantially bind to a nucleic acid other than a target nucleic acid, but only binds to a target nucleic acid. The primer according to the present application is a target Chicken anemia virus according to the present application under high stringency conditions, Marek's disease virus, Reticuloendotheliosis virus And It can specifically bind to the nucleic acid of the infectious bursal disease virus or its complementary sequence, and can be determined by considering the length of the primer sequence, buffer, type of nucleic acid, pH, magnesium concentration, and reaction temperature in template-based DNA or RNA synthesis. will be.

In the present application, target genes for each virus are as disclosed in Tables 1 to 4, and were designed to detect each virus with high sensitivity by specifically recognizing each gene site. In the present application, as described above, in the LAMP reaction, at least 4 to 6 primers are designed to detect a target gene, and primers and reaction conditions are disclosed to satisfy certain specificity and sensitivity.

The primer according to the present application is Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And It is designed to specifically detect the infectious bursal disease virus through the LAMP method, and specifically binds to the target nucleic acid or its complementary sequence.

As described above, at least four types of primers are used in the standard LAMP reaction: FIP (forward inner primer), BIP (backward inner primer), F3 (forward outer primer), and B3 (backward outer primer). And for rapid reaction (Accelerated LAMP), a total of 6 primers can be used, including additionally LF (Loop F) and LB (Loop B). For the characteristics and functions of each F3 primer, B3 primer, FIP primer, BIP primer, Loop F primer (LF), and Loop B (LB) primer, previously mentioned documents Notomi et al., 2000 and Nagamine et al., 2002 You can refer to.

The primer specifically binding to the target nucleic acid or its complementary sequence according to the present application is at least 10 nucleotides in length, at least 15 nucleotides in the case of F3 and B3, at least 30 nucleotides in the case of FIP and BIP, and the minimum in the case of LF and LB. It is 15 nucleotides. It has at least 70% complementarity, in particular 80% complementarity, more 90% complementarity, even more particularly 95%, 96%, 97%, 98%, 99% or 100% complementarity with the corresponding portion of the target nucleic acid.

In one embodiment, the primers according to the present application include primers represented by SEQ ID NOs: 1 to 78 and substantially the same, and specifically binds to a target nucleic acid or a complementary sequence thereof. Primers of substantially the same sequence have 70% complementarity, in particular 80% complementarity, more particularly 90% complementarity, even more particularly 95%, 96%, 97%, 98%, 99% or It has 100% complementarity.

The LAMP primer according to the present application is a target Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And Infectious bursal disease virus It specifically binds to nucleic acids and detects them with high sensitivity. Infectious F cyst virus requires an additional step of synthesizing RNA into DNA with the LAMP reaction or in a separate reaction.

F3 primer, B3 primer, FIP primer, including the BIP primer according to the present application, chicken anemia virus (Chicken anemia virus), Primer for LAMP analysis for detection of at least one poultry immunosuppressive disease virus selected from the group consisting of Marek's disease virus, Reticuloendotheliosis virus, and chicken infectious bursal disease virus The set is as follows.

The chicken anemia virus For detection The F3 primer is represented by any one primer selected from the group consisting of SEQ ID NO: 1, 7, or 13; The B3 primer is represented by any one primer selected from the group consisting of SEQ ID NO: 2, 8, or 14; The FIP primer is represented by one primer selected from the group consisting of SEQ ID NO: 3, 9, or 15; And the BIP primer is represented by any one primer selected from the group consisting of SEQ ID NO: 4, 10, or 16, and the F3 primer for detecting the Marek's disease virus is selected from the group consisting of SEQ ID NO: 19, 25, or 31 Indicated by any one primer, the B3 primer is represented by one primer selected from the group consisting of SEQ ID NO: 20, 26, or 32, and the FIP primer is any selected from the group consisting of SEQ ID NO: 21, 27, or 33 Represented by one primer; And, the BIP primer is represented by any one primer selected from the group consisting of SEQ ID NO: 22, 28, or 34; The reticuloendothelial virus The F3 primer for detection is represented by any one primer selected from the group consisting of SEQ ID NO: 37, 43, 49, or 55; The B3 primer is represented by any one primer selected from the group consisting of SEQ ID NO: 38, 44, 50, or 56; The FIP primer is represented by any one primer selected from the group consisting of SEQ ID NO: 39, 45, 51, or 57; And the BIP primer is represented by any one primer selected from the group consisting of SEQ ID NO: 40, 46, 52, or 58, the chicken infectious F cyst virus The F3 primer for detection is represented by one primer selected from the group consisting of SEQ ID NOs: 61, 67, or 73; The B3 primer is represented by one primer selected from the group consisting of SEQ ID NO: 62, 68, or 74; The FIP primer is represented by one primer selected from the group consisting of SEQ ID NOs: 63, 69, or 75; In addition, the BIP primer is represented by one primer selected from the group consisting of SEQ ID NOs: 64, 70, or 76, and may be used in various combinations for each primer for each strain.

Optionally, the primer set may further include an LF primer and an LB primer. The LF primer for detecting the chicken anemia virus is a primer represented by one selected from the group consisting of SEQ ID NO: 5, 11, or 17, and the LB primer is a primer represented by one selected from the group consisting of SEQ ID NO: 6, 12 or 18 Is; The LF primer for detecting the Marek's disease virus is a primer represented by one selected from the group consisting of SEQ ID NO: 23, 29, or 35, and the LB primer is represented by one selected from the group consisting of SEQ ID NO: 24, 30, or 36. Is a primer; The reticuloendothelial virus The LF primer for detection is a primer represented by one selected from the group consisting of SEQ ID NO: 41, 47, 53, or 59, and the LB primer is a primer represented by one selected from the group consisting of SEQ ID NO: 42, 48, 54, or 60 Is; And the chicken infectious cyst virus The LF primer for detection is a primer represented by one selected from the group consisting of SEQ ID NO: 65, 71, or 77, and the LB primer is a primer represented by one selected from the group consisting of SEQ ID NO: 66, 72, or 78, each It can be used in various combinations for each strain.

In one embodiment, the LAMP primer set that can specifically detect Chicken anemia virus herein is a FIP selected from SEQ ID NO: 3, 9 or 15, BIP selected from SEQ ID NO: 4, 10 or 16, SEQ ID NO: 1, 7 Or F3 selected from 13 and B3 selected from SEQ ID NO: 2, 8 or 14. In another embodiment, the LAMP primer set capable of specifically detecting Chicken anemia virus is FIP selected from SEQ ID NO: 3, 9 or 15, BIP selected from SEQ ID NO: 4, 10 or 16, SEQ ID NO: 1, 7 or 13 F3 selected from and B3 selected from SEQ ID NO: 2, 8 or 14, LF selected from SEQ ID NO: 5, 11 or 17, and LB selected from SEQ ID NO: 6, 12 or 18.

In one embodiment, Marek's disease virus herein The LAMP primer set that can be specifically detected is FIP selected from SEQ ID NO: 21, 27 or 33, BIP selected from SEQ ID NO: 22, 28 or 34, F3 selected from SEQ ID NO: 19, 25 or 31, and SEQ ID NO: 20 And B3 selected from 26 or 32. In another embodiment, Marek's disease virus The LAMP primer set that can be specifically detected is FIP selected from SEQ ID NO: 21, 27 or 33, BIP selected from SEQ ID NO: 22, 28 or 34, F3 selected from SEQ ID NO: 19, 25 or 31, and SEQ ID NO: 20 , B3 selected from 26 or 32, LF selected from SEQ ID NO: 23, 29 or 35, and LB selected from SEQ ID NO: 24, 30 or 36.

In one embodiment, the Reticuloendotheliosis virus herein A set of specifically detectable LAMP primers is selected from FIP selected from SEQ ID NO: 39, 45, 51 or 57, BIP selected from SEQ ID NO: 40, 46, 52 or 58, selected from SEQ ID NO: 37, 43, 49 or 55 F3 and B3 selected from SEQ ID NOs: 38, 44, 50 or 56. In another embodiment, Reticuloendotheliosis virus A set of specifically detectable LAMP primers is selected from FIP selected from SEQ ID NO: 39, 45, 51 or 57, BIP selected from SEQ ID NO: 40, 46, 52 or 58, selected from SEQ ID NO: 37, 43, 49 or 55 F3 and B3 selected from SEQ ID NO: 38, 44, 50 or 56, LF selected from SEQ ID NO: 41, 47, 53 or 59, and LB selected from SEQ ID NO: 42, 48, 54 or 60.

In one embodiment, the RT-LAMP primer set capable of specifically detecting the Infectious bursal disease virus herein is a FIP selected from SEQ ID NO: 63, 69 or 75, a BIP selected from SEQ ID NO: 64, 70 or 76, and SEQ ID NO: F3 selected from 61, 67 or 73 and B3 selected from SEQ ID NO: 62, 68 or 74. In another embodiment, Infectious bursal disease virus The specific detectable RT-LAMP primer set is FIP selected from SEQ ID NO: 63, 69 or 75, BIP selected from SEQ ID NO: 64, 70 or 76, F3 selected from SEQ ID NO: 61, 67 or 73 and sequence B3 selected from number 62, 68 or 74, LF selected from SEQ ID NO: 65, 71 or 77, and LB selected from SEQ ID NO: 66, 72 or 78.

Primer according to the present application and a method comprising the same is Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And Infectious bursal disease virus The detection of infection or contamination can be usefully used in various assays for screening for contamination or infection in various samples suspected of infection or contamination of virus, detection of presence or absence, and quantification.

Samples to which the primer set and method according to the present application can be used are samples of pathogenicity of poultry, slaughter products in slaughterhouses, etc., broilers in the poultry industry. It can be carried out on a variety of samples in the industry producing SPF chickens and equivalent breeding eggs, including samples from laying hens, breeders, and native chicken farms and environmental samples.

In addition, such a biological sample may be obtained directly by a conventional sample obtaining method immediately before the test, or may be separated and stored in advance.

In another embodiment, the sample according to the present application may be a nucleic acid separated from the above-described sample as a sample. Isolation refers to separation from a sample containing nucleic acids, and includes separation from various purity levels.

The primers according to the present application and the method including the same are used in LAMP and RT-LAMP analysis for detection of viruses in any sample in which the virus may be present.

In one embodiment, the LAMP reactant is 0.2 uM of each F3 and B3 primers, 1.6 uM of each FIP and BIP primer, 0.8 uM of each LF and LB primer, 0.4M betaine, 10 mM MgSO4, 1.4 mM dNTPs, 1× ThermoPol reaction buffer (Mmonitor, South Korea), 8 U Bst DNA polymerase (Mmonitor, South Korea), and samples that require analysis of 1.0~9.5µl are included.

In another embodiment, the RT-LAMP reactant is 0.2 uM of each F3 and B3 primers, 1.6 uM of each FIP and BIP primer, 0.8 uM of each LF and LB primer, 0.4M betaine, 10 mM MgSO4, 1.4 The samples required for analysis of mM dNTPs, 1× ThermoPol reaction buffer (Mmonitor, South Korea), 8 U Enzyme mix (Mmonitor, South Korea) and 1.0~9.5µl of analysis are included.

The reactants are then reacted at a temperature suitable for DNA polymerase activity. The reaction temperature can be determined without difficulty in consideration of the enzyme used in the reaction and the nucleic acid sequence of the target. The reaction is then reacted at a time suitable for amplification of the target nucleic acid.

After amplification, the amplification reaction product can be detected by various methods, for example, color change, turbidity change, fluorescence and/or electrophoresis of the reaction solution according to DNA synthesis, and the detected product is positive and/or It is compared with the product of the negative control sample and used for quantitative or qualitative analysis.

In addition, when detection of non-specific amplification due to the occurrence of non-specific priming of the primer is required, a non-specifically synthesized total amplification agent such as acid bromide or picogreen, which is a non-specific nucleic acid binding agent, is used in a control reaction that does not contain a template. The amount of product can be determined.

The primer set according to the present application may be usefully used in various assays for contamination, screening for infection, detection of presence, and quantification in various samples suspected of infection or contamination of a virus to detect infection or contamination.

Chicken anemia virus using a primer according to the present application, Marek's disease virus, Reticuloendotheliosis virus And Infectious bursal disease virus is detected at the nucleic acid level, and compared with the control or reference group, chicken anemia virus, depending on the presence of nucleic acid, increase in nucleic acid, and degree of change, Marek's disease virus, Reticuloendotheliosis virus And Infectious bursal disease virus infection, contamination, or antibiotic treatment can predict the effectiveness of treatment.

The present invention will be described in detail through the following examples, but this embodiment is only illustrative and does not limit the scope of the present application in any way.

The present invention can be practiced using conventional techniques within the skill level of those skilled in the art in cell biology, cell culture, molecular biology, gene transformation technology, microbiology, and DNA recombination technology unless otherwise stated. In addition, for a more detailed description of the general technique, see Molecular Biotechnology: (Bernard et al., ASM press 2014); Molecular Cloning: A Laboratory Manual, 4th Ed. (Sambrook et al., Cold Spring Harbor Laboratory Press 2012); Short Protocols in Molecular Biology, 5th Ed. (Ausubel F. et al. eds., John Wiley & Sons 2002).

Example 1. CAV, MDV, REV and IBDV sample preparation

Herein, DNA was extracted from the liver samples of chickens diagnosed with CAV, MDV and REV respectively using the QIAamp DNA Mini kit (Qiagen, Germany), and QIAamp RNA Mini kit (Qiagen, Germany) from the synovial sac of chickens identified as IBDV. RNA was prepared using.

Example 2. Preparation of primers specific to CAV, MDV, REV and IBDV

Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And In order to accurately detect the infectious bursal disease virus, various genes were obtained from NCBI and GenBank, and a final primer specific for the virus was selected. Specifically, the gene VP1 that forms the envelope protein of CAV as a target of CAV, the gene VP3 (GenBank accession number, AB031296) that induces apoptosis in chickens, and the gene that induces cancer in chickens as a target of MDV (GenBank accession number, KT768121) , F3, B3, FIP, based on the surface glycoprotein (GenBank accession number, GQ415645) constituting the envelope of the virus as a target of REV and the protein (GenBank accession number, EF070167) constituting the surface envelope of the virus particle as a target of IBDV Primers were prepared for each primer of BIP, LF and LB to complete a primer set for LAMP amplification (Tables 1 to 4). Each primer sequence was verified using NCBI (National Center for Biotechnology Information), Genbank, and Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi website). This did not exist. Each primer set was designed and combined to be optimized for LAMP or RT-LAMP reaction, and showed excellent effects in terms of specificity and sensitivity.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

Example 3. Confirmation of specificity and sensitivity of LAMP and RT-LAMP reactions

First, cross-reaction between Chicken anaemia virus, Fowl adenovirus, Infectious Laryngotracheitis virus, Marek's disease virus, Reticuloendotheliosis virus, and Avian leukosis virus using one of the six primer combinations (refer to Tables 1 to 4). Through the color change and electrophoresis results of the reactants confirmed the specificity (Figs. 1a, 2a, and 3a). In addition, through the cross-reaction using RT-LAMP between Infectious bursal disease virus, Infectious bronchitis virus, Newcastle disease virus, Avian Influenza A (H9N2) virus, Avian reovirus, Duck hepatitis A virus and Avian encephalomyelitis virus with RNA as genome The specificity was confirmed by color change and electrophoresis results (Fig. 4a).

To this end, genomes were extracted from the viruses of Chicken anaemia virus, Fowl adenovirus, Infectious Laryngotracheitis virus, Marek's disease virus, Reticuloendotheli-osis virus, and Avian leukosis virus obtained in Example 1, and each DNA was prepared at 500 ng/ul. Subsequently, for LAMP analysis, 0.2 μM of each F3 and B3 primer, 1.6 μM of each FIP and BIP primer, 0.8 μM of each LF and LB primer, using a Mmiso DNA amplification kit (Mmonitor, South Korea) in a total volume of 25 μl, 0.4M betaine, 10 mM MgSO ₄ , 1.4 mM dNTPs, 1× ThermoPol reaction buffer (Mmonitor, South Korea), 8 U Bst DNA polymerase (Mmonitor, South Korea), and a solution of a composition containing 2 μl of the isolated DNA Used. With the above composition, isothermal amplification was performed at 63° C. for 40 minutes and then reacted at 80° C. for 5 minutes to terminate the reaction.

In addition, RNA genomes were extracted from viruses of Infectious bursal disease virus, Infectious bronchitis virus, Newcastle disease virus, Avian Influenza A (H9N2) virus, Avian reovirus, Duck hepatitis A virus, and Avian encephalomyelitis virus, and each RNA was extracted at a concentration of 500 ng/ul. Prepared and performed RT-LAMP. For RT-LAMP analysis, 0.2 μM of each F3 and B3 primer, 1.6 μM of each FIP and BIP primer, 0.8 μM of each LF and LB primer in a total volume of 25 μl using an Mmiso RNA amplification kit (Mmonitor, South Korea). , 2XThermoPol reaction buffer (Mmonitor, South Korea), 8 U Enzyme mix (Mmonitor, South Korea), and 2μl of a solution containing the isolated RNA. After isothermal amplification at 58°C for 40 minutes at 80°C. The reaction was completed by reacting for 5 minutes.

The results showed that the primer set according to the present application did not cross-react with other similar viruses as described in FIGS. 1A, 2A, 3A, and 4A, and only detects the virus specifically. The above results can sufficiently exhibit specificity with different combinations of primer sets (Tables 1 to 4).

Next, using each combination of primer sets (Tables 1 to 4), Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And The sensitivity to the infectious bursal disease virus was confirmed. For this, DNA was extracted from liver tissue of chickens infected with CAV, MDV and REV, respectively, and samples of 500 pg/ul, 100 pg/ul, 20 pg/ul, 4 pg/ul, 800 fg/ul, 160 fg/ul, and 32 fg/ul were collected. After preparation, isothermal amplification was performed at 63° C. for 35 minutes at the same composition, and then reacted at 80° C. for 5 minutes, and the color change of the reactants was confirmed (FIGS. 1b, 2b and 3b ). In addition, after RNA was extracted from the F sac of a chicken infected with IBDV alone, samples of 500 pg/ul, 100 pg/ul, 20 pg/ul, 4 pg/ul, 800 fg/ul, 160 fg/ul, and 32 fg/ul were prepared and 40 at 58°C. After isothermal amplification was performed for a minute, the reaction was reacted at 80° C. for 5 minutes to confirm the color change of the reactant (FIG. 4b). In addition, based on the above sensitivity results, F3 of Primer 2 and B3 of Primer 1 specific to Chicken anemia virus, F3 of Primer 5 and B3 of Primer 6 specific to Marek's disease virus, and F3 of Primer 7 specific to Reticuloendotheliosis virus and primers Using B3 of 9, PCR (94°C, 5min; 94°C, 30sec, 55°C, 30sec, 72°C, 30sec, 35cycles; 72°C, 7min) was performed. And RT-PCR (45℃, 30min; 94℃, 5min; 94℃, 5min; 94℃, 30sec, 55℃, 30sec, 72) using F3 of primer 13 and B3 of primer 12 specific for infectious bursal disease virus. ℃, 30sec, 35cycles; 72 ℃, 7min). The product amplified through PCR and RT-PCR is subjected to electrophoresis on a 1% TAE agarose gel, followed by gel extraction (Qiagen, Germany) according to the manufacturer's method, and then pTOP TA V2 vector of TOPcloner TA kit (Enzynomics, Korea). Each was inserted into pTOP-CAV, pTOP-MDV, pTOP-REV, and pTOP-IBDV.

In addition, the prepared pTOP-CAV plasmid was added in µl reaction solution with 6.25 x 10 ³ copies, 1.25 x 10 ³ copies, 2.5 x 10 ² copies, 50copies, 10copies, 2copies, 0.4copy, pTOP-MDV plasmid in µl reaction solution. 1.25 x 10 ⁴ copies, 2.5 x 10 ³ copies, 5.0 x 10 ² copies, 1.0 x 10 ² copies, 20copies, 4copies, 0.8 copies, pTOP-REV plasmid in µl reaction solution 1.0 x 10 ⁴ copies, 2.0 x 10 ³ copies, 4.0 x 10 ² copies, 80copies, 16copies, 3.2copies, 0.64copy and pTOP-IBDV plasmid in µl reaction solution, 6.25 x 10 ³ copies, 1.25 x 10 ³ copies, 2.50 x 10 ² copies, 50copies, 10copies, 2copies , Isothermal amplification was performed at 63° C. for 40 minutes at the same composition using a concentration of 0.4 copies, and then reacted at 80° C. for 5 minutes, and the sensitivity was confirmed as a result of color change and electrophoresis of the reactants (Fig. 1c, Fig. 2c, Fig. 3c and 4c).

Therefore, it exists in purple before the reaction, but the concentration of Mg ²⁺ ions decreases as the DNA synthesis reaction occurs, and accordingly, the color change from purple to blue. It happens. A change in color is a positive reaction indicating that the nucleic acid has been amplified.

The method according to the present application has the advantage of enabling qualitative analysis by viewing results quickly and conveniently, and also enabling qualitative and quantitative analysis when absorbance is measured at a wavelength of 650 nm using a spectrophotometer.

As described in Figures 1c to 4c, detection of the virus using the primer set according to the present application is 50copies when the sensitivity is plasmid containing the genome of Chicken anemia virus; 20copies when using plasmid containing the genome of Marek's disease virus; 16copies when using plasmid containing the genome of Reticuloendotheliosis virus; In the case of using the plasmid containing the genome of the infectious bursal disease virus, it was found to be very high at 250 copies.

<110> REPUBLIC OF KOREA (Animal and Plant Quarantine Agency) <120> Primers for LAMP based detection of Marek's disease virus in poultry and its use <130> D201704003D1 <160> 78 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 1 accgagaggc cgatttta 18 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 2 agtcatagtt gattgggggt t 21 <210> 3 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 3 cctccgatgt cgaaatttat atcttcagaa gaggacggtg gcac 44 <210> 4 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 4 agcggtatcg tagacgagct ttagcctcac actatacgta cc 42 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 5 tcgtcgaagt cgcttgaggt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 6 ggaaggcctt tcacaacccc 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 7 tctccaagaa gatactccac c 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 8 tttctgaatt gtccgcagtt g 21 <210> 9 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 9 ctgcagtgag gggtttccaa cggaccatca acggtgttca gg 42 <210> 10 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 10 cggattggta tcgctggaat tacacgtggg agcgcgagca tt 42 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 11 ggccgtgaac ttgttggtg 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 12 cactctatcg ctgtgtggct 20 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 13 tttcaagaat gtgccggac 19 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 14 taaaatcggc ctctcggt 18 <210> 15 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 15 tttagctcgc ttaccctgta gaccgatcaa cccaagcct 39 <210> 16 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 16 cttgattacc actactccca gccgcgtctt gccatcttac agtcttat 48 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 17 ggtcgcagga tcgcttctt 19 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Chicken anaemia virus vp1, vp3 <400> 18 accccgaacc gcaagaag 18 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Marek's disease virus meq <400> 19 ggaaaagtca cgacatccc 19 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Marek's disease virus meq <400> 20 cagctcttca catgcttcat 20 <210> 21 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Marek's disease virus meq <400> 21 tttccagctt ctgtttctcc tcaacagccc ctccaaacac c 41 <210> 22 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Marek's disease virus meq <400> 22 agaaaaagga atcgtgacgc cgcgagtttg tctacatagt acgtctgc 48 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Marek's disease virus meq <400> 23 ctcagatagg ccgtcaggga a 21 <210> 24 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Marek's disease virus meq <400> 24 tcggagaaga cgcaggga 18 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Marek's disease virus meq <400> 25 tcgatctttc tctcgggtc 19 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Marek's disease virus meq <400> 26 tttgtctaca tagtacgtct gct 23 <210> 27 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Marek's disease virus meq <400> 27 tcagggaagg ggtgtttgga gacttcgaga cggaaaaaaa ggaa 44 <210> 28 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Marek's disease virus meq <400> 28 tatctgagga ggagaaacag ttctccgagc ggcgtcacga t 41 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Marek's disease virus meq <400> 29 ctgttgggga tgtcgtgact t 21 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Marek's disease virus meq <400> 30 gctggaaagg aggagaaaaa gga 23 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Marek's disease virus meq <400> 31 aggaggagaa acagaagct 19 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Marek's disease virus meq <400> 32 ctcatgacaa gccaactgt 19 <210> 33 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Marek's disease virus meq <400> 33 tggagtttgt ctacatagta cgtgaaaaag gaatcgtgac gccgct 46 <210> 34 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Marek's disease virus meq <400> 34 agcatgtgaa gagctgcaga acgtgcactc agtccttaga tct 43 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Marek's disease virus meq <400> 35 ctgctccctg cgtcttct 18 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Marek's disease virus meq <400> 36 ggccaatgaa cacctacgta agg 23 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 37 gctaggttcg tatgaagacg 20 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 38 tgcctgatga tttcctctag t 21 <210> 39 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 39 cggggtccca acatactggt ttcctaataa actgcttcaa gcctc 45 <210> 40 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 40 tagcccctgt gtatgtctct gattttcacg cacagattct tccc 44 <210> 41 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 41 ccctatggtt cccgtgcaa 19 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 42 gcggtcccac tgacatgatt 20 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 43 accaagagga gtagatctgg 20 <210> 44 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 44 ggctaagact gcaatttcca t 21 <210> 45 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 45 ttctctgcta gctggggatt accagacgtc tgacatactg ga 42 <210> 46 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 46 tgctggcttt gtatgactct tgagagtgac attgccattc gc 42 <210> 47 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 47 cgttaaggac ctggtgagta gct 23 <210> 48 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 48 aactccaatc cccgcagc 18 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 49 gtcaatagat gtcaactgct atg 23 <210> 50 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 50 gtgtaggcca tgttgttc 18 <210> 51 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 51 ctggatgcta gtgcagttag tgcaggggaa gcagacaata gg 42 <210> 52 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 52 gaggtctcta acgagacaag tcccacacac aaatacatga cccg 44 <210> 53 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 53 tgaacatacc ctatgggtat accag 25 <210> 54 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 54 aaatcttacg aggctatgtc ctcc 24 <210> 55 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 55 ccagctagca gagaactg 18 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 56 cagtcctatt gtctgcttcc 20 <210> 57 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 57 gagagtgaca ttgccattcg cggctttgta tgactcttgg aac 43 <210> 58 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 58 atggaaattg cagtcttagc ctccgcatag cagttgacat ctattgac 48 <210> 59 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 59 ggatggctgc ggggatt 17 <210> 60 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Reticuloendotheliosis virus gp90 <400> 60 cctttcgggt gcaaccca 18 <210> 61 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Infectious bursal disease virus vp2 <400> 61 tacaatttga ctgtggggg 19 <210> 62 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Infectious bursal disease virus vp2 <400> 62 gcttgacctc actgtgag 18 <210> 63 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Infectious bursal disease virus vp2 <400> 63 ttgtagttcc cattgctctg caggggctaa ttgtcttttt ccctgg 46 <210> 64 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Infectious bursal disease virus vp2 <400> 64 gttcgatcag atgctcctga ctccgactca ctagcctgca gta 43 <210> 65 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Infectious bursal disease virus vp2 <400> 65 gtagtgagca cccacaattg agc 23 <210> 66 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Infectious bursal disease virus vp2 <400> 66 agaacctacc ggccagcta 19 <210> 67 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Infectious bursal disease virus vp2 <400> 67 cataaacgcc gtgacctt 18 <210> 68 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Infectious bursal disease virus vp2 <400> 68 ggtcaccaag tctcacat 18 <210> 69 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Infectious bursal disease virus vp2 <400> 69 tgatgttggc tgttgcagac caaggaagcc tgagtgaact 40 <210> 70 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Infectious bursal disease virus vp2 <400> 70 aaaatcggga acgtcctagt agggacccaa gatcatatga tgtgggta 48 <210> 71 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Infectious bursal disease virus vp2 <400> 71 tcaacccatt gtagctaaca tctgtc 26 <210> 72 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Infectious bursal disease virus vp2 <400> 72 aaggggtaac cgtcctcag 19 <210> 73 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> F3 primer for LAMP detection of Infectious bursal disease virus vp2 <400> 73 agatcaaacc caacagattg t 21 <210> 74 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> B3 primer for LAMP detection of Infectious bursal disease virus vp2 <400> 74 cacaattgag ccagggaa 18 <210> 75 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> FIP primer for LAMP detection of Infectious bursal disease virus vp2 <400> 75 agggtgtcgt ccggaatggt ccgttcatac ggagccttct 40 <210> 76 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> BIP primer for LAMP detection of Infectious bursal disease virus vp2 <400> 76 gagaagcaca ctctcaggtc agaaaagaca attagccctg accctgt 47 <210> 77 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LF primer for LAMP detection of Infectious bursal disease virus vp2 <400> 77 cggtccggtt gttggcat 18 <210> 78 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LB primer for LAMP detection of Infectious bursal disease virus vp2 <400> 78 tcgacctaca atttgactgt gggg 24

Claims (10)

A primer set for LAMP analysis for detection of Marek's disease virus, including F3 primer, B3 primer, FIP primer, and BIP primer, wherein the primer set includes the F3, B3, FIP, and BIP primers, respectively.
A primer set comprising primers represented by SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22;
A primer set comprising primers represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; or
A primer set comprising primers represented by SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, a primer set for LAMP analysis for detection of Marek's disease virus.
The method of claim 1,
The primer set further includes an LF primer and an LB primer,
The primer set further comprising the LF primer and the LB primer is any one of the following, a primer set for LAMP analysis for detection of Marek's disease virus:
The F3, B3 FIP, BIP, LF, and LB primers are a primer set comprising primers represented by SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively;
A primer set comprising primers represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30; or
A primer set comprising a primer represented by SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.
delete delete The method according to claim 1 or 2,
One or more primers of each of the primer sets are labeled with a detectable labeling material, a primer set for LAMP analysis for Marek's disease virus detection.
A method of detecting Marek's disease virus in a LAMP method in vitro using the primer set according to claim 1 or 2, wherein the method comprises the following steps:
Providing a sample suspected of being infected or contaminated with Marek's disease virus;
Providing a primer set according to claim 1 or 2;
Performing a LAMP reaction using the sample and primer set; And
Analyzing the reaction result during or after the LAMP reaction, and comparing it with a negative control sample, if there is a difference, determining the sample as Marek's disease virus infection or contamination.
The method of claim 6,
The sample is a poultry disease test sample; Slaughter products; Broiler. Laying hens, breeders, native chickens, or specimens derived from these farms; SPF (Specific Pathogen Free) chicken or its broodstock or a method comprising a specimen derived from these kennels
The method of claim 6,
The analysis of the LAMP reaction result is determined through measurement of color change of the reaction result or measurement using at least one of turbidity.
A composition for detecting Marek's disease virus using the LAMP method comprising the primer set according to claim 1 or 2.
A kit for detecting Marek's disease virus using the LAMP method comprising the primer set according to claim 1 or 2.

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