KR102091280B1 - Primers for LAMP based detection of Chicken anemia virus and its use - Google Patents

Abstract

The present application discloses a primer set capable of detecting a virus that causes various diseases by suppressing immunity in poultry by an isothermal amplification method and the virus detection method using the same. The primer according to the present application can be widely used in the veterinary field, the food industry, the environment, and the human field because it can quickly identify whether the pathogen is infected or contaminated from an infectious or contaminant as well as high sensitivity and specificity. After the electrophoresis, it is possible to detect immediately without any additional processing, which increases convenience.

Description

Primer for chicken anemia virus detection using LAMP and its use {Primers for LAMP based detection of Chicken anemia virus and its use}

The present application relates to a primer and its use for accurately and rapidly detecting chicken anemia virus, which is a type of poultry immunosuppressive virus using the LAMP method.

Chicken infectious anemia (CIA) is a disease characterized by aplastic anemia, immunosuppression, and atrophy of the lymph nodes. When infected, the disease defense ability is poor, and secondary diseases such as viruses, bacteria, and fungi often occur. Chickens of young age infected with the disease have slow growth, mortality rates typically reach 10-20%, and in severe cases, up to 60%. The virus that causes CIA (CIAV) is a DNA virus belonging to the genus gyrovirus of the Circoviridae family. Methods for diagnosing chicken infectious anemia include isolation and identification of viruses, and serometry. Viruses are mainly isolated from liver, spleen, and thymus tissues, but inoculation into cells and separation are time-consuming, so gene amplification using PCR is widely used. Differentiation of chicken infectious anemia is important because the symptoms are similar to those of avian leukemia virus, chicken infectious epangitis, and Marek's disease. Since the damage caused by chicken infectious anemia usually occurs when egg infection or early infection occurs, it is best to prevent infection by searching for infected individuals and vaccinating them.

Marek's Disease (MD) is an oncological disease commonly found in chickens and is characterized by infiltration of monocytes in various tissues such as peripheral blood vessels, eyes and skin. In particular, it causes cytolytic infections in lymphocytes such as the thymus, F cyst, and spleen. Up to 60% can be killed in laying hens affected by the disease, and in broilers, the disease rate can be as high as 10%. Marek's disease virus (MDV) is a cell-associated Herpes virus, a DNA virus having affinity for lymphocytes. Marek's disease can be isolated and identified as a virus or diagnosed as a serological test. For the virus isolation, blood lymphocytes, blood, spleen and tumor cells are used, and the virus can be isolated by inoculating chicken kidney cells or embryo cells. It can be diagnosed using histopathology and genetic testing using PCR. Differentiation from avian leukemia, chicken infectious anemia, reticular endothelial disease, and chicken infectious F cyst is important because of similar symptoms during infection. There are no effective treatments for Marek's disease, and early detection and prevention are the best.

Reticuloendotheliosis (RE) is a disease in which immunity decreases and tumors appear in internal organs such as the liver, spleen, and kidneys. Infection causes dwarfism or chronic tumors, causing serious economic loss, and immunosuppression, which reduces the effectiveness of the vaccine. The virus that causes RE (REV) is a retrovirus belonging to the retroviridae family and is a DNA virus. There are virus isolation and identification, and serological examination methods to diagnose reticular endothelial disease. For the isolation of the virus, various tissues such as blood, liver, stomach, thymus, and F sac can be samples, and after inoculation using fetal fibroblasts, a fluorescent antibody method using a specific antibody or gene amplification method such as PCR Use Differentiation from diseases that lead to atrophy of the thymus and F sac is important. The damage caused by REV infection is mainly seen in the case of infection or premature infection, so preventive measures are important. However, since a vaccine for reticular endothelial virus has not been developed, it is necessary to hygienically manage to prevent infection of REV from the breeder, and it should not be used to search for infected families. In addition, REV can be transmitted by being contaminated with Marek's disease and chickenpox vaccine.

Infectious Bursal Disease (IBD) is an acute infectious disease of chickens that causes severe damage to the lymphatic tissue of young chickens, causing immunosuppression. This can increase the chance of secondary pathogen infection and reduce the immunogenicity of other vaccines, which is a disease that is causing great economic damage to the poultry industry worldwide. When infected with this disease, mortality rates of up to 60% in chickens older than 3 weeks, depending on the pathogenicity of the virus, cause severe immunity in young chickens, causing economic loss. The virus that causes chicken infectious F cyst (IBDV) is a double-stranded RNA virus composed of two segments. Diagnosis includes isolation and identification of viruses and serological screening methods. F-cyst tissue is used as a sample for virus isolation and is inoculated into chicken embryonic urine membrane (CAM). In addition, gene amplification methods such as RT-PCR have been widely used as a target gene for the VP protein portion of the virus. Although the atrophy of the F capsule is a significant lesion, it is important to distinguish it from other immunosuppressive diseases that cause similar symptoms. Since chicken infectious F cyst is an acutely advanced disease, treatment after the onset is of little significance and early detection and prevention are important.

The viral immunosuppressive disease is a single or multiple infection in the poultry industry, resulting in damage not only due to the virus infection itself, but also as a secondary disease infection for immunosuppression. Preventive measures against these immunosuppressive diseases can be applied effectively in the field by overcoming the limitations of the existing amplification, identification, and PCR methods of gene amplification methods in order to prepare effective countermeasures through early detection and differential diagnosis. Development of diagnostic methods is necessary.

U.S. Patent 5,605,792 United States Patent Publication 2006-0188871

The present application is intended to provide a method for detecting whether an infection of Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus, and Infectious bursal disease virus occurs quickly and easily while showing high sensitivity and specificity.

In one aspect the present application is selected from the group consisting of Chicken anemia virus, Marek's disease virus and Reticuloendotheliosis virus A primer set for LAMP analysis for detection is provided: Chicken anemia virus specific primer set including 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 set comprising the primers represented by SEQ ID NOs: 19-22, SEQ ID NOs: 25-28 or SEQ ID NOS: 31-34 and SEQ ID NOS: 37-40, SEQ ID NOS: 43-46, SEQ ID NOS: 49-52 OR Reticuloendotheliosis virus comprising primers represented by SEQ ID NOs: 55-58 Specific primer set.

Also provided is a primer set for RT-LAMP analysis for detecting Infectious bursal disease virus: 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 may be set to SEQ ID NOs: 5 and 6, SEQ ID NOs: 11 and 12, or SEQ ID NOs: 17 and 18 Primers displayed; 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; Reticuloendotheliosis virus Specific primer sets include 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.

The primer set according to the present application may be labeled with a detectable labeling substance in one or more primers included in each set, if necessary for detection of amplification products in real time or at the end of the reaction.

In another aspect, the present application also provides a chicken anemia virus in vitro using the primer set disclosed herein, Marek's disease virus, Reticuloendotheliosis virus And Infectious bursal disease virus detection method, the method is Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And Providing a sample of a subject suspected of being infected with 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 when analyzing the LAMP or RT-LAMP reaction result and comparing it with a control sample, if there is a difference, this is Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And And determining the infection or contamination of Infectious bursal disease virus.

The primer set according to the present application is Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And Various samples that require detection of Infectious bursal disease virus, for example, immune organs and tumor tissues such as the thymus, spleen, and F sac can be used, but are not limited thereto.

Analysis of the results of the LAMP or RT-LAMP reaction in the method according to the present application can be performed in real time or after the reaction is completed, and whether or not amplification is determined through measurement using one or more of color change measurement or turbidity, for example When compared to the color change or increased turbidity, the amplified chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And It can be judged as positive for Infectious bursal disease virus.

According to the present application, a set of primers capable of specifically detecting Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus and Infectious bursal disease virus in an isothermal amplification method, and detection methods of the viruses using the primers are highly sensitive and specific as well as various specimens Can quickly detect whether the pathogen is contaminated. It can be widely used in disease monitoring samples in veterinary fields and disease monitoring in poultry farms, and if necessary, it can be directly detected without any additional treatment such as electrophoresis after termination of the reaction, thereby increasing convenience.

1 is a result of detecting the specificity and sensitivity of the chicken anemia virus specific LAMP primer set for the primer set of the combination described in Table 1 according to an embodiment of the present application by color change and agarose gel electrophoresis.
2 is a Marek's disease virus for the primer set of the combination described in Table 2 according to another embodiment of the present application It is a result of detecting specificity and sensitivity of a specific LAMP primer set by color change and agarose gel electrophoresis.
Figure 3 is a Reticuloendotheliosis virus for the primer set of the combination described in Table 3 according to another embodiment of the present application It is a result of detecting specificity and sensitivity of a specific LAMP primer set by color change and agarose gel electrophoresis.
Figure 4 Infectious bursal disease virus for the primer set of the combination described in Table 4 according to another embodiment of the present application It is a result of detecting specificity and sensitivity of a specific RT-LAMP primer set by color change and agarose gel electrophoresis.
In each of the drawings, the sensitivity is indicated in red. Reactions were performed at a total of 7 different concentrations and negative controls for each set, each concentration being as follows: DNA concentration of chicken anemia virus is 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 copy 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 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: 80copies, 5: 16copies, 6: 3.2copies, 7: 0.64copy and 8: The DNA concentration of negative control and Infectious bursal disease virus is 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.

Chicken anemia virus using LAMP and RT-LAMP method, 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.

In the poultry industry and in the industry that produces SPF (Specific Pathogen Free) chickens and eggs, viral immunosuppressive diseases are very important. The cause of these immunosuppressive diseases is that they cause single or multiple infections and cause serious economic damage by acting as a major cause of secondary disease outbreaks as well as damages caused by the virus infection itself. An effective countermeasure against these immunosuppressive diseases is to quickly identify whether the causative virus is present through early detection and selection of these immunosuppressive causative agents, and differential diagnosis with similar diseases, and to prepare preventive measures such as vaccination. Therefore, we tried to develop a detection method that overcomes the limitations of existing amplification, identification, and PCR methods, and can be effectively applied in the field, and simultaneously detects the main cause of immunosuppressive diseases in poultry.

According to the LAMP-CIAV: MDV: REV: IBDV method according to the present application, specific primers for chicken infectious anemia virus, Marek's disease virus, reticuloendothelial virus and chicken infectious F cyst virus are prepared and Loop-Mediated Isothermal Amplification (LAMP) method It is possible to detect the above four types of viruses simultaneously.

Therefore, this application uses the LAMP method of 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 Disclosed is a detection method.

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 the Infectious bursal disease virus.

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

The term “nucleic acid” as used herein refers to a single-stranded or double-stranded oligonucleotide or polynucleotide, and refers to an RNA or DNA molecule comprising one or more nucleotides or analogs 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 a genome, and in one embodiment, DNA or RNA.

As used herein, the terms “oligonucleotide” and “polynucleotide” are used interchangeably, either of which includes both. The term “primer” can also be used interchangeably with “oligonucleotide” and “polynucleotide”, and includes nucleic acids (RNA or DNA), aptamers, and the like.

The term “detection” herein refers to Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And It analyzes the presence or absence of nucleic acid, whether it is contaminated or infected with Infectious bursal disease virus, and based on LAMP and RT-LAMP analysis.

As used herein, the term “target” or “target nucleic acid” refers to a nucleic acid sequence to which a 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 reticular endothelial virus are detected by LAMP method and infectious F cyst virus is detected by RT-LAMP method.

The primer according to the present application specifically binds to the target nucleic acid through complementarity and amplifies the target nucleic acid in a LAMP manner. In particular, only genes specific to each strain / virus described in Tables 1 to 4 can be specifically detected herein.

LAMP is a nucleic acid amplification method that can amplify target nucleic acids 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) DNA polymerase having activity and 6 sites of target nucleic acid, i.e., B3 region, B2 region, B1 region, F1c region, F2c region, and F3c region in 5 '→ 3' direction (regions complementary to the region are B3c , B2c, B1c, F1, F2, and F3), at least 4 to 6 primer sets specifically designed (Korea 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.). Standard LAMP reactions include 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 six primers may be used, including LF (Loop F) and LB (Loop B).

The LAMP reaction largely includes an initiation structure production step, a cycling amplification step, and an elongation and recycling step. In the initiation step, the F2 portion constituting the inner primer FIP binds to the F2c region of the target nucleic acid to initiate a reaction. Then, 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 F1 / F1c binding, and a second inner primer BIP and an outer primer B3 bind to DNA synthesis and strand replacement DNA synthesis. As a result, a single strand formed with stem-loops at both ends of the dumbbell shape is formed, and DNA synthesis starts from the 3 'end of the F1 region. Subsequently, in the cycling amplification step, only two inner primers are used, and at first, one inner primer binds to the ring and strand replacement DNA synthesis occurs. A single strand of formation is formed, and as a template, strand replacement DNA synthesis is continuously performed to amplify about 10 ⁹ products within 1 hour. For more detailed principles and features and functions of each F3 primer, B3 primer, FIP primer, BIP primer, Loop F primer (LF), and Loop B (LB) primer, the aforementioned references Notomi et al., 2000 and Nagamine et al., 2002. RT (reverse transcription) LAMP is a template that synthesizes RNA first into DNA and then performs LAMP. Methods for synthesizing RNA into DNA are known in the art.

The term “complementary” or “complementarity” herein refers to a state in which a primer or oligonucleotide is capable of specifically binding a target nucleic acid to a target nucleic acid through pairing of a Warson-Crick base at predetermined hybridization (hybridization), binding or annealing conditions. Refers to, and includes both partially complementary, 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 degree of complementarity to bind to the target nucleic acid and 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 the step of binding the target nucleic acid or template in a LAMP reaction in which the primer according to the present application is used. The degree of hybridization is influenced by various factors such as the degree of complementarity of any two nucleic acid molecules involved, their melting temperature (Tm) or stringency of the hybridization. Hybridization reaction conditions may 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 bind, complementarity, reaction temperature, ionic strength, and / or concentration of a general substance included in the hybridization reaction solution. And 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; 5th Ed, 2002 and the like.

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 non-target nucleic acid, but only to the 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 nucleic acid or complementary sequence of Infectious bursal disease virus, and can be determined by considering primer sequence length, buffer, nucleic acid type, pH, magnesium concentration and reaction temperature in template-based DNA or RNA synthesis. will be.

The target genes for each virus are as described in Tables 1 to 4, and each gene site was specifically recognized to detect each virus with high sensitivity. 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 Designed to specifically detect the Infectious bursal disease virus through the LAMP method, it 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: forward inner primer (FIP), backward inner primer (BIP), forward outer primer (F3) and backward outer primer (B3). In addition, for a rapid reaction (Accelerated LAMP), a total of six primers may be used, including 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, the aforementioned references Notomi et al., 2000 and Nagamine et al., 2002 Can be referenced.

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

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 bind to a target nucleic acid or its complementary sequence. Primers of substantially the same sequence have 70% complementarity with primers represented by SEQ ID NOS: 1-78, in particular 80% complementarity, more particularly 90% complementarity, even more particularly 95%, 96%, 97%, 98%, 99% or It has 100% complementarity.

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 along with the LAMP reaction or in a separate reaction.

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

The 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 It is represented 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 selected from the group consisting of SEQ ID NO: 21, 27, or 33 Marked with 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 reticular endothelial virus The F3 primer for detection is represented by any one primer selected from the group consisting of SEQ ID NOs: 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 NO: 63, 69, or 75; And the BIP primer is represented by one primer selected from the group consisting of SEQ ID NO: 64, 70, or 76, and can be used in various combinations for each primer for each strain.

Optionally, the primer set may further include LF primer and LB primer. The LF primer for detecting the 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 reticular endothelial 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 F cyst virus The LF primer for detection is a primer represented by one selected from the group consisting of SEQ ID NOs: 65, 71, or 77, and the LB primer is a primer represented by one selected from the group consisting of SEQ ID NOs: 66, 72, or 78, each It can be used in various combinations by strain.

In one embodiment, the LAMP primer set capable of specifically detecting Chicken anemia virus herein is a FIP selected from SEQ ID NO: 3, 9 or 15, a 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 NOs: 2, 8 or 14. In another embodiment, the LAMP primer set capable of specifically detecting Chicken anemia virus is a FIP selected from SEQ ID NO: 3, 9 or 15, a BIP selected from SEQ ID NO: 4, 10 or 16, SEQ ID NO: 1, 7 or 13 F3 selected from B3 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 the Marek's disease virus herein The specifically detectable LAMP primer set includes 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. In another embodiment the Marek's disease virus The specifically detectable LAMP primer set includes 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 The specifically detectable LAMP primer set is selected from FIPs selected from SEQ ID NOs: 39, 45, 51 or 57, BIPs selected from SEQ ID NOs: 40, 46, 52 or 58, and SEQ ID NOs: 37, 43, 49 or 55 F3 and B3 selected from SEQ ID NOs: 38, 44, 50 or 56. In another embodiment the Reticuloendotheliosis virus The specifically detectable LAMP primer set 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, 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 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, SEQ ID NO: F3 selected from 61, 67 or 73 and B3 selected from SEQ ID NOs: 62, 68 or 74. In another embodiment, Infectious bursal disease virus The specifically detectable RT-LAMP primer set includes 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 numbers 62, 68 or 74, LF selected from SEQ ID NOs: 65, 71 or 77 and LB selected from SEQ ID NOs: 66, 72 or 78.

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

Samples in which the primer set and method according to the present application can be used include poultry disease-sensitive samples, slaughter products in slaughterhouses, broilers in the poultry industry. It can be carried out on various samples from the industry that produce SPF chicken and equivalent eggs, including samples from the laying hens, broilers, and native chicken farms.

In addition, the biological sample may be obtained directly by a conventional method for obtaining a sample immediately before the test, or may be stored separately.

In another embodiment, the sample according to the present application may be a nucleic acid separated from the sample described above as a sample. Isolation means that it is isolated from a sample containing nucleic acid, and includes those separated in various purity.

The primer according to the present application and a method comprising the same are used in the analysis of the LAMP and RT-LAMP methods for the detection of the virus in any sample where the virus is likely to 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 in a total of 25 ul, Includes 1 × ThermoPol reaction buffer (Mmonitor, South Korea), 8 U Bst DNA polymerase (Mmonitor, South Korea) and samples requiring analysis of 1.0 to 9.5 μl.

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 in a total of 25 ul. Includes mM dNTPs, 1 × ThermoPol reaction buffer (Mmonitor, South Korea), 8 U Enzyme mix (Mmonitor, South Korea) and samples requiring analysis of 1.0 to 9.5 μl.

The reactants are then reacted at a temperature suitable for DNA polymerase activity. The reaction temperature can be determined without difficulty considering the enzyme and target nucleic acid sequences used in the reaction. The reactants are 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 used for quantitative or qualitative analysis compared to the product of the negative control sample.

In addition, when it is necessary to detect whether non-specific amplification is caused by the occurrence of non-specific priming of the primer, non-specifically synthesized total amplification is performed using a non-specific nucleic acid binding agent, e.g. bromide or picogreen, in a control reaction that does not include a template. The amount of product can be determined.

The primer set according to the present application can be usefully used for various analyzes for screening, presence / absence detection and quantification of contamination or infection in various samples suspected of infection or contamination of the virus for detection of infection or contamination.

Chicken anemia virus using the 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 group or reference group, chicken anemia virus, 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 the examples are merely illustrative and do not limit the scope of the present application in any way.

The present invention may be practiced using conventional techniques within the skill level of those skilled in the art of cell biology, cell culture, molecular biology, genetic transformation technology, microbiology, and DNA recombination technology, unless otherwise stated. In addition, a more detailed description of the general technology can be found in 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

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

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

Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And For accurate detection of Infectious bursal disease virus, various genes were obtained from NCBI and GenBank to select the final primer specific for the virus. Specifically, the gene VP1 that forms the envelope protein of CAV as a target of CAV and the gene VP3 (GenBank accession number, AB031296) that induces apoptosis in chickens, and the gene that causes cancer of chickens as a target of MDV (GenBank accession number, KT768121) , F3, B3, FIP, based on the surface glycoprotein (GenBank accession number, GQ415645) constituting the viral envelope as a target of REV and the protein (GenBank accession number, EF070167) that constitutes the viral particle surface envelope as a target of IBDV Primers were prepared for each primer of BIP, LF, and LB to complete the primer set for LAMP amplification (Tables 1 to 4). Each primer sequence was verified using the National Center for Biotechnology Information (NCBI), 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 reactions 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 is performed using one combination of red primers (see Tables 1 to 4) among six primer combinations. Through the color change of the reactants and the specificity was confirmed as a result of electrophoresis (Fig. 1a, 2a and 3a). In addition, reactants can be reacted through 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, which have RNA as a genome. The specificity was confirmed as a result of color change and electrophoresis (FIG. 4A).

To this end, genomes were extracted from viruses of Chicken anaemia virus, Fowl adenovirus, Infectious Laryngotracheitis virus, Marek's disease virus, Reticuloendotheliosis virus and Avian leukosis virus obtained in Example 1, and each DNA was prepared at 500 ng / ul. Subsequently, 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 at a total volume of 25 μl, using a Mmiso DNA amplification kit (Mmonitor, South Korea) for LAMP analysis, 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 the composition comprising 2 μl of the isolated DNA Used. After the isothermal amplification at 63 ° C for 40 minutes with the above composition, the reaction was terminated by reacting at 80 ° C for 5 minutes.

In addition, after extracting the RNA genome 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, each RNA was extracted at a concentration of 500 ng / ul. Prepared to perform RT-LAMP. For RT-LAMP analysis, 0.2 μM of each F3 and B3 primer, 1.6 μM of each FIP and BIP primer, and 0.8 μM of each LF and LB primer at a total volume of 25 μl using the Mmiso RNA amplification kit (Mmonitor, South Korea). , 2XThermoPol reaction buffer (Mmonitor, South Korea), 8 U Enzyme mix (Mmonitor, South Korea) and a solution of the composition containing 2 μl of the isolated RNA was subjected to isothermal amplification at 58 ° C. for 40 minutes and then at 80 ° C. The reaction was terminated 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 the corresponding virus was specifically detected. The above results can sufficiently express specificity with different combination primer sets (Tables 1 to 4).

Next, using the primer set of each combination (Table 1 to Table 4) Chicken anemia virus, Marek's disease virus, Reticuloendotheliosis virus And Sensitivity to Infectious bursal disease virus was confirmed. To do this, after extracting DNA from liver tissue of chickens infected with CAV, MDV and REV alone, samples of 500pg / ul, 100pg / ul, 20pg / ul, 4pg / ul, 800fg / ul, 160fg / ul, 32fg / ul After the preparation, the mixture was subjected to isothermal amplification at 63 ° C for 35 minutes at the same composition, and then reacted at 80 ° C for 5 minutes to confirm the color change of the reactants (FIGS. 1B, 2B and 3B). In addition, after extracting RNA from F sac of chickens infected with IBDV alone, samples of 500 pg / ul, 100 pg / ul, 20 pg / ul, 4 pg / ul, 800 fg / ul, 160 fg / ul, 32 fg / ul were prepared, and 40 at 58 ° C. After isothermal amplification for a minute, the color change of the reactants was confirmed by reacting at 80 ° C. for 5 minutes (FIG. 4B). In addition, based on the above sensitivity results, F3 of primer 2 and B3 of primer 1 specific for Chicken anemia virus, F3 of primer 5 specific for Marek's disease virus and B3 of primer 6 and F3 and primer of primer 7 specific for Reticuloendotheliosis virus PCR (94 ° C, 5min; 94 ° C, 30sec, 55 ° C, 30sec, 72 ° C, 30sec, 35cycles; 72 ° C, 7min) was performed using B3 of 9. 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) was carried out. The products amplified through PCR and RT-PCR were electrophoresed 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 the TOPcloner TA kit (Enzynomics, Korea) Into each of them to prepare pTOP-CAV, pTOP-MDV, pTOP-REV and pTOP-IBDV.

In addition, the prepared pTOP-CAV plasmid was added to the reaction solution of 6.25 x 10 ³ copies, 1.25 x 10 ³ copies, 2.5 x 10 ² copies, 50copies, 10copies, 2copies, 0.4copy, and pTOP-MDV plasmid in the 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, 0.8copy, 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 ℃ for 40 minutes at the same composition using a concentration of 0.4copy, and then reacted for 5 minutes at 80 ℃, and the sensitivity was confirmed as a result of color change and electrophoresis of the reactants (FIGS. 1C, 2C, and FIG. 3c and 4c).

Thus, while present in purple before the reaction, while up the DNA synthesis reaction is Mg ²⁺ ion concentration in the reduced accordingly takes place in the purple to blue color change (s). A change in color is a positive response indicating that the nucleic acid has been amplified.

The method according to the present application can quickly and conveniently look at the results and perform qualitative analysis. Also, when measuring absorbance at a wavelength of 650 nm using a spectrophotometer, there is an advantage of qualitative and quantitative analysis.

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

<110> REPUBLIC OF KOREA (Animal and Plant Quarantine Agency) <120> Primers for LAMP based detection of virus causing immune          suppression in poultry and its use <130> DP201704003P <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 chicken anemia virus, including F3 primer, B3 primer, FIP primer, and BIP primer,
The primer set includes a primer set comprising the F3, B3, FIP, and BIP primers represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; A primer set comprising primers represented by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10; Or a primer set for LAMP analysis for chicken anemia virus detection, which is a primer set comprising primers represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.
According to claim 1,
The primer set further includes LF primer and 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 chicken anemia virus detection:
A primer set comprising the F3, B3 FIP, BIP, LF, and LB primers represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively;
A primer set comprising primers represented by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12; or
A primer set comprising primers represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.
delete delete The method of claim 1 or 2,
At least one primer of each primer set is labeled with a detectable labeling material, a primer set for LAMP analysis for chicken anemia virus detection.
A method for detecting chicken anemia virus using the LAMP method in vitro using the primer set according to claim 1 or 2, the method comprising the following steps:
Providing a sample suspected of infection or contamination with chicken anemia 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, determining the sample as chicken anemia virus infection or contamination.
The method of claim 6,
The sample is a sample of the disease evaluation of chickens; Slaughter products; Broiler. Spawning, breeding, free-range chickens or samples from these breeding sites; SPF (Specific Pathogen Free) A method for detecting chicken anemia virus using the LAMP method, which includes a chicken or its eggs or a sample derived from these kennels.
The method of claim 6,
Analysis of the result of the LAMP reaction is determined by measurement using at least one of color change measurement or turbidity (turbidity) of the reaction result, chicken anemia virus detection method using the LAMP method.
A composition for detecting anemia virus of chickens using the LAMP method comprising the primer set according to claim 1 or 2.
Kit for chicken anemia virus detection using the LAMP method comprising the primer set according to claim 1 or claim 2.

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