KR102525970B1 - Primers for multiplex PCR based detection of Infectious laryngotracheitis virus, Fowlpox virus, and Reticuloendotheliosis virus in poultry and its use - Google Patents

Abstract

The present application discloses a primer set and method capable of simultaneously and accurately discriminating fowlpox virus, transmissible laryngotracheitis virus, and reticuloendotheliosis virus. The primer set and method according to the present application enable early detection of the virus with high specificity and sensitivity, allowing accurate differential diagnosis between fowlpox and infectious laryngeal tracheitis, which show similar clinical symptoms, and fowlpox virus into which the reticuloendothelial virus gene has been inserted. Through this, rapid follow-up measures such as vaccination can minimize the damage of infection, thereby contributing to reducing economic damage to farmhouses due to productivity decline.

Description

Primers for multiplex PCR based detection of Infectious laryngotracheitis virus, Fowlpox virus, and Reticuloendotheliosis virus in poultry and its use}

The present application is a technology related to a genetic diagnosis method capable of simultaneously detecting Infectious laryngotracheitis virus (ILTV), Fowlpox virus (FWPV), and Reticuloendotheliosis virus (REV).

Infectious laryngotracheitis (ILT) is a respiratory disease of birds caused by an infectious laryngotracheitis virus (Gallid herpesvirus type I, GaHV-1 or ILTV), the main lesions of which are laryngitis and tracheitis. In chickens infected with ILTV, the trachea becomes inflamed, causing respiratory symptoms such as sneezing and coughing. The morbidity rate of the disease is around 5%, but it appears up to 100% in the case of severe epidemic ILTV, and the mortality rate is usually 20%.

Fowlpox is a disease in which nodules are formed on the skin, oral and respiratory mucous membranes of chickens and turkeys caused by Fowlpox virus (FWPV). Depending on the susceptibility of the host, the pathogenicity of the virus, the site of occurrence, and the route of transmission, the clinical symptoms are the dermatological type (dry type) in which nodular proliferation is formed on the hairless skin and the mucosal type (wet type) in which nodular growth is formed in the mucous membranes of the upper respiratory tract, oral cavity, and esophagus. ) are separated. The skin type is common, and nodular lesions appear on the hairless areas of the comb, eyelids, mouth, wings and legs. do. In addition, the skin type has a low mortality rate, but the mucosal type has a high mortality rate due to difficulty in breathing, and it is known that the mortality rate increases when the breeding environment is poor or combined with other diseases.

Reticuloendotheliosis is caused by gamma-retrovirus, and when infected with this virus, dwarfism may appear due to a severe decrease in weight gain and a decrease in immune function due to damage to the immune system. As a result, chickens infected with reticuloendothelial disease may develop secondary infection by bacteria and parasites such as colibacillosis or coccidiosis, and are also easily infected with viral diseases such as chicken pox or infectious laryngotracheitis.

The clinical symptoms and lesions of fowlpox (mucosal type) are very similar to transmissible laryngotracheitis, and the multi-infection of fowlpox virus and transmissible laryngotracheitis virus is increasing, so differential methods are important. As a commonly known histopathological differential diagnosis, in the case of transmissible laryngotracheitis, inclusion bodies are observed in the nucleus of infected cells, but in the case of fowlpox, inclusion bodies are observed in the cytoplasm. In addition, the infected tissue is inoculated into the chorioallantoic membrane (CAM) of 9- to 11-day-old chicken fetuses, and the CAM is collected after 5 to 7 days and the virus can be identified through polymerase chain reaction. Methods for detecting reticuloendotheliosis virus include virus isolation and identification using animal cells, gene-based detection method, serological test (enzyme immunoassay), and the like. These methods require skilled personnel and a lot of time, and thus have limitations in diagnosing viruses quickly and accurately. In addition, there are many known cases in which the existing vaccine does not sufficiently exert a protective effect due to the appearance of a variant fowlpox virus in which the gene of the fowlpox virus has been inserted into the gene of the fowlpox virus, the transmissible laryngotracheitis virus, and the reticuloendotheliosis virus. There is a need for a method capable of simultaneously and accurately detecting fowlpox virus and reticuloendotheliosis virus into which the gene of .

Prior literature

Song, H., Y. Bae, S. Park, H. Kwon, H. Lee, and S. Joh. 2018. Loop-mediated isothermal amplification assay for detection of four immunosuppressive viruses in chicken. J Virol Methods. 256:6-11.

Wozniakowski, G., M. Frant, and A. Mamczur. 2018. Avian Reticuloendotheliosis in Chickens - An Update on Disease Occurrence and Clinical Course. J Vet Res. 62: 257-260.

The present application aims to provide a method that can quickly and conveniently detect the infection of ILTV, FWPV, REV-integrated FWPV and REV in one reaction with high sensitivity and specificity.

In one aspect, the present application is a primer pair for multiplex PCR analysis for detecting Infectious laryngotracheitis virus (ILTV), Fowlpox virus (FWPV), Reticuloendotheliosis virus-integrated Fowlpox virus (REV-integrated FWPV) and Reticuloendotheliosis virus (REV) as follows Provided is a primer set for simultaneous multiplex detection of avian co-infection virus comprising a combination of: an ILTV-specific primer pair including primers represented by SEQ ID NOs: 1 and 2; A FWPV-specific primer pair comprising the primers represented by SEQ ID NOs: 3 and 4; REV-integrated FWPV containing primers represented by SEQ ID NOs: 5 and 6 specific primer pairs; REV comprising primers represented by SEQ ID NOs: 7 and 8 specific primer pairs.

The ILTV, FWPV, REV-integrated FWPV and REV detection, diagnosis, or discrimination primer set according to the present application may be provided in the form of a composition or kit containing the same.

In another aspect, the present application is a method for detecting or diagnosing infection of ILTV, FWPV, REV-integrated FWPV and / or REV in vitro using the disclosed primer set, the method comprising ILTV, FWPV, REV-integrated FWPV and REV Providing a sample of a bird suspected of being infected; providing a primer set according to the present disclosure; Performing simultaneous multi-gene polymerase chain reaction (multiplex PCR) using the sample and the primer set; And analyzing the simultaneous multiplex PCR result and comparing it with a control sample to determine if there is a difference as ILTV, FWPV, REV-integrated FWPV and REV infection or contamination.

The primer set according to the present disclosure can be used in a variety of avian-derived and/or environmental samples, such as poultry pathology samples, where detection of ILTV, FWPV, REV-integrated FWPV and REV is required; broiler. Specimens derived from laying hens, breeders, native chickens or their breeding grounds; It includes SPF (Specific Pathogen Free) chickens or their eggs, or specimens derived from these farms, and samples such as organs or combs may be used, but are not limited thereto.

Analysis of the simultaneous multiple gene polymerase chain reaction results in the method according to the present invention is to confirm the size of the product through electrophoresis after completion of the reaction, the PCR result is 877bp for the ILTV, 630bp for the FWPV, 429bp for the REV-integrated FWPV , And if the REV is 207 bp, ILTV, FWPV, REV-integrated FWPV and REV, respectively can be judged positive.

According to the present application, a primer set capable of specifically detecting ILTV, FWPV, REV-integrated FWPV and REV by a simultaneous multiplex PCR method and a method for detecting the viruses using the same have high sensitivity and specificity As a result, it is possible to quickly check the contamination of pathogens in various samples, including disease analysis samples in the veterinary field, and also contribute to the prevention of immunosuppressive diseases and establishment of countermeasures through early diagnosis of immunosuppressive diseases in farms. can

1 is a result of detecting the specificity of ILTV, FWPV, REV-integrated FWPV and REV-specific primer sets with respect to the primer sets of the combinations described in Table 1 according to one embodiment of the present application by agarose gel electrophoresis.
Figure 2 is a result of detecting the sensitivity of ILTV, FWPV, REV-integrated FWPV and REV-specific primer sets to the primer sets of the combinations described in Table 1 according to one embodiment of the present application by agarose gel electrophoresis.
In the figure above, reactions were performed at a total of seven different concentrations and negative control groups for each set, and each concentration was as follows: The concentration of plasmid DNA containing the ILTV gene was 1: 1.0 x 5 ⁶ copies, 2: 1.0 x 5 ⁵ copies, 3: 1.0 x 5 ⁴ copies, 4: 1.0 x 5 ³ copies, 5: 1.0 x 5 ² copies, 6: 1.0 x 5 ¹ copies, 7: 1.0 x 5 ⁰ copies, and 8: negative control; The concentration of plasmid DNA containing the gene of FWPV was 1: 1.0 x 5 ⁶ copies, 2: 1.0 x 5 ⁵ copies, 3: 1.0 x 5 ⁴ copies, 4: 1.0 x 5 ³ copies, 5: 1.0 x 5 ² copies, 6: 1.0 x 5 ¹ copies, 7: 1.0 x 5 ⁰ copies, and 8: negative control; The concentration of plasmid DNA containing the gene of REV-integrated FWPV was 1: 1.0 x 5 ⁶ copies, 2: 1.0 x 5 ⁵ copies, 3: 1.0 x 5 ⁴ copies, 4: 1.0 x 5 ³ copies, 5: 1.0 x 5 ² copies, 6: 1.0 x 5 ¹ copies, 7: 1.0 x 5 ⁰ copies, and 8: negative control; The concentration of plasmid DNA containing the REV gene was 1: 1.0 x 5 ⁶ copies, 2: 1.0 x 5 ⁵ copies, 3: 1.0 x 5 ⁴ copies, 4: 1.0 x 5 ³ copies, 5: 1.0 x 5 ² copies, 6: 1.0 x 5 ¹ copies, 7: 1.0 x 5 ⁰ copies, and 8: negative control.

In this hospital, ILTV (Infectious laryngotracheitis virus), FWPV (Fowlpox virus), FWPV (Fowlpox virus), REV-inserted FWPV (Reticuloendotheliosis virus-integrated Fowlpox virus) and REV (Reticuloendotheliosis virus) infection, which cause complex infections in birds, are tested once. It is based on the discovery of genetic diagnostic technology that can be detected quickly and accurately with high sensitivity and specificity at the same time.

Accordingly, in one aspect, the present application provides a pair of primers of SEQ ID NO: 1 and SEQ ID NO: 2 for detecting ILTV, which can specifically detect ILTV, FWPV, REV-integrated FWPV and REV of birds, including poultry, respectively, in one reaction; Primer pairs of SEQ ID NOs: 3 and 4 for detecting FWPV; Primer pairs of SEQ ID NOs: 5 and 6 for detecting REV-integrated FWPV; It relates to a primer set for simultaneous multiplex detection of an avian co-infecting virus comprising primer pairs of SEQ ID NOs: 7 and 8 for detecting REV in combination.

Primer sets according to the present application are listed in Table 1.

[Table 1]

As used herein, a primer refers to ribonucleotides or deoxyribonucleotides of any length, includes both single-stranded and double-stranded, and includes both primers and probes, and a gene specific to each strain to be detected. It refers to an oligonucleotide having a sequence complementary to at least a portion of, and is the starting point of polymerization in a polymerization reaction using a polymerase such as a polymerase chain reaction.

Primer sets according to the present disclosure can be used in a variety of nucleic acid amplification techniques.

In one embodiment the primer set according to the present application is used in particular for PCR.

As used herein, the term PCR (Polymerase Chain Reaction) is a method widely known in the art as a method of exponentially amplifying a starting nucleic acid material by sequentially synthesizing nucleic acids using a polymerase, and determining the presence or absence of a specific nucleic acid. It is a concept that includes both qualitative analysis PCR to confirm, quantitative PCR to measure the amount of a specific nucleic acid, and real-time PCR to enable qualitative and quantitative analysis by tracking the PCR process in real time.

As used herein, the term real-time PCR is a method capable of tracking the process in real time, and the numerical value used for qualitative or quantitative analysis is Ct or Cq. The terms are used interchangeably, and the term indicates the number of PCR cycles at the point where the amplification curve reaches a specific threshold value in the amplification process, and the threshold value is usually the point at which the curvature of the amplification curve is maximized. determined and located in the exponential phase. Quantification or quantitative analysis of nucleic acids using this value can be performed according to the manual of the manufacturer of the real-time PCR device. The step of checking whether a specific DNA is amplified in this PCR can be performed by checking the size of the amplified DNA product through electrophoresis or the like. Alternatively, the real-time polymerase chain reaction is a polymerase chain reaction using a fluorescent material such as SYBR Green on the market, and the degree of fluorescence can be measured simultaneously with amplification without an additional confirmation procedure after amplification, or fluorescence resonance energy transfer , FRET) can be used to determine whether or not amplification is amplified, and additionally, it can be performed by a method including checking the melting temperature at which the double helix of the amplified DNA product is separated.

As used herein, detection means detecting the presence or amount of a nucleic acid, and in particular PCR (quantitative and qualitative PCR, real-time (including quantitative and qualitative PCR).

Simultaneous multiple detection herein means that each strain can be specifically detected without cross-reactivity to other strains in one amplification reaction by including a specific primer set capable of detecting each strain in one reaction. . For example, when one sample is infected with two or more strains to be detected herein, each strain can be specifically detected at the same time in one reaction.

As mentioned above, each primer set according to the present application can specifically detect a corresponding virus in a poultry sample suspected of being contaminated with ILTV, FWPV, REV-integrated FWPV and/or REV in one reaction.

In this respect, the present application relates to a composition or kit for simultaneous multiplex detection of avian complex infection viruses of ILTV, FWPV, REV-inserted FWPV and REV comprising a combination of the above primer sets.

In one embodiment, such a composition includes a buffer necessary for the reaction of PCR, Taq polymerase and MgCl ₂ . Various buffers known in the art may be used, for example, Tris-HCl, pH 9.0 buffer may be used, but is not limited thereto. Taq polymerase is commercially available, for example, AmpliTaq Gold (Applied Biosystems, USA) may be used, and an appropriate concentration, for example, 1.5 mM to 2.5 mM MgCl ₂ may be included.

In one embodiment, the kit further includes a positive control, a negative control, and instructions for use. The negative control group may use DNA of a different type of virus other than the detection target. For example, the positive control group may use a plasmid containing the gene of the detection target virus.

In another aspect, the present application is a simultaneous multiple detection method of avian complex infection virus for specifically detecting the virus in a poultry sample suspected of being contaminated with ILTV, FWPV, REV-inserted FWPV and REV using a combination of primer sets disclosed in Table 1 It is about.

In one embodiment, the method according to the present disclosure includes the steps of providing an algae-derived sample or environmental sample requiring simultaneous multiple detection of ILTV, FWPV, REV-inserted FWPV and REV: and mixing the sample with a combination of primer sets disclosed herein and performing a nucleic acid amplification reaction.

Birds or poultry applied to the primer, composition, kit or method according to the present application include various birds such as chickens, ducks, geese, quails, turkeys, canaries, parrots, pheasants, pigeons, seagulls, and ostriches. One embodiment includes farmed birds, particularly chickens or turkeys.

In one embodiment, the algae-derived samples include avian pathology samples, poultry industry broilers, laying hens, breeders, egg, SPF (Specific Pathogen Free) chickens and eggs.

In one embodiment, an avian disease sample is used, which includes nasal cavity, oral cavity, tracheal lung, skin including comb, liver or spleen.

In another embodiment, an environmental sample is used, and the environmental sample includes various samples from the industry, such as samples from farms, mites, mosquitoes, contaminated feathers or hides, chicken dander, and dust from barn floors.

The method according to the present invention includes determining the size of the amplification product after completion of the nucleic acid amplification reaction; And further comprising determining whether ILTV, REV-inserted FWPV and / or REV in the sample are present or infected based on the size of the amplification product, and the presence of each virus according to the size of the amplification product Alternatively, the infection results are shown in Table 2 below.

[Table 2]

In the table above, ○ indicates the presence of an amplification product of the corresponding size, and X indicates the absence of an amplification product of the corresponding size.

The product (amplicon) amplified by PCR has a sequence corresponding to the molecule used as a template, and can be analyzed by various methods known in the art. These methods are known in the art and, for example, agarose gel electrophoresis, real-time CR analysis, SSCP (single strand conformational polymorphism), RFLP (restriction fragment length polymorphism), CZE (capillary zone electrophoresis), WAVE (HPLC-based nucleic acid analyzing echnology), microchip, but is not limited thereto. In one embodiment of the present application, the PCR product along with the size marker is analyzed by electrophoresis on an agarose gel.

In one embodiment, the method according to the present application is a total of 20 μl of reaction of simultaneous multiplex PCR in one embodiment, ILTV-specific SEQ ID NOs: 1 and 2 in Black PCR premix QM (Ventech Science, South Korea) of primers, primer pairs of FWPV specific SEQ ID NOs: 3 and 4, primer pairs of REV-integrated FWPV specific SEQ ID NOs: 5 and 6, primer pairs of REV specific SEQ ID NOs: 7 and 8, and 3.0 to 5.0 μl of analysis Necessary samples are included. The size of the PCR product or reaction product is 877 bp for the ILTV, 630 bp for the FWPV, 429 bp for the REV-integrated FWPV, and 207 bp for the REV.

In one embodiment, in the method according to the present application, the nucleic acid amplification reaction includes heating at 94 ° C for 5 minutes, denaturation at 94 ° C for 45 seconds, annealing at 58 ° C for 45 seconds, and polymerization at 72 ° C for 45 seconds, repeated 38 times in one cycle. and polymerization at 72° C. for 5 minutes.

Hereinafter, examples are presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, but the present invention is not limited to the following examples.

Example

Example 1. ILTV, FWPV and REV sample preparation

DNA was extracted from organ, comb, and liver tissue samples of chickens diagnosed with ILTV, FWPV, and REV herein using a QIAamp DNA Mini kit (Qiagen, Germany) according to the manufacturer's method.

Example 2. Construction of Primers Specific to ILTV, FWPV, REV-integrated FWPV and REV

For accurate detection of ILTV, FWPV, REV-integrated FWPV and REV, the sequences of target genes of each virus selected herein were obtained from NCBI and GenBank, and final primers specific to each virus were selected.

There is no known diagnostic method that can simultaneously detect FWPV with REV insertion, FWPV, and REV. FWPV forward primers and REV reverse primers were designed to select corresponding sites to confirm that REV is present in FWPV. In addition, in order to confirm that the entire genome including the LTR portion of REV is inserted, primers specific to REV were designed in the corresponding gene (gp90). NPH-I of FWPV is a nucleoside triphosphatephosphohydrolase-I, which is an essential gene for terminating transcription by binding to DNA-dependent ATPase, and is a specific site for detecting FWPV.

The target of ILTV is envelope glycoprotein G (GenBank accession number, JN804826) involved in viral replication, the target of FWPV is transcription termination factor (GenBank accession number, AF198100), which terminates transcription, and the target of REV-integrated FWPV is FWPV. To detect REV inserted into the gene of FWPV, forward primers based on ORF 201 of FWPV, long terminal repeat of REV (GenBank accession number, AF246698), and envelope glycoprotein (GenBank accession number, MG471384) as the target of REV ) and reverse primers, respectively, to complete simultaneous multiplex PCR detection primers and detection methods (Table 3).

Each primer sequence was verified using NCBI (National Center for Biotechnology Information), Genbank, and Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi website), and as a result, the base sequence matched except for the virus. this didn't exist

[Table 3] Primer pairs and reaction conditions for multiplex PCR

Example 3. Confirmation of specificity and sensitivity of simultaneous multi-gene polymerase chain reaction

Simultaneous multi-gene polymerase chain reaction was performed on ILTV, FWPV, REV-integrated FWPV and REV using a combination of four types of primer pairs in Table 3, followed by agarose gel electrophoresis and sequencing of the amplified products As a result, it was confirmed that the primers specifically amplified only the target virus in sizes of 877bp, 630bp, 429bp, and 207bp, respectively (FIG. 1).

To this end, Black PCR premix QM (Ventech Science, South Korea) was used, and 1 μl each of ILTV, FWPV, REV-integrated FWPV and REV DNA extracted in Example 1 as a template, 4 pairs of primers (Table 1 concentration of) or a pair of primers for each virus (see concentration in Table 1), and sterile distilled water were added to a total volume of 20 μl, followed by PCR reaction, and the reaction conditions were as follows: at 94 ° C. Heating for 5 minutes, denaturation at 94°C for 45 seconds, annealing at 58°C for 45 seconds, and polymerization at 72°C for 45 seconds repeated 38 times in one cycle, polymerization at 72°C for 5 minutes.

When the 4 types of primer sets designed herein are used for simultaneous multiple detection, a total of 4 types of PCR products with different sizes can be detected, and the infection results according to the types of PCR products detected are shown in Table 4 below.

[Table 4]

Simultaneous multiple PCR results are as described in FIG. 1 . As shown in Figure 1, the primer set according to the present application can specifically detect ILTV, FWPV, REV-integrated FWPV and REV in simultaneous multi-gene polymerase chain reaction, and also specific primers for each virus In the gene polymerase chain reaction used, it was shown that only the target virus was specifically detected. The amplified product above was separated from the agarose gel and as a result of requesting an external sequencing (Cosmogenetech, South Korea), it is a sequence specific to the virus, which shows that the primers according to the present application are specific for each virus without cross-reactivity. detection was confirmed.

Next, the sensitivity to ILTV, FWPV, REV-integrated FWPV and REV was confirmed using each combination of primer sets (Table 3). To this end, DNA was extracted from organs, livers, and combs of chickens infected with ILTV, REV-integrated FWPV, and REV alone, and then each target product amplified through PCR using the primer set according to the present application was subjected to 1% TAE agarose gel. After electrophoresis was performed, gel extraction (Qiagen, Germany) was performed according to the manufacturer's method, and pGEM-T Easy Vector (promega, USA) was inserted according to the manufacturer's method, respectively, and pGEM-ILTV, pGEM-FWPV, pGEM-REV- Integrated FWPV and pGEM-REV were produced.

In addition, 1.0 x 5 ⁶ copies, 1.0 x 5 ⁵ copies, 1.0 x 5 ⁴ copies, 1.0 x 5 of the prepared pGEM-ILTV, pGEM-FWPV, pGEM-REV-integrated FWPV and pGEM-REV in μl reaction solution, respectively. ³ copies, 1.0 x 5 ² copies, 1.0 x 5 ¹ copies, 1.0 x 5 ⁰ copies Diluted to a concentration of 1.0 x 5 0 copy As a result of simultaneous multi-gene polymerase chain reaction, pGEM-ILTV, pGEM-FWPV at a concentration of 1.0 x 5 ⁴ copies or more , it was confirmed that pGEM-REV-integrated FWPV and pGEM-REV could be simultaneously detected, and as a result of performing individual PCR for one virus using primers specific to each virus, the concentration of 1.0 x 5 or more than ² copies Viruses were detected in each (Fig. 2).

As shown in Figure 2, the detection of viruses using the primer set according to the present application has a sensitivity of 1.0 x 5 when using all four genomes of pGEM-ILTV, pGEM-FWPV, pGEM-REV-integrated FWPV, and pGEM-REV. ⁴ copies; 1.0 x 5 ² copies when plasmid containing ILTV genome is used; 1.0 x 5 ² copies when plasmid containing FWPV genome is used; 1.0 x 5 ² copies when plasmid containing REV-integrated FWPV genome is used; In the case of using a plasmid containing the REV genome, the sensitivity was very high with 1.0 x 5 ² copies.

<110> REPUBLIC OF KOREA (Animal and Plant Quarantine Agency) <120> Primers for multiplex PCR based detection of Infectious laryngotracheitis virus, Fowlpox virus, and Reticuloendotheliosis virus in poultry and its use <130> DP202012004P <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> artificial sequence <220> <223> ILTV forward <400> 1 tgagcggctt cagtaacata g 21 <210> 2 <211> 19 <212> DNA <213> artificial sequence <220> <223> ILTV Reverse <400> 2 ctactgctgg agcgtagag 19 <210> 3 <211> 23 <212> DNA <213> artificial sequence <220> <223> FWPV Forward <400> 3 agttctcttc taggacgaag gat 23 <210> 4 <211> 24 <212> DNA <213> artificial sequence <220> <223> FWPV Reverse <400> 4 gtaggcttat cgggtaatac tgtg 24 <210> 5 <211> 24 <212> DNA <213> artificial sequence <220> <223> REV- FWPV Forward <400> 5 actacctatg cctcttattc cact 24 <210> 6 <211> 24 <212> DNA <213> artificial sequence <220> <223> REV- FWPV Reverse <400> 6 ggctcagtat gatagttcga tctc 24 <210> 7 <211> 22 <212> DNA <213> artificial sequence <220> <223> REV Forward <400> 7 ggatttagac aacagtggga gt 22 <210> 8 <211> 22 <212> DNA <213> artificial sequence <220> <223> REV Reverse <400> 8 cgtcttcata cgaacctagc tt 22

Claims (13)

Infectious laryngotracheitis virus (ILTV), Fowlpox virus (FWPV), REV-inserted Reticuloendotheliosis virus-integrated Fowlpox virus (FWPV) and Reticuloendotheliosis virus (REV) Containing a combination of the following primer pairs for simultaneous multiplex detection of an avian complex infection virus Primer sets for simultaneous multiplex detection:

According to claim 1,
The primer set is used for nucleic acid amplification or real-time nucleic acid amplification, a primer set for simultaneous multiplex detection of avian complex infection virus.
A composition for simultaneous multiple detection of ILTV, FWPV, REV-inserted FWPV and REV avian complex infection viruses comprising the primer set according to claim 1 or 2.
According to claim 3, wherein the composition is used for nucleic acid amplification or real-time nucleic acid amplification, avian multi-infectious virus simultaneous multiple detection composition.
A kit for simultaneous multiple detection of ILTV, FWPV, REV-inserted FWPV and REV avian complex infection viruses comprising the primer set according to claim 1 or 2.
The kit according to claim 5, wherein the kit is used for nucleic acid amplification or real-time nucleic acid amplification.
Providing a sample and / or an environmental sample derived from algae requiring simultaneous multiple detection of ILTV, FWPV, REV-inserted FWPV and REV: and
Simultaneous multiplex detection method of avian complex infection virus, comprising mixing the sample with a primer set including a combination of primer pairs in the table below to perform a nucleic acid amplification reaction:

According to claim 7,
The algae-derived sample includes avian disease analysis samples, broilers, laying hens, breeders, chickens, SPF (Specific Pathogen Free) chickens and eggs in the poultry industry, avian complex infection virus simultaneous multiple detection method.
According to claim 8,
The algae-derived sample is a pathology sample, and the pathology sample is skin, liver, or spleen including nasal cavity, oral cavity, trachea, lung, and comb. Method for simultaneous multiplex detection of avian complex infection virus.
According to claim 7,
The environmental sample includes farm samples, mites, mosquitoes, contaminated feathers or scabs, chicken dander, and livestock dust.
According to claim 7,
The method includes determining the size of the amplification product after completion of the nucleic acid amplification reaction; and
Further comprising determining whether ILTV, FWPV, REV-inserted FWPV and / or REV of the sample is present or infected based on the size of the amplification product of each primer pair,
The size of the amplification product according to each primer pair and the presence or infection result of each virus according to the result are as shown in the following table, simultaneous multiplex detection method of avian complex infection virus:

According to claim 11,
The size of the nucleic acid amplification product is performed by electrophoresis, an avian multi-infection virus simultaneous multiplex detection method.
According to any one of claims 7 to 12,
The nucleic acid amplification reaction includes heating at 94 ° C for 5 minutes, denaturation at 94 ° C for 45 seconds, annealing at 58 ° C for 45 seconds, and polymerization at 72 ° C for 45 seconds 38 times in one cycle, and polymerization at 72 ° C for 5 minutes A simultaneous multiple detection method of avian multi-infection virus comprising the step of doing.

Images