KR20220112394A - 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 distinguishing fowlpox virus, infectious laryngotracheitis virus, and reticuloendotheliosis virus. The primer set and method according to the present application can detect the virus at an early stage with high specificity and sensitivity, so that through accurate differential diagnosis between fowlpox and transmissible laryngotracheitis, which show similar clinical symptoms, and fowlpox virus into which the reticuloendothelial virus gene has been inserted, the damage of infection is minimized through prompt follow-up measures such as vaccination, thereby reducing the economic cost of farms due to reduced productivity.

Description

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

The present application relates to a genetic diagnostic 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 (Gallid herpesvirus type I, GaHV-1, or ILTV), and laryngitis and tracheitis are the main lesions. In chickens infected with ILTV, the trachea becomes inflamed, causing respiratory symptoms such as sneezing and coughing, and in severe cases, breathing difficulties due to exudate in the bronchial tubes and suffocation may occur. The morbidity of the disease is around 5%, but when severe epidemic ILTV occurs, it appears up to 100%, and the mortality rate is usually 20%.

Fowlpox is a disease in which nodules are formed on the skin, oral and respiratory mucosa of chickens and turkeys by the Fowlpox virus (FWPV). Clinical symptoms include a skin type (dry) that forms nodular growths on hairless skin, and a mucosal type (wet) that forms nodules on the mucous membranes of the upper respiratory tract, oral cavity and esophagus, depending on the susceptibility of the host, the pathogenicity of the virus, the site of occurrence and the route of transmission. ) are separated. The skin type is common, and nodular lesions appear on the hairless areas of the crown, eyelids, mouth, wings and legs, and the mucous membrane type has yellowish-white nodules of various sizes in the oral cavity, esophagus, tongue, and trachea, leading to respiratory difficulties. do. In addition, the skin type has a low mortality rate, but the mucosal type has a high mortality rate due to respiratory difficulties.

Gamma-retrovirus is the causative agent of reticuloendotheliosis, and when infected with this virus, dwarf syndrome 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 reticuloendotheliosis may develop secondary infections caused by bacteria and parasites such as Escherichia coliosis or coccidiosis, and are easily infected with viral diseases such as chickenpox or infectious laryngeal tracheitis.

The clinical symptoms and lesions of fowlpox (mucosal type) are very similar to those of infectious laryngeal tracheitis, and the differentiating method is important because the complex infection of fowlpox virus and infectious laryngeal tracheitis virus is increasing. In the case of infectious laryngotracheitis, inclusion bodies are observed in the nucleus of infected cells, but in the case of chickenpox, inclusion bodies are observed in the cytoplasm in the case of infectious laryngotracheitis. In addition, the infected tissue is inoculated into the chorioallantoic membrane (CAM) of a 9-11-day-old fetus, the CAM is collected 5-7 days later, and the virus can be identified through a polymerase chain reaction. Methods for detecting reticuloendotheliosis virus include isolation and identification of the virus using animal cells, gene-based detection, and serology (enzyme immunoassay). These methods require skilled manpower and a lot of time, so there is a limit to quickly and accurately diagnosing the virus. In addition, there are many known cases in which a mutant fowlpox virus in which a reticuloendotheliosis virus gene is inserted into the fowlpox virus gene appeared and the existing vaccines did not sufficiently exert a protective effect. There is a need for a method that can accurately detect fowlpox virus and reticuloendotheliosis virus at the same time.

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.

An object of the present application is to provide a method for rapidly and conveniently detecting ILTV, FWPV, REV-integrated FWPV and REV infection with a single reaction with high sensitivity and specificity.

In one embodiment, the present application provides a pair of primers for multiplex PCR analysis for the following Infectious laryngotracheitis virus (ILTV), Fowlpox virus (FWPV), Reticuloendotheliosis virus-integrated Fowlpox virus (REV-integrated FWPV) and Reticuloendotheliosis virus (REV) detection. It provides a primer set for simultaneous multiple detection of avian complex infection virus comprising a combination of: ILTV-specific primer pair comprising primers represented by SEQ ID NOs: 1 and 2; FWPV-specific primer pair comprising primers represented by SEQ ID NOs: 3 and 4; REV-integrated FWPV comprising 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 primer set for ILTV, FWPV, REV-integrated FWPV and REV detection, diagnosis or discrimination according to the present application may be provided in the form of a composition or kit comprising the same.

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

The primer set according to the present application includes various algal-derived and/or environmental samples requiring detection of ILTV, FWPV, REV-integrated FWPV and REV, for example, poultry pathology samples; Broiler. Specimens from laying hens, breeders, native chickens or these pens; SPF (Specific Pathogen Free) includes, but is not limited to, chickens or eggs thereof, or specimens derived from these breeding grounds, and samples such as organs or plants may be used.

In the method according to the present application, analysis of the results of the simultaneous multi-gene polymerase chain reaction is performed after the reaction is completed to confirm the size of the product through electrophoresis. , and if the REV is 207bp, 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 in a simultaneous multiple gene polymerase chain reaction (multiplex PCR) method and a method for detecting the viruses using the same have high sensitivity and specificity With this method, it is possible to quickly check whether pathogens are contaminated in various specimens including pathology samples in the veterinary field. 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 an embodiment of the present application by agarose gel electrophoresis.
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 an embodiment of the present application by agarose gel electrophoresis.
In the figure, reactions were performed at a total of 7 different concentrations and negative controls for each set, and the concentrations were 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 FWPV gene is 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 is 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 REV gene is 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.

This study examines whether or not infection with ILTV (Infectious laryngotracheitis virus), FWPV (Fowlpox virus), FWPV (Fowlpox virus), REV-inserted FWPV (Reticuloendotheliosis virus-integrated Fowlpox virus) and REV (Reticuloendotheliosis virus) It is based on the discovery of genetic diagnostic technology that can detect rapidly and accurately with high sensitivity and specificity at the same time as a test.

Accordingly, in one embodiment, the present application provides a primer pair 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; a pair of primers of SEQ ID NOs: 3 and 4 for FWPV detection; a pair of primers of SEQ ID NOs: 5 and 6 for detecting REV-integrated FWPV; It relates to a primer set for simultaneous multiple detection of an avian complex infection virus comprising the primer pair of SEQ ID NOs: 7 and 8 for detecting REV.

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

[Table 1]

As used herein, a primer refers to a ribonucleotide or deoxyribonucleotide of any length, including both single-stranded and double-stranded, and a concept including both primers and probes, and a gene specific to each strain to be detected. Refers to an oligonucleotide having a sequence complementary to at least a portion of

The primer set according to the present disclosure can be used in various nucleic acid amplification techniques.

In one embodiment the primer set according to the invention 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 for exponentially amplifying a starting nucleic acid material by synthesizing a nucleic acid in a chain using a polymerase. 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 that can track 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 terms represent the number of PCR cycles at the point where the amplification curve reaches a specific threshold in the amplification process. It is determined and is in exponential phase. Quantification or quantitative analysis of nucleic acids using this value may be performed according to the manual of a real-time PCR device manufacturer. The step of confirming whether a specific DNA is amplified in such a PCR may be performed by a method of confirming 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 in 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 checked for amplification, and in addition, it can be carried out by a method comprising the step of checking the melting temperature at which the double helix of the amplified DNA product is separated.

As used herein, detection refers to detecting the presence or absence of a nucleic acid or the amount thereof, if present. For example, specifically PCR (quantitative and qualitative PCR, real-time PCR) based on a nucleic acid-specific DNA-DNA, RNA-DNA hybridization reaction including quantitative and qualitative PCR).

Simultaneous multiple detection herein means that a specific primer set capable of detecting each strain is included in one reaction, so that each strain can be specifically detected without cross-reactivity to other strains in a single amplification reaction. . For example, when one sample is infected with two or more strains to be detected in the present application, 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 aspect, the present application relates to a composition or kit for simultaneous multiple detection of ILTV, FWPV, REV-inserted FWPV and REV avian complex infection viruses, including a combination of the primer sets.

In one embodiment, such a composition comprises a buffer solution 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 can be purchased commercially, for example, such as AmpliTaq Gold (Applied Biosystems, USA) can be used, and an appropriate concentration, for example, 1.5mM to 2.5mM MgCl ₂ It may be included.

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

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

In one embodiment, the method according to the present disclosure comprises the steps of providing a sample or environmental sample derived from an alga requiring simultaneous multiplex 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 disclosure include various birds such as chickens, ducks, geese, quails, turkeys, canaries, parrots, pheasants, pigeons, seagulls, ostriches and the like. One embodiment particularly includes domestic birds such as chickens or turkeys.

In one embodiment, the sample derived from the avian includes a disease test sample of birds, broilers in the poultry industry, laying hens, breeders, eggs, SPF (Specific Pathogen Free) chickens and eggs.

In one embodiment, an avian pathology sample is used, which includes the nasal cavity, oral cavity, trachea and lungs, skin, liver or spleen including crest.

In another embodiment, an environmental sample is used, and the environmental sample includes a variety of samples from the industry, such as farm samples, mites, mosquitoes, contaminated feathers or scabs, chicken dander, and dust from barn floors.

The method according to the present application comprises the steps of determining the size of the amplification product after completion of the nucleic acid amplification reaction; and determining the presence or infection of ILTV, REV-inserted FWPV and/or REV in the sample based on the size of the amplification product, wherein 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, ○ 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. Such methods are known in the art, for example, agarose gel electrophoresis, real-time CR analysis, single strand conformational polymorphism (SSCP), restriction fragment length polymorphism (RFLP), capillary zone electrophoresis (CZE), WAVE (HPLC-based nucleic acid) acid analyzing echnology), including, but not limited to, a microchip. In one embodiment of the present application, the PCR product together with the size marker is analyzed by electrophoresis on an agarose gel.

In one embodiment, the method according to the present invention, in one embodiment, the reaction product of the simultaneous multiple gene polymerase chain reaction (multiplex PCR) is a total of 20 μl of ILTV-specific SEQ ID NOs 1 and 2 in Black PCR premix QM (Ventech Science, South Korea) of primers, FWPV-specific primer pairs of SEQ ID NOs: 3 and 4, REV-integrated FWPV-specific primer pairs of SEQ ID NOs: 5 and 6, REV-specific primer pairs of SEQ ID NOs: 7 and 8, and 3.0-5.0 μl of analysis Required 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 disclosure, 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. Repeat 38 times in one cycle and polymerization at 72° C. for 5 minutes.

Hereinafter, examples are provided to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the present invention is not limited thereto.

Example

Example 1. ILTV, FWPV and REV Sample Preparation

Herein, DNA was extracted using the QIAamp DNA Mini kit (Qiagen, Germany) according to the manufacturer's method from the tissue samples of the organs, glands, and livers of chickens diagnosed with ILTV, FWPV, and REV, respectively.

Example 2. Preparation of primers specific to ILTV, FWPV, REV-integrated FWPV and REV

For accurate detection of ILTV, FWPV, REV-integrated FWPV and REV, the sequence of the target gene of each virus selected herein was obtained from NCBI and GenBank, and final primers specific for each virus were selected.

There is no known diagnostic method capable of simultaneously detecting FWPV, FWPV, and REV with REV inserted. By designing a forward primer for FWPV and a reverse primer for REV, a corresponding site was selected to confirm that REV is present in FWPV. In addition, to confirm that the full-length genome including the LTR portion of REV is inserted, a primer specific to REV was 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 ILTV target is envelope glycoprotein G (GenBank accession number, JN804826) involved in viral replication, the FWPV target is the transcription termination factor (GenBank accession number, AF198100), and the REV-integrated FWPV target is FWPV. To detect the REV inserted into the gene of ) and reverse primers were prepared, respectively, to complete a primer and a detection method for simultaneous multiplex PCR detection (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). As a result, the sequence is identical except for the corresponding virus. This did not exist.

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

Example 3. Confirmation of Specificity and Sensitivity of Simultaneous Multigene Polymerase Chain Reaction

Simultaneous multi-gene polymerase chain reaction for ILTV, FWPV, REV-integrated FWPV and REV was performed using a combination of four primer pairs in Table 3, followed by agarose gel electrophoresis and nucleotide sequence analysis of the amplified product As a result, it was confirmed that the primer specifically amplifies only the target virus to a size of 877bp, 630bp, 429bp, and 207bp, respectively (FIG. 1).

For this purpose, 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) (see the concentration of ) or a pair of primers for each virus (see the concentration in Table 1), and sterile distilled water was added to a total volume of 20 μl, followed by PCR reaction, and the reaction conditions are as follows: at 94°C Heating for 5 minutes, denaturing at 94°C for 45 seconds, annealing at 58°C for 45 seconds, repeating the polymerization reaction at 72°C for 45 seconds 38 times in one cycle, polymerization at 72°C for 5 minutes.

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

[Table 4]

Simultaneous multiplex 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, respectively, in a simultaneous multi-gene polymerase chain reaction, and a specific primer for each virus It was shown that only the target virus was specifically detected in the used gene polymerase chain reaction. The amplified product was separated from the agarose gel, and as a result of requesting an external sequencing analysis (Cosmogenetech, South Korea), it was a sequence specific only to the virus. 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 the organs, livers and head of chickens infected with ILTV, REV-integrated FWPV and REV, respectively, and each target product amplified by PCR using the primer set according to the present application was prepared using 1% TAE agarose gel. After electrophoresis, gel extraction (Qiagen, Germany) was performed according to the manufacturer's method, and then inserted into pGEM-T Easy Vector (promega, USA) according to the manufacturer's method, respectively, and pGEM-ILTV, pGEM-FWPV, pGEM-REV- Integrated FWPV and pGEM-REV were fabricated.

In addition, 1.0 x 5 ⁶ copies, 1.0 x 5 ⁵ copies, 1.0 x 5 ⁴ copies, 1.0 x 5 As a result of simultaneous multi-gene polymerase chain reaction by diluting to a concentration of ³ copies, 1.0 x 5 ² copies, 1.0 x 5 ¹ copies, 1.0 x 5 ⁰ copies, 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 detected simultaneously, and as a result of performing individual PCR for one virus using primers specific for each virus, the concentration of 1.0 x 5 ² copies or more Viruses were detected in each ( FIG. 2 ).

As described in Figure 2, the detection of virus 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; In case of using plasmid containing ILTV genome 1.0 x 5 ² copies; 1.0 x 5 ² copies when using a plasmid containing the genome of FWPV; When using a plasmid containing the REV-integrated FWPV genome 1.0 x 5 ² copies; When a plasmid containing the REV genome was used, the sensitivity was found to be very high at 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)

Contains combinations of the following primer pairs for simultaneous multiplex detection of Infectious laryngotracheitis virus (ILTV), Fowlpox virus (FWPV), Fowlpox virus (FWPV), Reticuloendotheliosis virus-integrated Fowlpox virus (REV) and Reticuloendotheliosis virus (REV). A primer set for simultaneous multiple detection of avian complex infection virus:

The method of claim 1,
Wherein the primer set is used for nucleic acid amplification or real-time nucleic acid amplification, a primer set for simultaneous multiple detection of avian complex infection virus.
A composition for simultaneous multiple detection of avian complex infection virus of ILTV, FWPV, REV-inserted FWPV and REV comprising the primer set according to claim 1 or 2.
The composition for simultaneous multiple detection of avian complex infection virus according to claim 3, wherein the composition is used for nucleic acid amplification or real-time nucleic acid amplification.
A kit for simultaneous multiple detection of ILTV, FWPV, REV-inserted FWPV and REV avian complex infection virus comprising the primer set according to claim 1 or 2.
The kit of claim 5, wherein the composition is used for nucleic acid amplification or real-time nucleic acid amplification.
providing an algal-derived sample and/or an environmental sample requiring simultaneous multiplex detection of ILTV, FWPV, REV-inserted FWPV and REV; and
A method for simultaneous multiple detection of avian complex infection virus, comprising the step of performing a nucleic acid amplification reaction by mixing the sample with a primer set including a combination of primer pairs in the table below:

8. The method of claim 7,
The avian-derived sample includes a disease test sample of birds, broilers, laying hens, breeders, eggs, SPF (Specific Pathogen Free) chickens and eggs of the poultry industry, simultaneous multiple detection method of avian complex infection virus.
9. The method of claim 8,
The avian-derived sample is a sensibility sample, and the morbidity assessment sample is a skin, liver or spleen including nasal, oral cavity, trachea, lung, and spleen.
8. The method of claim 7,
The environmental sample includes a farm sample, mites, mosquitoes, contaminated feathers or scabs, chicken dander, and livestock dust.
8. The method of claim 7,
The method comprises the steps of determining the size of the amplification product after completion of the nucleic acid amplification reaction; and
Further comprising the step of determining the presence or infection of ILTV, REV-inserted FWPV and/or REV in the sample 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 accordingly are as shown in the following table, a method for simultaneous multiple detection of avian complex infection virus:

12. The method of claim 11,
The size of the nucleic acid amplification product will be performed by electrophoresis, a simultaneous multiple detection method of avian complex infection virus.
13. The method according to any one of claims 7 to 12,
The nucleic acid amplification reaction is a step of heating at 94°C for 5 minutes, denaturing at 94°C for 45 seconds, annealing at 58°C for 45 seconds, repeating the polymerization reaction at 72°C for 45 seconds 38 times in one cycle, and polymerization at 72°C for 5 minutes. Simultaneous multiple detection method of avian complex infection virus comprising the step of.

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