KR20240130215A - Primer set and probe for diagnosing Porcine lymphotropic herpesvirus type 1 and uses thereof - Google Patents

Abstract

The present invention relates to a primer set and a probe for diagnosing PLHV1 (Porcine lymphotropic herpesvirus type 1) infection, and uses thereof. Using the primer and probe for diagnosing PLHV1 (Porcine lymphotropic herpesvirus type 1) infection and a positive control vector according to the present invention, PLHV1 infection can be confirmed. Specifically, by using a real-time polymerase chain reaction method, sensitivity and specificity are increased, so that infection with PLHV1, known to be capable of latent infection, can be detected with higher sensitivity than with a general polymerase chain reaction method. In addition, the present invention is expected to be applicable not only to health monitoring of germ-free pigs in designated pathogen control breeding facilities, but also to confirmation of PLHV1 infection in xenograft preparations and patients who have received xenografts.

Description

Primer set and probe for diagnosing PLHV1(Porcine lymphotropic herpesvirus type 1) infection and uses thereof {Primer set and probe for diagnosing Porcine lymphotropic herpesvirus type 1 and uses thereof}

The present invention relates to a primer set and probe for diagnosing PLHV1 (Porcine lymphotropic herpesvirus type 1) infection, and uses thereof.

In 2021, the number of people waiting for transplants in Korea reached 45,855, including 39,261 solid organ transplants, 2,073 eyes, and 4,496 bone marrow transplants, but only 5,674 cases of organ and tissue transplants were performed. The imbalance between the demand and supply of organs is not limited to Korea but is an international problem, and researchers around the world are working to realize xenotransplantation, which transplants cells, tissues, and organs obtained from animals other than humans into humans as an alternative. Pigs are used as source animals for xenotransplantation because they are physiologically and anatomically similar to humans, have rapid sexual maturity, and produce a large number of eggs. Source animals for xenotransplantation must be raised in a designated pathogen-free facility, and infection with designated pathogens must be proven through a specific and sensitive test method for the source of infection.

The recently published "Guidelines for the Quality, Nonclinical, and Clinical Evaluation of Xenotransplantation Products" by the Ministry of Food and Drug Safety has established designated pathogen tests for raw animal products, and PLHV1 is included. In addition, the characterization and quality control of xenotransplantation products generally require safety, identity, purity, and potency tests. The test methods can be used for the characterization and quality control of advanced biopharmaceuticals and biological products. For specific xenotransplantation products, the measurement of cell populations within the xenotransplantation product may be included.

Porcine lymphotropic herpesvirus (PLHV) is a gammaherpesvirus found in pigs and is generally known to be non-pathogenic. However, in the case of PLHV1 (Porcine lymphotropic herpesvirus type 1), post-transplantation lymphoproliferative disorder (PTLD) has been reported in transplant experiments using minipigs, which is known to be similar to PTLD caused by Epstein-Barr virus in human allogeneic organ transplantation. Since it is a non-pathogenic virus in the swine industry, a specific and sensitive test method for PLHV1 has not been commercialized.

Accordingly, the present inventors have completed the invention by designing a primer set and probe for a specific and sensitive real-time polymerase chain reaction (real-time PCR) test for PLHV1; and a positive control vector that can be used as a positive control.

Republic of Korea Patent Publication No. 10-2081965

The purpose of the present invention is to provide a primer set comprising a forward primer represented by sequence number 2 and a reverse primer represented by sequence number 3; and

A composition for diagnosing PLHV1 (Porcine lymphotropic herpesvirus type 1) infection is provided, comprising a probe represented by sequence number 4.

Another object of the present invention is to provide a kit for diagnosing PLHV1 infection, comprising the composition of the present invention.

Another object of the present invention is (a) a step of amplifying the PLHV1 gene using the primer set; and

(b) provides a method for diagnosing PLHV1 infection in a pig, comprising a step of detecting the product amplified in step (a).

Another object of the present invention is to provide a PLHV1 positive control gene comprising a sequence represented by SEQ ID NO: 1.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

The present invention comprises a primer set comprising a forward primer represented by SEQ ID NO: 2 and a reverse primer represented by SEQ ID NO: 3; and

A composition for diagnosing PLHV1 (Porcine lymphotropic herpesvirus type 1) infection is provided, comprising a probe represented by sequence number 4.

In the present invention, "diagnosis" means finding out the presence or absence of a chemical species or microorganism, etc., in a sample, an experimental sample, a source animal, a xenograft preparation, or a patient who received a xenograft, preferably a pig, a xenograft preparation, or a patient who received a xenograft, and diagnosis or detection in the present invention preferably means finding out the presence or absence of PLHV1. That is, the composition may be for diagnosing PLHV1 infection in a pig, a xenograft preparation, or a patient who received a xenograft, but is not limited thereto. The infection diagnosis may be used to mean including detecting the presence or absence of PLHV1.

In the present invention, "PLHV1 (Porcine lymphotropic herpesvirus type 1)" is a type of porcine lymphotropic herpesvirus (PLHV) belonging to gammaherpesvirus, and is related to post-transplantation lymphoproliferative disorder (PTLD). The PLHV1 is included in the Designated Pathogen Free (DPF) test items of source animals for xenotransplantation.

In the present invention, "xenotransplantation" means a process in which living cells, tissues or organs obtained from a non-human animal are transplanted, inserted or infused into a human, or a process in which human body fluids, cells, tissues or organs are transplanted, inserted or infused into a human after coming into contact with living cells, tissues or organs of a non-human animal in vitro.

In the present invention, the "xenotransplantation product" refers to a cell, tissue or organ manufactured by physically, chemically or biologically manufacturing a living organ of an animal. The xenotransplantation product may be used immediately after collection from a source animal, or may be stored or processed, but is not limited thereto. In addition, the xenotransplantation product may be obtained under aseptic conditions designed to minimize contamination in a designated facility. In order to control the quality of the xenotransplantation product, virus tests may be performed on the source animal and the xenotransplantation product, and the virus may include PLHV1 (Porcine lymphotropic herpesvirus type 1).

In the present invention, the "primer" means a short nucleic acid sequence having a short free 3-terminal hydroxyl group, which can form base pairs with a complementary template and functions as a starting point for copying the template strand. The primer can initiate DNA synthesis in the presence of a reagent for polymerization reaction (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. The primer may be a primer set including a forward primer represented by SEQ ID NO: 2 and a reverse primer represented by SEQ ID NO: 3, and the primer may include a sequence having 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 2 or 3, but is not limited thereto.

In the present invention, the "probe" means a nucleic acid fragment such as RNA or DNA, which is short for a few bases to long for several hundred bases and can specifically bind to mRNA, and is labeled so as to be able to confirm the presence or absence of a specific mRNA. The probe can be produced in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, etc. The selection of an appropriate probe and hybridization conditions can be modified based on those known in the art. The probe can be represented by SEQ ID NO: 4.

In addition, the probe can be coupled with a reporter and a quencher, and the reporter can be FAM (6-carboxyfluorescein), Texas red, fluorescein, HEX (5-hexachlorofluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC (fluorescein isothiocyanate), Oregon green, alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'- Dimethoxyfluorescein), ROX (6-Carboxyl-X-Rhodamine), TET (Tetrachloro-Fluorescein), TRITC (tertramethylrodamine isothiocyanate), The quencher may be selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), cyanine series dyes, and thiadicarbocyanine, but is not limited thereto. In addition, the quencher may be selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), NFQ (nonfluorescent quencher), dabcyl, Eclipse, DDQ (Deep Dark Quencher), Blackberry Quencher, and Iowa black, but is not limited thereto.

In the present invention, the primer or probe can be chemically synthesized using a phosphoramidite solid support synthesis method or other widely known methods. In addition, the primer or probe can be variously modified according to methods known in the art as long as it does not interfere with the diagnosis of PLHV1 infection. Examples of such modifications include methylation, capping, substitution with one or more homologues of natural nucleotides, modification between nucleotides, for example, uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.), and binding of labeling materials using fluorescent or enzymes.

In the present invention, the primer set and the probe can target the DNA polymerase of PLHV1, and the detection limit can be 15 to 25 copies/rxn, preferably 25 copies/rxn. In addition, the lowest quantitation limit can be 1x10 ² copies/rxn, but is not limited thereto. In addition, the primer set and the probe of the present invention have specificity for PLHV1.

Therefore, the primer set and probe of the present invention have excellent analytical sensitivity and can specifically detect only PLHV1, excluding other bacteria and viruses, even in low concentration samples.

The present invention provides a kit for diagnosing PLHV1 infection, comprising a composition of the present invention. The kit may include reagents for performing an amplification reaction. In addition, the kit may take the form of a bottle, a tub, a sachet, an envelope, a tube, an ampoule, and the like, which may be formed partly or wholly from plastic, glass, paper, foil, wax, and the like. The container may be equipped with a completely or partially detachable stopper, which may initially be part of the container or may be attached to the container by mechanical, adhesive, or other means. The container may also be equipped with a stopper, which may allow access to the contents by means of a syringe needle. The kit may include an outer package, which may include instructions for use of the components. In addition, the instructions for use may correspond to a user guide describing optimal reaction performance conditions, which explains how to use the kit, for example, the reaction conditions presented, etc. In addition, the instructions for use may include a guide booklet in the form of a pamphlet or leaflet, a label attached to the kit, and a description on the surface of a package including the kit, and may correspond to information disclosed or provided through an electronic medium such as the Internet. In addition, the instructions for use may describe, but are not limited to, contents related to a step of amplifying a PLHV1 gene using the primer set of the present invention; and a step of detecting the amplified product. In addition, the kit may include a primer set including a forward primer represented by SEQ ID NO: 2 and a reverse primer represented by SEQ ID NO: 3; and a probe represented by SEQ ID NO: 4, as well as one or more kinds of other component compositions, solutions, or devices suitable for an analysis method.

In addition, the present invention comprises: (a) a step of amplifying the PLHV1 gene using the primer set of the present invention; and

(b) A method for diagnosing PLHV1 infection in a pig is provided, comprising a step of detecting the product amplified in step (a).

The amplification of step (a) may be performed using any one method selected from the group consisting of polymerase chain reaction (Polymerase Chain Reaction), duplex polymerase chain reaction (duplex PCR), multiplex polymerase chain reaction (multiplex PCR), reverse transcription polymerase chain reaction (RT-PCR), competitive polymerase chain reaction, real-time polymerase chain reaction, quantitative polymerase chain reaction, DNA chip, loop-mediated isothermal amplification, and combinations thereof. Preferably, the amplification of step (a) may be performed using real-time polymerase chain reaction, but is not limited thereto.

The detection of step (b) above can be performed by any one method selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement, phosphorescence measurement, and combinations thereof, but is not limited thereto.

In addition, the present invention provides a PLHV1 positive control gene consisting of a sequence represented by SEQ ID NO: 1. The PLHV1 positive control may include, but is not limited to, a forward primer for amplifying a DNA polymerase gene of PLHV1 consisting of a sequence represented by SEQ ID NO: 2; a reverse primer for amplifying a DNA polymerase gene of PLHV1 consisting of a sequence represented by SEQ ID NO: 3; and a probe consisting of a sequence represented by SEQ ID NO: 4.

In the present invention, the "positive control group" refers to a positive control group used for standardization in detecting PLHV1 in a sample using a molecular biological method such as RT-PCR or Real-time PCR. By securing such a positive control group, a reliable test can be performed on a raw animal or xenograft product suspected of being infected with PLHV1.

In addition, the present invention provides a recombinant vector comprising a PLHV1 positive control gene. In the present invention, a "recombinant vector" is a genetic construct comprising essential regulatory elements operably linked to allow expression of a genetic insert, and may be, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, or a viral vector.

The present invention confirmed the excellent analytical sensitivity by confirming the lower limit of quantitation and the limit of detection for a composition for diagnosing PLHV1 (Porcine lymphotropic herpesvirus type 1) infection including the primer set and probe of the present invention, and confirmed that it can specifically detect only PLHV1. By utilizing the present invention, not only can the health of germ-free pigs in a designated pathogen control breeding facility be monitored, but also the presence or absence of PLHV1 infection in xenograft preparations and patients who have received xenografts can be quickly and accurately confirmed.

Using the primers and probe for diagnosing PLHV1 (Porcine lymphotropic herpesvirus type 1) infection and the positive control vector according to the present invention, PLHV1 infection can be confirmed. Specifically, by using a real-time polymerase chain reaction method, the sensitivity and specificity are increased, so that infection with PLHV1, known to be capable of latent infection, can be detected with higher sensitivity than with a general polymerase chain reaction method. In addition, the present invention is expected to be applicable not only to the health monitoring of germ-free pigs in designated pathogen control breeding facilities, but also to confirm infection with PLHV1 in xenograft preparations and patients who have received xenografts.

Figure 1 shows the results of the lower limit of quantitation test independently repeated three times for standard samples for the calibration curve (1x10 ⁷ , 1x10 ⁶ , 1x10 ⁵ , 1x10 ⁴ , 1x10 ³ , 1x10 ² copies/rxn).
Figure 2 shows the results of confirming the detection limit by performing 24 repeated tests by selecting LOD1 (50 copies/rxn), LOD2 (25 copies/rxn), LOD3 (12.5 copies/rxn), and LOD4 (6.25 copies/rxn) outside the quantitative range.
Figure 3 shows the results of confirming the accuracy of the primer and probe set of the present invention by adding a small amount of standard template DNA at a concentration of 1x10 ³ /rxn to human NC (negative control) and pig NC, respectively.
Figure 4 shows the results of confirming the detection specificity of the primer and probe set of the present invention for bacteria 1 to 18 (Table 9 below).
Figure 5 shows the results of confirming the detection specificity of the primer and probe set of the present invention for viruses 1 to 18 (Table 10 below).
Figure 6 shows a positive control vector manufactured by synthesizing the DNA polymerase gene sequence of PLHV1 (Porcine lymphotropic herpesvirus type 1).

Hereinafter, the following examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

[ Example ]

1. Standard Template DNA

The DNA polymerase gene sequence of porcine lymphotropic herpesvirus type 1 (PLHV1) was synthesized to prepare a positive control vector (SEQ ID NO: 1, Figure 6) and a standard template DNA (Bionics Co., Ltd.). The synthesized standard template DNA was quantified, dispensed into an amount for a single analysis, stored at -20 ± 5°C, and used after thawing at 4°C, respectively, by preparing the standard template DNA for calibration curve and the concentration of the quality control sample. Primers and probes targeting DNA polymerase; reagents used in the experiment; and test equipment and software used in the experiment are shown in Tables 1 to 3 below.

Target Primer Type manufacturing company Sequences (5’->3’) Sequence number DNA Polymerase Forward Thermofisher TCT CAG AGA TTG CCA AAA TTG C 2 Reverse Thermofisher CGG ATC TGT TGC CCA TCT GT 3 Probe Thermofisher FAM-ATA TTC CAA TGC GAA GAG T-MGB/NFQ 4

Reagent and material names manufacturing company Cat No. TaqMan™ Fast Advanced Master Mix Thermofischer Scientific 4444557 High-Capacity cDNA Reverse Transcription Kit Thermofischer Scientific 4368814 PCR 96 well plate Bio-Rad 64475823 PCR plate adhesive film Bio-Rad MSB1001

equipment name manufacturing company Cat.No CFX 96 OPUS Real-Time PCR
Detection System Bio-RAD 12011319 NanoDrop™ 2000 Thermofisher ND-2000 Heating Block Thermofisher 13687712 CFX Maestro Software for CFX Real-Time PCR Instruments BIO-RAD 12013758 Primer Express™ Software v3.0.1 Applied Biosystems 4363993

2. Analysis method Verification test design

A real-time polymerase chain reaction (real-time PCR) assay for detecting PLHV1 (Porcine lymphotropic herpesvirus type 1) was validated by evaluating its analytical sensitivity and specificity. This was validated according to Table 4 below.

item Sample name Analytical Sensitivity
Lower limit of quantitation (LLOQ)/
Detection limit
(LOD) NTC No Template Control Standard sample for calibration curve 1x10 ⁷ , 1x10 ⁶ , 1x10 ⁵ , 1x10 ⁴ , 1x10 ³ , 1x10 ² copies/rxn Detection limit standard sample 50, 25, 12.5, 6.25 copies/rxn Specificity
(Specificity) Standard sample for calibration curve 1x10 ⁷ , 1x10 ⁶ , 1x10 ⁵ , 1x10 ⁴ , 1x10 ³ , 1x10 ² copies/rxn NC Human NC, Pig NC 18 types of bacteria ^5x104 copies/rxn for each bacteria 18 types of viruses ^5x104 copies/rxn for each virus

3. For calibration curve Preparation of standard samples, quality control samples, and control materials

Standard samples for calibration curves (STD 1 to STD 6) and quality control samples (HQC, MQC, LQC) were prepared by diluting the standard sample stock solution to the concentrations specified in Table 5 below and used. In addition, control substances were prepared and used as shown in Table 6 below.

Standard sample for calibration curve Standard sample concentration for final calibration curve
(Copies/ul) STD 1 2x10 ⁶ STD 2 2x10 ⁵ STD 3 2x10 ⁴ STD 4 2x10 ³ STD 5 2x10 ² STD 6 2x10 ¹ Quality Control Samples Final quality control sample concentration
(Copies/ul) HQC 1.6x10 ⁶ MQC 8x10 ³ LQC 2x10 ¹

Contrast material definition NTC PCR master mix + Nucleic acid free water
Control to check for contamination from reagents used in PCR reactions NC DNA that is not amplified by PCR master mix + primer and probe
Control to confirm that amplification by PCR reaction is specific to standard template DNA
1. Human NC (using HEK293 cell gDNA)
2. Pig NC (using pig tissue gDNA)
3. Use of gDNA from 18 bacterial species
4. Viral genomes of 18 different types of viruses. However, in the case of RNA viruses, reverse transcription was performed on a measured amount of viral RNA genome and the cDNA was used.

4. Confirm analytical sensitivity and specificity

PCR (polymerase chain reaction) was performed according to the conditions in Tables 7 and 8 below. Analytical sensitivity was confirmed through the lowest quantitation limit and detection limit, and specificity was confirmed using Human NC, Pig NC, other bacteria, and heterologous viruses.

Components Volume (ul) Final concentration TaqMan™ Fast Advanced Master Mix 10 1x Forward primer 1 0.5 uM Reverse primer 1 0.5 uM Probe 0.5 0.2 uM Template 5 D.W(Distilled water) 1.5

Step Temp. (℃) Time (mm:ss) Cycle Incubation 50 02:00 1 Denaturation 95 10:00 1 Amplification 95 00:20 45 60 00:30

4-1. Analytical Sensitivity

The lower limit of quantification (LLOQ) is the lowest concentration at which an analyte can be quantified within the range where accuracy and precision are demonstrated, and generally refers to the lowest concentration of the calibration curve. In order to set the LLOQ, standard template DNA at each concentration was analyzed by real-time PCR to confirm. It was performed three times in repetition, and the lowest concentration that met the accuracy and precision criteria was set as the LLOQ. Accuracy was calculated by Equation 1 below. The results are shown in Figure 1.

As shown in Fig. 1, the detection limit test was independently repeated three times, and the accuracy and precision of all standard samples for the calibration curve (1x10 ⁷ , 1x10 ⁶ , 1x10 ⁵ , 1x10 ⁴ , 1x10 ³ , 1x10 ² copies/rxn) were confirmed. In addition, the lower limit of quantitation was confirmed to be 1x10 ² copies/rxn.

[Formula 1]

In addition, the limit of detection (LOD) is the minimum amount at which one can say with considerable confidence that an analyte is detected. In addition to the quantitation range, LOD1 (50 copies/rxn), LOD2 (25 copies/rxn), LOD3 (12.5 copies/rxn), and LOD4 (6.25 copies/rxn) were selected, and the lowest concentration showing a 95% Positive cut-off value through 24 repeated tests was selected. The results are shown in Fig. 2.

As shown in Figure 2, the cut-off ranges were confirmed as 100%, 100%, 91.67%, and 75.00% at LOD1, LOD2, LOD3, and LOD4, respectively. Therefore, the detection limit was confirmed as 25 copies/rxn.

4-2. Specificity

To confirm whether the primer & probe set of the present invention detects only PLHV1 (Porcine lymphotropic herpesvirus type 1), a small amount of standard template DNA was added (spiked) to the human NC (negative control) and pig NC described in Table 7 at a concentration corresponding to 1x10 ³ /rxn, respectively, and the accuracy was confirmed. The results are shown in Fig. 3.

As shown in Figure 3, the accuracy was confirmed to be 109.66% and 109.67%, respectively, which is within the acceptable standard of 100±15%.

In addition, it was confirmed whether the primer & probe set of the present invention has the possibility of detecting other bacteria or heterologous viruses. In order to confirm the detection specificity of the primer & probe set of the present invention, the detection effect for the bacteria or virus concentration of 5x10 ⁴ copies/rxn shown in Tables 9 and 10 was confirmed, and the results are shown in Figs. 4 and 5. The list of bacteria and viruses used in this experiment is shown in Tables 9 and 10 below, and the numbers of the bacteria and viruses in Tables 9 and 10 are matched and shown in the same manner as in Figs. 4 and 5.

As shown in Figures 4 and 5, it was confirmed that it was not detected in the gDNA of 18 bacterial species provided by the Korea Center for Biological Resources (KCTC) and the viral genome of 18 viral species provided by the Korean Pathogenic Virus Bank (KBPV).

This means that the primer & probe set of the present invention has specificity for PLHV1 (Porcine lymphotropic herpesvirus type 1).

Bacteria No. Bacteria KCTC Resource Number 1 Bacillus cereus 1093 2 Bacillus subtilis 33068 3 Campylobacter coli 15212 4 Campylobacter jejuni 5327 5 Clostridium perfringens 5014 6 Enterobacter cloacae 2361 7 Escherichia coli 2441 8 Klebsiella pneumoniae 12385 9 Legionella pneumophila 12009 10 Listeria monocytogenes 3710 11 Mycobacterium intracellulare 9514 12 Pseudomonas aeruginosa 1750 13 Salmonella enterica subsp . Enterica 2424 14 Shigella boydii 22528 15 Shigella sonnei 22530 16 Staphylococcus aureus 1621 17 Vibrio parahaemolyticus 2729 18 Vibrio vulnificus 2959

Virus No. Virus KBPV Cat No. 1 Human adenovirus KBPV-VR-1D 2 Human coronavirus 229E KBPV-VR-9D 3 Human coronavirus NL63 KBPV-VR-88D 4 Human coronavirus OC43 KBPV-VR-8D 5 Human Influenza virus A H1N1 KBPV-VR-33D 6 Human Influenza virus A H3N2 KBPV-VR-32D 7 Human Influenza virus B Yamagata-ikeB/Korea/94/2017 KBPV-VR-94D 8 Human metapneumovirus KBPV-VR-86D 9 Human parainfluenza virus 1 KBPV-VR-44D 10 Human parainfluenza virus 2 KBPV-VR-45D 11 Human parainfluenza virus 3 KBPV-VR-46D 12 Human rhinovirus A KBPV-VR-81D 13 Human rhinovirus B KBPV-VR-39D 14 Measles virus KBPV-VR-35D 15 Mumps virus KBPV-VR-51D 16 Respiratory syncytial virus A (RSV A) KBPV-VR-41D 17 Respiratory syncytial virus B (RSV B) KBPV-VR-42D 18 Rubella virus KBPV-VR-1401D

Through the above results, the present invention synthesized a standard template DNA and produced primers and a probe to detect the PLHV1 (Porcine lymphotropic herpesvirus type 1) gene, and confirmed the lower limit of quantitation and the limit of detection, thereby confirming excellent analytical sensitivity and verifying that only PLHV1 can be specifically detected.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Attach electronic file of sequence list

Claims (10)

A primer set comprising a forward primer represented by sequence number 2 and a reverse primer represented by sequence number 3; and
A composition for diagnosing PLHV1 (Porcine lymphotropic herpesvirus type 1) infection, comprising a probe represented by sequence number 4.
In the first paragraph,
The composition above is a composition for diagnosing PLHV1 infection in pigs or xenograft preparations.
In the first paragraph,
A composition wherein the above primer set and probe target the DNA polymerase of PLHV1.
In the first paragraph,
The above probe is a composition in which a reporter and a quencher are combined.
In paragraph 4,
The above reporters are FAM (6-carboxyfluorescein), Texas red, fluorescein, HEX (5-hexachlorofluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC (fluorescein isothiocyanate), Oregon green, alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'- Dimethoxyfluorescein), ROX (6-Carboxyl-X-Rhodamine), TET (Tetrachloro-Fluorescein), TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl)-1,2-Dimethoxyfluorescein). A composition comprising at least one selected from the group consisting of ethylenediamine, cyanine series dyes, and thiadicarbocyanine.
In paragraph 4,
A composition wherein the quencher is at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), NFQ (nonfluorescent quencher), dabcyl, Eclipse, DDQ (Deep Dark Quencher), Blackberry Quencher, and Iowa black.
A kit for diagnosing PLHV1 infection, comprising the composition of claim 1.
(a) a step of amplifying the PLHV1 gene using the primer set of paragraph 1; and
(b) A method for diagnosing PLHV1 infection in a pig, comprising a step of detecting the product amplified in step (a).
In Article 8,
The amplification of step (a) above is a method for diagnosing PLHV1 infection in pigs using real-time polymerase chain reaction.
In Article 8,
A method for diagnosing PLHV1 infection in a pig, wherein the detection in step (b) is performed by any one method selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement, phosphorescence measurement, and combinations thereof.