KR20240165494A - Primer sets development for the detection of African Swine Fever Virus and Classical Swine Fever virus and their uses - Google Patents

Abstract

The present invention relates to a method for diagnosing African swine fever virus and swine fever virus simultaneously. By performing PCR using the primers according to the present invention, African swine fever virus and swine fever virus can be detected with high sensitivity using a small amount of sample, thereby enabling rapid confirmation of infection with each disease, and differential diagnosis of the two diseases is also possible. In addition, by rapidly and accurately diagnosing with one-step real-time genetic diagnosis, it is possible to reduce time, labor, and costs, as well as prevent the spread of diseases through rapid quarantine measures, and consequently contribute to the protection of the domestic livestock industry.

Description

Method and primer sets for simultaneous diagnosis of African swine fever virus and classical swine fever virus {Primer sets development for the detection of African swine fever virus and classical swine fever virus and their uses}

The present invention relates to a method for diagnosing African swine fever virus and swine fever virus simultaneously.

African swine fever virus (ASFV) is the only virus belonging to the Asfarviridae family, and has a very large dsDNA genome of 170–193 kbp, containing 150–167 ORFs (open reading frames). Based on the base sequence variation in the C-terminal region of the B646L structural protein gene encoding the capsid protein p72 among the African swine fever virus-specific proteins, it is classified into 24 genotypes. All 24 genotypes do not infect humans, and most of the viruses that have spread worldwide outside Africa are highly pathogenic viruses of genotype II.

Meanwhile, since the clinical symptoms of African swine fever virus are very similar to those of classical swine fever, and the two diseases should generally be differentiated in laboratory diagnosis, many studies have been conducted to confirm infection with the African swine fever virus. Conventional virus diagnosis methods mainly used electron microscopy or serological methods. However, although the diagnosis method using electron microscopy can confirm the presence of the virus, it is almost impossible to diagnose the species based on morphological characteristics. The current generalized method is a test using a serological method, which detects the genetic material (DNA) of African swine fever virus in blood, urine, cerebrospinal fluid, or amniotic fluid, or detects blood antibodies produced in response to African swine fever virus infection. Among serological methods, the ELISA (Enzyme-Linked Immunosorbent Assay) method is the most commonly used diagnostic method, but its detection sensitivity is about 1,000 times lower than that of the polymerase chain reaction (PCR) diagnostic method, and there are frequent cases where accurate diagnosis fails due to unexpected nonspecific reactions between antibodies and test samples. In addition, since the virus must be mass-produced and purified to inactivate the virus, and then used as an antigen to detect antibodies to the African swine fever virus, there is a problem that it takes a long time of several weeks or months and a lot of money to produce the antigen. In addition, since testing using the serological method requires a specialist with specialized equipment to collect samples from pigs, it takes additional time and money, so it was necessary to develop a method that can be easily collected at pig farms. Therefore, it is necessary to develop a diagnostic method that is specific and highly sensitive, capable of rapidly detecting the African swine fever virus and having no cross-reactivity with other viruses other than the African swine fever virus.

Patent Document 1: Korean Patent Publication No. 10-2022-0112623 (Published on August 11, 2022)

The purpose of the present invention is to provide a composition that simultaneously detects African swine fever virus and swine fever virus.

Another object of the present invention is to provide a method for differentially diagnosing African swine fever virus and swine fever virus with high sensitivity.

Accordingly, the present invention provides a primer set for African swine fever virus (ASFV) comprising a forward primer consisting of a base sequence of sequence number 1 and a reverse primer consisting of a base sequence of sequence number 2; and

A composition for diagnosing African swine fever virus and classical swine fever virus at the same time is provided, comprising a primer set for classical swine fever virus, the primer set comprising a forward primer consisting of a base sequence of sequence number 3 and a reverse primer consisting of a base sequence of sequence number 4.

In addition, the present invention provides a kit for diagnosing African swine fever virus and swine fever virus simultaneously, comprising the composition.

In addition, the present invention provides a method for diagnosing African swine fever virus and swine fever virus simultaneously, comprising the following steps:

(a) a step of preparing nucleic acid from a separated sample; (b) a step of amplifying a target nucleotide sequence in the specimen sample by RPA using a composition comprising a primer pair comprising nucleotide sequences of SEQ ID NOs: 1 and 2 and a primer pair comprising nucleotide sequences of SEQ ID NOs: 3 and 4; and

(c) A step for confirming the above amplification result is provided.

By performing PCR using the primers according to the present invention, African swine fever virus and swine fever virus can be detected with high sensitivity using a small amount of sample, so that not only can the infection of each disease be quickly confirmed, but also a differential diagnosis of the two diseases is possible. In addition, by quickly and accurately diagnosing with one-step real-time genetic diagnosis, it can not only save time, labor, and costs, but also prevent the spread of diseases through rapid quarantine measures, and consequently contribute to the protection of the domestic livestock industry.

Figure 1 is a diagram showing the sensitivity of real-time real-time PCR for CSFV.
Figure 2 is a diagram confirming the sensitivity of CSFV detection using the primer set of the present invention.
Figure 3 is a diagram evaluating the optimal temperature and time for RPA reaction using a primer set for CSFV of the invention.
Figures 4 and 5 are diagrams confirming the detection of CSFV virus in a sample using the primer set of the present invention.
Figure 6 is a diagram showing the sensitivity of real-time PCR for ASFV.
Figure 7 is a diagram evaluating the optimal temperature and time for RPA reaction using a primer set for ASFV of the invention.
Figure 8 is a diagram showing the sensitivity of detecting ASFV using the primer set of the present invention.
Figure 9 is a diagram confirming the detection of ASFV in a sample using the primer set of the present invention.

Hereinafter, the invention according to the present invention will be described in detail with respect to a novel Bacillus velezensis strain and its use, but the scope of the invention is not limited by the following description.

In the present invention, the primer may be one used in recombinase polymerase amplification (RPA) or reverse transcriptase-recombinase polymerase amplification (RT-RPA), but is not limited thereto as long as it corresponds to a method capable of amplifying and detecting a gene in the field in a short period of time.

In the present invention, the "recombinase polymerase amplification (RPA)" is a method for rapid isothermal amplification of nucleic acids. Amplification is performed depending on a specific combination of enzymes and proteins (recombinase, single-strand binding protein, and strand displacing DNA polymerase) used at a constant temperature. Real-time detection of RPA amplification products is possible through an exo-probe. Fluorescent substances can use fluorophores and quenchers, etc. through exonuclease III cleavage at an internal abasic site mimic (e.g., tetrahydrofuran (THF)) of a hybridized exo-probe. The fluorescent signal is measured in real time through a point-of-care scanner weighing 1.2 kg including a laptop (ESEQuant Tubescanner device, Qiagen Lake Constance GmbH, Stockach, Germany).

For example, the use of different labeling strategies has been investigated to reduce the number of steps required for the analysis, using reverse primers labeled with biotin or horseradish peroxidase. One approach has been to optimize the amplification temperature used and to use surface blocking agents. The combination of these changes has facilitated a much more rapid amplification and detection protocol, lowering the limit of detection (LOD) to 1 x 10 -15 M (J. Clin. Microbiol., 50 (2012), pp. 2234-2238). Reverse transcription recombinase polymerase amplification (RPA) is the addition of reverse transcriptase to the RPA method described above.

The term "real-time RT-PCR" used in the present invention refers to the sum of PCR and real-time (RT-) PCR, each of which has its own meaning. When examining each meaning, PCR refers to a technology for amplifying a specific region of DNA in large quantities in a test tube, and RT-PCR refers to a technology for reverse transcribing RNA into DNA in a test tube and then amplifying a specific region of the complementary DNA (cDNA) thus produced in large quantities. Real-time (RT-)PCR means that the results of amplifying a specific region are monitored in real time.

The term "primer" used in the present invention means a short nucleic acid fragment having a free 3'-hydroxyl group that complementarily binds to a template nucleic acid and functions as a starting point for synthesis. The primer can initiate the synthesis of a strand complementary to the template in the presence of an enzyme for polymerization reaction [DNA polymerase, reverse transcriptase, etc.] and four different deoxynucleoside triphosphates (dNTPs) at an appropriate reaction buffer and temperature, and the primer length and reaction conditions can be modified based on those known in the art. The primer may include a label that is directly or indirectly detectable by spectroscopic, photochemical, biochemical, immunochemical or chemical means, if necessary, and examples of the label include enzymes, radioisotopes, fluorescent molecules, chemical groups, etc.

In the present invention, the primer is not particularly limited to those capable of detecting African swine fever virus and swine fever virus, but preferably, oligonucleotides of SEQ ID NOs: 1 to 2 and SEQ ID NOs: 3 to 4 can be used.

The term "probe" used in the present invention refers to a DNA or RNA nucleic acid fragment, ranging from a few bases to several hundred bases, which is used to detect the presence of a complementary target nucleic acid sequence by hybridization, and can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, an RNA probe, etc. The probe may include markers used for detection at the 5' and 3' ends of the corresponding base sequence, respectively, and such markers may include a fluorophore and a quencher labeled at the 5' and 3' ends, respectively. Accordingly, when the fluorophore and quencher exist in the probe in a form in which they are bound to each other, luminescence is suppressed due to mutual interference, and when the probe is decomposed through a PCR reaction or the like, the interference between the fluorophore and quencher labeled on the probe disappears, allowing luminescence to occur. The fluorophore that can be labeled at the 5' end may be any fluorescent substance commonly used in the art without limitation, and examples thereof include 6-carboxyfluorescein (FAM), TET, hexachloro-6-carboxyfluorescein (HEX), JOE, ROX, TMR, Cyanine-5 (Cy5), Texas Red (TxR), and CAL Red 610.

The quencher that can be labeled at the 3' end can also be any quencher commonly used in the art without limitation, and for example, BHQ-1, BHQ-2, and TARMA can be used. When real-time (RT-)PCR is performed using a probe labeled with a fluorescent substance in this way, the amplified product can be monitored in real time, DNA and RNA can be quantified, and there is an advantage in that accurate diagnosis is possible quickly and easily because electrophoresis is not required. In addition, the primer set of the present invention can be used in a recombinase polymerization assay.

In the present invention, the probe is not particularly limited as long as it can detect African swine fever virus and swine fever virus.

The above primers and probes can incorporate additional features that do not change their basic properties. That is, the primers and probes can be modified using conventional means known in the art, as long as they exhibit the effect of detecting African swine fever virus and swine fever virus, which are the targets of the present invention.

In this specification, the International Union of Biochemistry (IUB) ambiguity code is used to indicate the base sequences of primers and probes. In the base sequence, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, R represents a position where both adenine and guanine are possible, and Y represents a position where both cytosine and thymine are possible. This was equally applied to presenting all base sequences in this specification.

The diagnostic kit of the present invention is a real-time RT-PCR diagnostic kit for performing African swine fever virus and swine fever virus economically, quickly, and easily, and may include not only one or more primers and probes among sequence numbers 1 to 4 included in the composition, but also one or more other component compositions, solutions, or devices suitable for the analysis method.

As a specific example, the diagnostic kit of the present invention may include essential elements necessary for performing reverse transcription reaction and PCR analysis. In addition to each primer/probe set specific for each detection site, the diagnostic kit may include reverse transcriptase for synthesizing cDNA from African swine fever virus and swine fever virus RNA, DNA polymerase for amplifying a specific region of cDNA in large quantities, a reaction buffer solution, dNTPs, an RNase inhibitor, sterile water, a test tube or other appropriate container, etc.

Ingredients included in each test tube in the present invention include, but are not limited to, a multi-oligo mixture composition for simultaneous testing of African swine fever virus and swine fever virus, DNA polymerase, reverse transcriptase, RNAi inhibitor, dATP, dCTP, dGTP, dTTP, Tris-HCl, MgCl2, KCl, BSA, and nuclease-free water, and the optimal amount of the component or reactant used for a specific reaction can be determined by a person skilled in the art.

According to another aspect of the present invention, the present invention provides a method for differentially detecting African swine fever virus and swine fever virus in a sample using the diagnostic kit.

Specifically, the method for testing African swine fever virus of the present invention comprises: (a) a step of isolating nucleic acid from a specimen; and (b) a step of specifically amplifying and detecting a target sequence through a real-time RT-PCR reaction using a composition or diagnostic kit including a primer pair including nucleotide sequences of SEQ ID NOS: 1 and 2 as a target of the isolated nucleic acid.

In addition, the method for testing the swine fever virus of the present invention comprises: (a) a step of isolating nucleic acid from a specimen; and (b) a step of specifically amplifying and detecting a target sequence through a PCR reaction using a composition or diagnostic kit including a pair of primers including nucleotide sequences of SEQ ID NOS: 3 and 4 as a target of the isolated nucleic acid.

In addition, the method for simultaneously testing for African swine fever virus and swine fever virus of the present invention specifically includes: (a) a step of isolating nucleic acid from a specimen from an animal suspected of being infected with African swine fever virus and swine fever virus; and (b) a step of specifically amplifying and detecting a target sequence through a PCR reaction using the diagnostic kit for detecting African swine fever virus and swine fever virus as the target of the separated nucleic acid.

According to a preferred embodiment of the present invention, the sample may be selected from the group consisting of domesticated pigs and wild boars, and includes blood, serum, plasma, feces, tissues, etc. isolated therefrom.

According to the present invention, nucleic acid is extracted from a specimen according to a method known in the art, and then RT-PCR is performed in a single tube using a diagnostic kit including a composition including primer/probe sets of the present invention on an extract liquid containing the extracted nucleic acid. The results of amplification and detection are confirmed in real time. For example, if luminescence of a fluorescent substance labeled with a specific probe is confirmed, this means that African swine fever virus is present in the specimen, and in a similar manner, if luminescence of a fluorescent substance labeled with a specific probe is confirmed, this means that African swine fever virus is present in the specimen. In other words, it is possible to detect African swine fever virus and swine fever virus in a specimen, as well as perform differential diagnosis.

In the present invention, novel primer/probe sets capable of specifically amplifying and detecting African swine fever virus and novel primer/probe sets capable of specifically amplifying and detecting African swine fever virus are designed, and it is confirmed that African swine fever virus and swine fever virus can be selectively diagnosed simultaneously using a composition including the primer/probe sets and a diagnostic kit including the same. The composition of the present invention and the diagnostic kit including the same can be used in a single tube real-time RT-PCR or recombinase polymerization assay. It is configured to enable testing with optimal amplification efficiency and specificity through reaction, and is manufactured so that non-specific reactions do not exist for viral infectious diseases that must be differentially diagnosed with African swine fever virus in pigs, such as PRRSV Type I (E38), PRRSV Type II (LMY), PEDV CU777, Aujeszky's Disease virus, BVDV 1, and BVDV 2, so it can be usefully used for differential diagnosis of African swine fever virus and swine fever virus.

Hereinafter, the present invention will be described in detail.

[Example 1] Cell and virus identification of African swine fever virus (ASF virus)

Primary porcine alveolar macrophages (PAMs) were collected from porcine lungs and cultured in RPMI 1640 medium (Thermo Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum at 37°C, 5% CO2, and adherent cell populations were harvested after overnight culture according to the OIE guidelines. ASFV titer was performed by HAD assay and expressed as 50% HAD dose/ml (HAD50).

That is, primary PAMs cells were seeded in 96-well plates, and then samples were added to the plates and titrated in triplicate using 10x dilutions. The amount of ASFV was determined by confirming the characteristic rosette formation indicating hemadsorption of erythrocytes around infected cells according to the OIE guidelines (OIE, 2012). HAD was observed for 4 days and HAD50 was calculated using the method of Reed and Muench (Reed and Muench, 1938).

ASFV genomic DNA was extracted from cell supernatants or tissue homogenates using the QIAamp DNA Mini Kit (QIAgen, Hilden, Germany). Real-time PCR was performed according to the OIE recommendations with specific primers as listed in Table 1. Real-time PCR was performed on an Agilent AriaMx Real-Time PCR System (Agilent) according to the OIE recommended procedures. The positive control consisted of known ASFV-positive DNA and the negative control was sterile water without nuclease.

Name Sequence (5'-3') Note King's primer F CTG CTC ATG GTA TCA ATC TTA TCG A [7]
King's primer R GAT ACC ACA AGA TC(AG) GCC GT OIE Probe FAM-CCA CGG GAG GAA TAC CAA CCC AGT G-TAMRA

[Example 2] Cell and virus identification of swine fever virus (CSF virus)

CSFV-VN91 virus was isolated from a dead pig in Hung Yen province in 1991 (GenBank # LC374604). The CSFV isolate was cultured in PK15a cells (ATCC, Manassas, VA, USA) in DMEM (Thermo Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Thermo Scientific) and 1% penicillin-streptomycin solution (Thermo scientific) for four passages. The 50% median tissue culture infectious dose (TCID50) of CSFV was calculated as previously described by OIE. Briefly, dilutions of CSFV in phosphate-buffered saline (PBS) were prepared and the dilutions were serially diluted 10-fold. 100 μl of each dilution was inoculated into PK15a cells (ATCC, Manassas, VA, USA) cultured in DMEM (Thermo Scientific, Waltham, MA, USA) medium containing 10% fetal bovine serum (FBS; Thermo Scientific) and 1% penicillin-streptomycin solution (Thermo Scientific).

Each titer measurement was repeated 40 times, including the negative control. The cells were cultured at 37°C for 48 h, and the replicated CSFV (TCID50) titer in each type of PK15 cells was detected by the IPMA method. The EID50/mL of the virus suspension was calculated using the Reed and Muench mathematical technique.

Immunoperoxidase monolayer assay (IPMA) for CSFV

TN5 cells were seeded in 96-well cell culture plates and inoculated with CSFV VN91 for 24 h at room temperature. The medium was then removed, the cells were washed with PBS, fixed in 4% paraformaldehyde for 10 min, and then washed twice with PBS. A 1% H2O2 solution in methanol was added to the cells for 5 min, and then the cells were washed twice with PBS. Anti-CSFV antibodies in phosphate-buffered saline supplemented with 1% Tween 80 (PBS-T) and negative SPF swine serum were added to the infected TN5 cells, and the cells were incubated for 1 h at 37°C. The serum dilution was then removed, and the cells were washed with PBS-T. The cells were then incubated with peroxidase-labeled goat anti-swine IgG antibodies (Sigma-Aldrich) for 1 h at 37°C. Afterwards, the plates were washed with PBS-T, 0.05% H2O2 (Sigma-Aldrich) was added to each well, and incubated at room temperature for 20 min. The reaction was stopped by replacing the substrate solution with Na-acetate buffer, and the staining was analyzed by light microscopy.

[Example 3] Differentiation and diagnosis of ASFV and CSFV

ASFV and CSFV sample collection

Based on clinical signs and postmortem lesions, pigs suspected of having ASF and CSF were used from several provinces in Northern Vietnam. Tissues including mesenteric lymph nodes, spleen, kidney, small intestine and liver were collected according to the guidelines of the OIE (OIE 2012). Negative samples were collected in Northern Vietnam in early 2018. The negative samples were negative for porcine porcine circovirus type 2 (PVC2), PCV3, porcine reproductive and respiratory syndrome virus (PRRS), swine influenza virus (SIV), PEDV and other pathogens including TGEV, ASFV and CSFV.

RNA extraction for field samples, one-step real-time RT-PCR and cDNA synthesis

Total RNA was extracted from cell supernatants and tissue homogenates using a High Pure Viral Nucleic Acid Kit (Roche Diagnostics, Rotkreuz, Switzerland). CSFV RNA in the samples was detected by real-time one-step reverse transcriptase-polymerase chain reaction (qRT-PCR) using a Taqman probe. The Superscript III Platinum Onestep qRT-PCR kit (Invitrogen) was used for qRT-PCR (forward primer: ATG CCC WTA GTA GGA CTA GCA; reverse primer: TCA ACT CCA TGT GCC ATG TAC; and Taqman probe: TGA TGG GGG TAC GAC CTG ATA GGGT). Each primer at a concentration of 40 μM and the probe at 10 μM were used in the reaction. A Ct value less than 35 was considered positive, a Ct value ≥35 was suspected to be positive, and a case where no Ct value was obtained was considered negative. Once CSFV was confirmed, complementary DNA was produced from the RNA extract using the M-MLV Reverse Transcription System (Promega) and sequenced.

In the same manner, qRT-PCR for ASFV was performed. More specifically, a pair of primers and probe targeting the conserved region of the P72 gene was designed and used based on the reference sequence of ASFV (GenBank: AY578706) (forward primer, 5'-GCCGAAGGG AATGGATACTGAGGGAATAGCAA-3'; reverse primer 5'-TCCC GAGAACTCTCACAATATCCAAACAGCAG-3 primer).

ASFV and CSFV RPA responses

For the RPA reaction, primer sets for ASFV of sequence numbers 1 and 2 in the present invention were designed and used, and primers for CFV of sequence numbers 3 and 4 were designed and used. The sequences are shown in Tables 2 and 3 below.

Sequence name 5'Modification Sequence (5'-3') RPA product size ASFV_151F FITC CCACAAGATCAGCCGTAGTGAGATAGACCCCAC 151bp ASFV_151R Biotin GATGATCCGGTGTGCGATGATGATTACCTTTG

Sequence name 5' Modification Sequence (5'-3') RPA product size CSFV_200F FITC TGAAGCTGGTAAGCCCCAAAAATTACAGAAGG 200bp CSFV_200R Digoxigenin TACCCCTCTCACCACTGGAATCTAACCTTGTA

RPA reactions were performed in a 50 μL reaction volume using the TwistAmp exo kit (TwistDX, Cambridge, UK). The reaction components included 420 nM of each RPA primer, 14 mM magnesium acetate, and nucleic acid template. All reagents except the viral template and magnesium acetate were prepared from a master mix dispensed into individual 0.2 mL reaction tubes containing lyophilized enzyme pellets. After viral nucleic acid was added to each tube, the magnesium acetate was pipetted into the tube cap, the cap was carefully closed, and the magnesium acetate was centrifuged using a minispin centrifuge to rehydrate the material. The samples were briefly vortexed and spun again, and the tubes were immediately placed in a Genie III scanner device (Optigene, Horsham, UK) and the reaction was performed at 39°C for 20 min. Fluorescence signals were collected in real time. To evaluate the sensitivity of the RPA analysis, 1.56 X 10 ⁷ copies of the pET30a-CSFV template were serially diluted 10-fold to concentrations ranging from 1.56 X 10 ⁷ to 1.56 X 10 ² , and 1 μL of each dilution was subjected to RPA analysis and real-time PCR. For the ASFV RPA analysis, 1.65 X ^{10 7} copies of the pET30a-ASFV template were serially diluted 10-fold to concentrations ranging from 1.65 X 10 ⁷ to 1.65 X 10 ² copies/μL, and 1 μL of each dilution was subjected to RPA analysis and real-time PCR.

result

As a result of synthesizing the above results, as shown in Figures 1 to 9, it was confirmed that the primer set of the present invention can be used for simultaneous diagnosis of CSFV and ASFV because the sensitivity is superior compared to real-time PCR.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single component may be implemented in a distributed manner, and likewise, components described as distributed may be implemented in a combined manner.

The scope of the present application is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present application.

Attach electronic file of sequence list

Claims (6)

A primer set for African swine fever virus (ASFV) comprising a forward primer consisting of the base sequence of sequence number 1 and a reverse primer consisting of the base sequence of sequence number 2; and
A composition for diagnosing African swine fever virus and classical swine fever virus simultaneously, comprising a primer set for classical swine fever, the primer set comprising a forward primer consisting of a base sequence of sequence number 3 and a reverse primer consisting of a base sequence of sequence number 4.
A composition according to claim 1, wherein the composition is performed by genetic amplification.
A composition according to claim 2, wherein the gene amplification is performed by PCR.
A composition in claim 3, wherein the gene amplification is performed by a recombinase polymerase amplification (RPA) reaction.
A kit for diagnosing African swine fever virus and swine fever virus simultaneously, comprising a composition of any one of claims 1 to 4.
A method for diagnosing African swine fever virus and swine fever virus simultaneously, comprising the following steps:
(a) a step of preparing nucleic acid from a separated sample;
(b) a step of amplifying the target nucleotide sequence in the test sample using a composition comprising a primer pair comprising the nucleotide sequences of SEQ ID NOs: 1 and 2 and a primer pair comprising the nucleotide sequences of SEQ ID NOs: 3 and 4; and
(c) A step of confirming the above amplification results.