KR20250147776A - Realtime genetic diagnosis system for simultaneous detection of Canine and Feline parvovirus(CPV and FPV) and Canine and Feline coronavirus(CCoV and FCoV) - Google Patents

Abstract

The present invention relates to a real-time genetic diagnostic system capable of simultaneously differentiating and detecting canine and feline parvovirus (CPV) and feline parvovirus (FPV), and canine and feline coronavirus (CCoV) and feline coronavirus (CPV). The present invention provides a composition, a kit, and a differential diagnostic method for the simultaneous differential diagnosis of canine / feline parvovirus (CPV/FPV) and canine / feline coronavirus (CCoV/FCoV). When the composition, kit, and method of the present invention are used, the effect of distinguishing and detecting canine/feline parvovirus (CPV/FPV) and canine/feline coronavirus (CCoV/FCoV) with high sensitivity and specificity is exhibited in a single PCR reaction, thereby allowing for rapid and accurate confirmation of infection with canine/feline parvovirus and canine/feline coronavirus.

Description

Realtime genetic diagnosis system for simultaneous detection of canine and feline parvovirus (CPV and FPV) and canine and feline coronavirus (CCoV and FCoV)

The present invention relates to a real-time genetic diagnostic system capable of simultaneously differentiating and detecting canine / feline parvovirus (CPV/FPV) and canine / feline coronavirus (CCoV/FCoV).

As of the end of 2022, the number of domestic households owning pets reached 5.52 million, representing a roughly 2.8% increase compared to 2020 and a continued year-over-year increase. Dogs and cats account for a significant portion of this number.

These recent social trends have also led to a growth in the market for infectious disease diagnosis for companion animals. In particular, with growing concern over companion animal abuse and the need for veterinary forensic examinations at the Animal and Plant Quarantine Agency and provincial animal health testing laboratories, diagnostic methods for identifying pathogens that can identify the underlying causes of death in companion animals are essential.

Canine parvovirus (CPV) and canine coronavirus (CCoV) are known as representative agents that cause viral diarrhea in dogs, and are also major pathogens that cause acute gastroenteritis. The two viruses have high transmissibility and morbidity, and show susceptibility to infection in all age groups, but puppies under 6 weeks to 6 months of age, or those with immunodeficiency or unvaccinated conditions are reported to be more susceptible (Chaeyeong MIN, et al. , 2023). Meanwhile, feline parvovirus (FPV) and feline coronavirus (FCoV) are also known as highly contagious viruses that cause serious intestinal diseases in feline animals. Like CPV and CCoV, infection can occur in all age groups, but it has been reported that they show a high mortality rate in young animals.

In general, combined infections by CPV/FPV and CCoV/FCoV are common in dogs and cats and can be more fatal than single infections. Unlike bacterial infections, viral infections have no specific treatment, making faster and more accurate diagnosis even more important.

Therefore, the present invention aims to develop a real-time genetic diagnostic method capable of simultaneously detecting canine/feline parvovirus (CPV/FPV) and canine/feline coronavirus (CCoV/FCoV), which exhibit high mortality rates due to complex infections in dogs and cats, and to utilize the method to establish a rapid and accurate diagnostic system, thereby contributing to public health.

Domestic Patent No. 10-1258614 (registration date: April 29, 2013) Domestic Publication Patent No. 10-2014-0131624 (published on November 14, 2014)

The present inventors have made extensive research efforts to develop a diagnostic method capable of simultaneously differentiating CPV/FPV and CCoV/FCoV from complex infections, not only to effectively detect canine / feline parvovirus (CPV/FPV) and canine / feline coronavirus (CCoV/FCoV), which are the causative agents of viral diarrhea and acute gastroenteritis in dogs/cats, based on rapid and accurate diagnosis, but also to contribute to public health in companion animal breeding, which has recently been rapidly increasing. As a result, the present inventors have manufactured a composition for real-time simultaneous differential diagnosis of canine/feline parvovirus (CPV/FPV) and canine/feline coronavirus (CCoV/FCoV), and have elucidated that a kit and a genetic diagnostic method including the same can enable rapid and accurate differential diagnosis, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for simultaneous genetic diagnosis of canine/feline parvovirus (CPV/FPV) and canine/feline coronavirus (CCoV/FCoV).

Another object of the present invention is to provide a kit for simultaneous differential diagnosis of canine/feline parvovirus and canine/feline coronavirus comprising a CPV/FPV and CCoV/FCoV simultaneous differential genetic composition.

Another object of the present invention is to provide a method for simultaneous differential diagnosis of canine/feline parvovirus and canine/feline coronavirus.

The present inventors have made extensive research efforts to develop a diagnostic method capable of not only effectively detecting canine/feline parvovirus (CPV/FPV) and canine/feline coronavirus (CCoV/FCoV), which are the causative agents of viral diarrhea and acute gastroenteritis in dogs and cats, based on rapid and accurate diagnosis, but also simultaneously differentiating canine/feline parvovirus (CPV/FPV) and canine/feline coronavirus (CCoV/FCoV) from complex infections, which can contribute to public health in companion animal breeding, which has recently been rapidly increasing. As a result, the present inventors have manufactured a real-time simultaneous differential diagnosis composition for canine/feline parvovirus and canine/feline coronavirus, and have found that a kit containing the same and a genetic diagnostic method can enable rapid and accurate differential diagnosis.

According to one aspect of the present invention, the present invention provides a composition for simultaneous differential diagnosis of canine and feline parvovirus (CPV) and feline parvovirus (FPV) and canine and feline coronavirus (CCoV) and feline coronavirus (FCoV), comprising (i) a first primer set consisting of nucleotide sequences of SEQ ID NOs: 1 and 2; and ( ii ) a second primer set consisting of nucleotide sequences of SEQ ID NOs : 4 and 5.

As used herein, the term " parvovirus " means a single-stranded DNA virus belonging to the Parvoviridae family and Parvovirus genus, which is the virus that causes canine or feline parvovirus infection.

Specifically, the term " Feline parvovirus (FPV)" as used herein refers to a parvovirus that causes feline panleukopenia (feline infectious enteritis), a type of viral enteritis. The feline parvovirus (FPV) specifically causes infection in feline animals, and infection mainly occurs through contact with the body fluids and excrement of infected animals, but infection can occur through a vector without contact. It is characterized by causing a severe decrease in white blood cells in infected animals, and in addition, it can migrate to the internal organs and destroy intestinal cells, forming ulcers and exhibiting various symptoms such as bloody stool, diarrhea, and severe dehydration.

Also, specifically, the term " canine parvovirus (CPV)" used herein is derived from feline parvovirus (FPV), and refers to an infectious virus that causes canine parvovirus infection. It is characterized by strong pathogenicity and high contagiousness in dogs, and is transmitted from dog to dog through feces, saliva, etc., either directly or indirectly. CPV infection causes digestive symptoms such as severe vomiting and diarrhea, and in the case of young animals, i.e. puppies, it can cause heart disease and is characterized by a very high mortality rate.

As described above, canine parvovirus (CPV) is derived from feline panleukopenia virus (FPLV), i.e., feline parvovirus (FPV), and in particular, is presumed to be a mutant strain of FPV. CPV and FPV are morphologically and genetically very similar and have been reported to be closely related, and therefore, the genetic diagnostic method according to the present invention can detect both canine parvovirus (CPV) and feline parvovirus (FPV).

Meanwhile, the term " coronavirus " used in this specification refers to an enveloped positive-strand RNA virus belonging to the Coronaviridae family and Alphacoronavirus genus.

Specifically, the term " Feline coronavirus (FCoV)" as used herein refers to a coronavirus commonly found in cats and the main causative agent of gastroenteritis. The Feline coronavirus (FCoV) exists in nature as two distinct biotypes, namely Feline enteric coronavirus (FECV) and Feline infectious peritonitis virus (FIPV), a mutant form of FECV. According to current research, FIPV is known to be caused by genetic mutations and changes in tissue tropism as cats become chronically infected with FECV. In this regard, infection with FCoV usually only causes mild enteritis, but it can cause inflammation in the body of some infected cats, leading to the highly fatal Feline infectious peritonitis.

Also, specifically, the term " canine coronavirus (CCoV)" as used herein refers to a virus that causes a highly contagious and severe enteric disease in dogs, and is characterized by causing a digestive infectious disease with symptoms such as vomiting, diarrhea, and dehydration. The canine coronavirus (CCoV) infects puppies and adult dogs, but is particularly prevalent in puppies, and susceptibility is virtually independent of breed or age. In addition, due to its rapid transmission and high morbidity rate, it presents a problem of transmission occurring within a short period of time in group breeding dogs.

Feline coronavirus (FCoV) and canine coronavirus (CCoV) are classified as the same species belonging to the Alphacoronavirus 1 species of the Alphacoronavirus genus along with Porcine transmissible gastroenteritis virus (TGEV), and are reported to show high sequence homology between them. Therefore, the genetic diagnostic method according to the present invention can detect both canine coronavirus (CCoV) and feline coronavirus (FCoV).

In general, canine parvovirus (CPV) and feline parvovirus (FPV), or canine coronavirus (CCoV) and feline coronavirus (FCoV), occur in the form of mixed infections, causing more serious damage than single infections.

The term "primer" as used herein means an oligonucleotide capable of acting as an initiation point of synthesis under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid strand (template), i.e., in the presence of nucleotides and a polymerizing agent such as DNA polymerase, and at a suitable temperature and pH.

In one embodiment of the present invention, the composition further comprises a probe for differential diagnosis of CPV and FPV consisting of a nucleotide sequence of SEQ ID NO: 3; a probe for differential diagnosis of CCoV and FCoV consisting of a nucleotide sequence of SEQ ID NO: 6; or a combination thereof.

The term "probe" as used herein refers to a single-stranded nucleic acid molecule comprising a portion or portions substantially complementary to a target nucleic acid sequence. The "target nucleic acid," "target nucleic acid sequence," or "target sequence" refers to a nucleic acid sequence to be detected, which anneals or hybridizes with a primer or probe under hybridization, annealing, or amplification conditions.

More specifically, the probe and primer are single-stranded deoxyribonucleotide molecules. The probe or primer used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides. The probe or primer may also include ribonucleotides.

The primer must be sufficiently long to prime the synthesis of the extension product in the presence of the polymerization agent. The exact length of the primer will depend on several factors, including temperature, application, and the source of the primer.

The term "annealing" or "priming" as used herein means the juxtaposition of an oligodeoxynucleotide or nucleic acid to a nucleic acid that serves as a template (hereinafter, template nucleic acid), wherein the juxtaposition causes a polymerase to polymerize the nucleotides to form a nucleic acid molecule complementary to the template nucleic acid or a portion thereof.

The primer used in the present invention hybridizes or anneals to a portion of the template to form a double-stranded structure. Conditions for nucleic acid hybridization suitable for forming such a double-stranded structure are disclosed in Joseph Sambrook, et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al ., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).

The term "hybridization," as used herein, refers to the formation of a double-stranded nucleic acid from complementary single-stranded nucleic acids. Hybridization can occur between two nucleic acid strands that are completely identical or substantially identical with some mismatch. The degree of complementarity for hybridization may vary depending on hybridization conditions, particularly temperature. The terms "annealing" and "hybridization" are used interchangeably herein and are not distinct from each other.

In general, a 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 fluorescent reporter dye and a quencher labeled at the 5' and 3' ends, respectively. Accordingly, when the fluorescent reporter dye and the quencher exist in a form in which they are bound to each other in the probe, luminescence is suppressed due to mutual interference, and when the probe is decomposed through a PCR reaction or the like, the interference between the fluorescent reporter dye and the quencher labeled on the probe disappears, allowing luminescence to occur. The fluorescent reporter dye that may be labeled at the 5' end may be any fluorescent substance commonly used in the art without limitation. For example, FAM (6-Carboxyfluorescein), TET (Carboxy-2,4,7,7-tetrachlorofluorescein), HEX (Hexachloro-6-carboxyfluorescein), JOE (2,7-dimethoxy-4,5-dichloro-6-carboxyfluoroscein)), ROX ((6-Carboxy-X-rhodamine)), TMR (Tetramethylrhodamine), Cy-5 (Cyanine-5), TxR (Texas Red), and CAL Red 610 can be used. The quencher that can be labeled at the 3' end above can also be used without limitation as a quencher commonly used in the art, for example, BHQ-1 (Black Hole Quencher 1), BHQ-2 (Black Hole Quencher 2), BHQ-3 (Black Hole Quencher 3), MGB (minor-groove binding), TAMRA (5-Carboxytetramethylrhodamine), DDQ1 (deep dark Quencher 1), and DDQ2 (deep dark Quencher 2) 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 an accurate diagnosis can be made quickly and easily because there is no need for electrophoresis.

In one embodiment of the present invention, the 5'-end of the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 6 is labeled with one different fluorophore selected from the group consisting of FAM, TET, HEX, JOE, ROX, TMR, Cy5, TxR, and Cal Red 610, and the 3'-end is modified with a quencher of BHQ-1, BHQ-2, or BHQ-3, but is not limited thereto.

Additionally, the primers and probes can incorporate additional features that do not alter 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 simultaneously differentiating between canine/feline parvovirus (CPV/FPV) and canine/feline coronavirus (CCoV/FCoV), as desired in the present invention.

The primers and probes of sequence numbers 1 to 3 of the present invention are characterized in that they target the NS1 gene of canine/feline parvovirus (CPV/FPV).

The primers and probes of sequence numbers 4 to 6 of the present invention are characterized in that they target the 3'-UTR gene of canine/feline coronavirus (CCoV/FCoV).

According to another aspect of the present invention, the present invention provides a kit for simultaneous differential diagnosis of canine and feline parvovirus and canine and feline coronavirus, comprising a composition for simultaneous differential diagnosis of canine and feline parvovirus (CPV and FPV) and canine and feline coronavirus (CCoV and FCoV) of the present invention.

Since the kit of the present invention comprises the composition as a component, the contents common to both inventions also apply equally to the kit invention. In addition, to avoid redundant description in this specification, common elements between the two inventions will be omitted.

In one embodiment of the present invention, the kit is performed by gene amplification.

In one specific embodiment of the present invention, the gene amplification is performed by PCR (polymerase chain reaction).

Polymerase chain reaction (PCR) is the most well-known nucleic acid amplification method, and many variations and applications have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying the traditional PCR procedure to increase the specificity or sensitivity of PCR. Furthermore, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (IPCR), vectorette PCR, thermal asymmetric interlaced PCR (TAIL-PCR), and multiplex PCR have been developed for specific applications. For more information on PCR, see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York, Berlin, Heidelberg, N.Y. (2000), whose teachings are incorporated herein by reference.

The kit of the present invention enables simultaneous differentiation and diagnosis of canine/feline parvovirus (CPV/FPV) and canine/feline coronavirus (CCoV/FCoV) through a polymerase chain reaction method using the above primers and probes.

The kit of the present invention may additionally include essential elements necessary for performing polymerase chain reaction (PCR). In addition to a specific primer/probe set for each detection target region, the kit may include a reverse transcriptase for synthesizing cDNA from a nucleic acid template extracted from a specimen, a DNA polymerase for amplifying a specific region of cDNA in large quantities, a reaction buffer, dNTPs, an RNase inhibitor, sterile water, a test tube, or other appropriate container, etc.

Various DNA polymerases can be used in the above polymerase chain reaction (PCR), including the 'Klenow' fragment of E. coli DNA polymerase I, thermostable DNA polymerase, and bacteriophage T7 DNA polymerase. Specifically, the polymerase is a thermostable DNA polymerase that can be obtained from various bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis , and Pyrococcus furiosus (Pfu).

Specifically, in one embodiment of the present invention, the kit of the present invention comprises Taq DNA polymerase or Pfu DNA polymerase.

More specifically, in one embodiment of the present invention, the kit of the present invention comprises Taq DNA polymerase.

The kit of the present invention may additionally contain dUTP (deoxyuridinetriphosphate) and UDG (Uracil DNA Glycosilase) to block carryover or crossover contamination by polymerase chain reaction products. UDG recognizes and cleaves uracil contained in the DNA template strand of the previous amplification product, and the cleaved DNA template strand no longer functions as a template strand, thereby preventing the amplification reaction from occurring. By using dUTP and UDG, the present invention fundamentally blocks the generation of amplification products due to contamination of the previous amplification product, thereby eliminating false positive results due to laboratory contamination of the previous amplification product, thereby improving the accuracy of the test.

When conducting a polymerization reaction, it is preferable to provide the reaction vessel with an excess of the components required for the reaction. The excess of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited by the concentration of the components. Additionally, cofactors such as Mg ²⁺ , dATP, dCTP, dGTP, and dTTP may be added to the reaction mixture to an extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction can be active under the same reaction conditions. In fact, the buffer allows all enzymes to approach their optimal reaction conditions. Therefore, the amplification process of the present invention can be performed in a single reaction without changing conditions such as the addition of reactants.

In one embodiment of the present invention, the gene amplification is performed by real-time PCR.

The above real-time PCR is a technology that monitors and analyzes the increase of PCR amplification products in real time (Levak KJ, et al. , PCR Methods Appl. , 4(6): 357-62(1995)). The PCR reaction can be monitored by recording the fluorescence emission at each cycle during the exponential phase, where the increase in PCR product is proportional to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the faster the fluorescence increase is observed and the lower the Ct value (cycle threshold). A marked increase in fluorescence above the reference value measured between 3 and 15 cycles indicates the detection of accumulated PCR products. Compared to conventional PCR methods, real-time PCR has the following advantages: (a) data can be obtained during the exponential growth phase, whereas conventional PCR is measured at a plateau; (b) the increase in reporter fluorescence signal is directly proportional to the number of generated amplicons; (c) the digested probe provides permanent record amplification of the amplicon; (d) an increased detection range; (e) more than 1,000 times less nucleic acid is required compared to conventional PCR methods; (f) detection of the amplified DNA is possible without separation by electrophoresis; (g) increased amplification efficiency can be achieved by utilizing small amplicon sizes; and (h) there is a reduced risk of contamination.

When the amount of PCR amplification product reaches a detectable amount by fluorescence, an amplification curve begins, with the signal increasing exponentially before reaching a plateau. The higher the initial amount of DNA, the fewer cycles it takes for the amplification product to reach a detectable amount, resulting in a faster amplification curve. Therefore, when a real-time PCR reaction is performed using a serially diluted standard sample, an amplification curve is obtained that is evenly spaced in descending order of initial DNA amount. Setting a threshold at an appropriate point yields the Ct value, the point at which the threshold intersects the amplification curve.

In real-time PCR, PCR amplification products are detected via fluorescence. Detection methods include the interchelating method (SYBR Green I method) and the method using fluorescently labeled probes (TaqMan probe method). The interchelating method detects all double-stranded DNA, eliminating the need for gene-specific probes and allowing for low-cost reaction system construction. While the method using fluorescently labeled probes is more expensive, it offers high detection specificity, allowing for the discrimination and detection of even similar sequences.

First, the interchelating method is a method that utilizes a double-stranded DNA binding dye, and quantifies non-specific amplification and amplicon production including primer-dimer complexes using a non-sequence-specific fluorescent intercalating reagent (SYBR Green I or ethidium bromide). This reagent does not bind to ssDNA. SYBR Green I is a fluorescent dye that binds to the minor groove of double-stranded DNA. It is an interchelator that shows little fluorescence in solution but exhibits strong fluorescence when bound to double-stranded DNA (Morrison TB, Biotechniques. , 24(6): 954-8, 960, 962(1998)). Therefore, the amount of amplification product produced can be measured because fluorescence is emitted through the binding between SYBR Green I and double-stranded DNA. SYBR Green real-time PCR involves optimization steps, such as melting point or dissociation curve analysis, for amplicon identification. Normally, SYBR Green is used in singleplex reactions, but it can also be used in multiplex reactions when accompanied by melting curve analysis (Siraj AK, et al. , Clin Cancer Res. , 8(12): 3832-40(2002); and Vrettou C., et al. , Hum Mutat. , Vol 23(5): 513-521(2004)).

The above Ct (cycle threshold) value refers to the cycle number at which the fluorescence generated in the reaction exceeds the threshold, and is inversely proportional to the logarithm of the initial copy number. Therefore, the Ct value assigned to a specific well reflects the number of cycles at which a sufficient number of amplicons has accumulated in the reaction. The Ct value is the cycle at which an increase in △ Rn is first detected. Rn refers to the magnitude of the fluorescence signal generated during PCR at each time point, and △ Rn refers to the fluorescence emission intensity of the reporter dye (normalized reporter signal) divided by the fluorescence emission intensity of the reference dye. The Ct value is also referred to as Cp (crossing point) in LightCycler. The Ct value represents the point at which the system begins to detect an increase in the fluorescence signal associated with the exponential growth of the PCR product in the log-linear phase. This point provides the most useful information about the reaction. The slope of the log-linear step represents the amplification efficiency (Eff) (www.appliedbiosystems.co.kr/).

Meanwhile, TaqMan probes are typically longer oligonucleotides (e.g., 20-30 nucleotides) than primers that contain a fluorophore at the 5'-end and a quencher (e.g., TAMRA or non-fluorescent quencher (NFQ)) at the 3'-end. The excited fluorophore transfers energy to a nearby quencher rather than fluorescing (FRET = FRET or fluorescence resonance energy transfer; Chen, X., et al. , Proc Natl Acad Sci USA , 94(20): 10756-61(1997)). Therefore, when the probe is normal, no fluorescence is emitted. TaqMan probes are designed to anneal to internal sites of PCR products.

TaqMan probes specifically hybridize to template DNA during the annealing step, but fluorescence is suppressed by a quencher on the probe. During the extension reaction, the TaqMan probe hybridized to the template is degraded by the 5' to 3' nuclease activity of Taq DNA polymerase, releasing the fluorescent dye from the probe, thereby releasing the suppression by the quencher and displaying fluorescence. At this time, the 5'-end of the TaqMan probe must be located downstream of the 3'-end of the extension primer. That is, when the 3'-end of the extension primer is extended by a template-dependent nucleic acid polymerase, the 5'-end of the TaqMan probe is cleaved by the 5' to 3' nuclease activity of the polymerase, resulting in the generation of a fluorescent signal from the reporter molecule.

According to an embodiment of the present invention, the kit of the present invention utilizes the TaqMan probe method.

The reporter molecule and quencher molecule bound to the TaqMan probe include a fluorescent substance and a non-fluorescent substance.

Any fluorescent reporter molecule and quencher molecule that can be used in the present invention can be used as long as they are known in the art, and examples thereof are as follows (the numbers in parentheses are the maximum emission wavelengths expressed in nanometers): Cy2 ^TM (506), YOPRO ^TM -1 (509), YOYO ^TM -1 (509), Calcein (517), FITC (518), FluorX ^TM (519), Alexa ^TM (520), rhodamine 110 (520), 5-FAM (522), Oregon Green ^TM 500 (522), Oregon Green ^TM 488 (524), RiboGreen ^TM (525), RhodamineGreen ^TM (527), Rhodamine 123 (529), Magnesium Green ^TM (531), Calcium Green ^TM (533), TO-PRO ^TM -1 (533), TOTO1 (533), JOE(548), BODIPY530/550(550), Dil(565), BODIPY TMR(568), BODIPY558/568(568), BODIPY564/570(570), Cy3 ^TM (570), Alexa ^TM 546(570), TRITC(572), Magnesium Orange ^TM (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium Orange ^TM (576), Pyronin Y (580), RhodamineB (580), TAMRA (582), Rhodamine Red ^TM (590), Cy3.5 ^TM (596), ROX (608), Calcium Crimson ^TM (615), Alexa ^TM 594(615), Texas Red(615), Nile Red(628), YO-PRO ^TM -3(631), YOYO ^TM -3(631), R-phycocyanin(642), CPhycocyanin(648), TO-PRO ^TM -3(660), TOTO3(660), DiD DilC(5)(665), Cy5 ^TM (670), Thiadicarbocyanine(671), Cy5.5(694), HEX(556), TET(536), VIC(546), BHQ-1(534), BHQ-2(579), BHQ-3(672), BiosearchBlue(447), CAL Fluor Gold 540(544), CAL Fluor Orange 560(559), CAL Fluor Red 590(591), CAL FluorRed 610(610), CAL Fluor Red 635(637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705), Quasar 705 (610) and TxR (592).

Suitable reporter-quencher pairs are described in many references: Pesce et al. , editors, FLUORESCENCE SPECTROSCOPY (Marcel Dekker, New York, 1971); White et al. , FLUORESCENCE ANALYSIS: A PRACTICAL APPROACH (Marcel Dekker, New York, 1970); Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATIC MOLECULES, 2nd EDITION (Academic Press, New York, 1971); Griffiths, COLOUR AND CONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York, 1976); Bishop, editor, INDICATORS (Pergamon Press, Oxford, 1972); Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes, Eugene, 1992); Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (Interscience Publishers, New York, 1949); Haugland, RP, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, Sixth Edition, Molecular Probes, Eugene, Oreg., 1996; US Pat. Nos. 3,996,345 and 4,351,760.

According to another aspect of the present invention, the present invention provides a method for simultaneous differential diagnosis of canine and feline parvovirus (CPV and FPV) and canine and feline coronavirus (CCoV and FCoV), comprising the following steps:

(a) a step of preparing nucleic acid from a specimen sample collected from a dog or cat or the environment;

(b) a step of amplifying the target nucleotide sequence in the specimen sample using a composition for simultaneous differential diagnosis of canine and feline parvovirus (CPV/FPV) and canine and feline coronavirus (CCoV/FCoV), comprising (i) a first primer set consisting of nucleotide sequences of SEQ ID NOs: 1 and 2; and (ii) a second primer set consisting of nucleotide sequences of SEQ ID NOs: 4 and 5, using the nucleic acid extracted in the step (a) as a template; and

(c) A step of confirming the above amplification results.

In one embodiment of the present invention, the composition comprising the primer sets of (i) and (ii) in step (b) further comprises a probe for differential diagnosis of CPV and FPV comprising a nucleotide sequence of SEQ ID NO: 3, a probe for differential diagnosis of CCoV and FCoV comprising a nucleotide sequence of SEQ ID NO: 6, or a combination thereof.

The above method is explained step by step below.

(a) Step of preparing nucleic acid from a specimen

The term "specimen sample" as used herein encompasses various samples. More specifically, the specimen sample may be, but is not limited to, a sample isolated from a dog or cat suspected of being infected with the virus, and may be an environmental sample isolated from the surrounding environment of the subject (dog or cat). The sample isolated from the subject may be a cell, tissue, feces, urine, or body fluid, and the body fluid may be, but is not limited to, blood, serum, plasma, lymph, ocular fluid, saliva, or tissue extract. The environmental sample may include, but is not limited to, drinking water, feed, etc.

The step of preparing nucleic acids from the above specimen sample is a step of processing the specimen sample to apply the PCR method. The method of processing the specimen sample may apply a DNA extraction method or an RNA extraction method, and more specifically, a DNA extraction method may be applied. Extracting DNA or RNA from the sample may be performed according to a conventional method known in the art (reference: Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere, C. et al., Plant Mol. Biol. Rep., 9:242 (1991); Ausubel, FM et al., Current Protocols in Molecular Biology, John Willey & Sons (1987); and Chomczynski, P. et al., Anal. Biochem. 162:156 (1987)).

(b) a step of amplifying a sequence using the primer set and probe described above;

In one embodiment of the present invention, the amplification is performed by PCR (polymerase chain reaction).

In one specific embodiment of the present invention, the amplification is performed by real-time PCR.

More specifically, in one embodiment of the present invention, the real-time PCR is performed using the TaqMan probe method.

In one embodiment of the present invention, the probe is bound to a label, and the confirmation in step (c) is performed by detecting a signal generated from the probe.

Since the method of the present invention utilizes the kit for simultaneous differential diagnosis of canine and feline parvovirus (CPV and FPV) and canine and feline coronavirus (CCoV and FCoV), description of common contents between the two is omitted to avoid excessive complexity of this specification.

(C) Step for confirming the above amplification results

When amplifying a sequence using the traditional PCR method, the amplification results can be confirmed by agarose gel or polyacrylamide gel electrophoresis. More specifically, but not limited to, agarose gel electrophoresis. After electrophoresis, the electrophoresis results can be analyzed by methods such as ethidium bromide staining.

The present invention pertains to a real-time genetic diagnostic method, and when a sequence is amplified using a real-time PCR method, regardless of whether the unknown sample is positive or negative, the change in fluorescence intensity of the labeling substance over the entire PCR amplification period of the internal control is measured, and the number of PCR reactions (Cp value or Ct value) that reach a certain fluorescence intensity is analyzed, and a standard curve of fluorescence intensity can be obtained. This standard curve of Cp value or Ct value and fluorescence intensity of the internal control is clearly distinguished from the curve pattern of Cp value or Ct value and fluorescence intensity of the negative control, and can provide a standard for determining whether the unknown sample is positive or negative. Therefore, by performing PCR amplification on a specimen sample, measuring the change in fluorescence intensity of the labeling substance over the entire PCR amplification period, and analyzing the number of PCR reactions (Cp value or Ct value) that reach a certain fluorescence intensity, the standard curve of fluorescence intensity can be obtained, and then comparing it with the curve pattern of Cp value or Ct value and fluorescence intensity of the internal control and the negative control, it is possible to determine whether the sample contains a target.

The above method is an exemplary method for confirming the amplification result, and the method for confirming the amplification result is not limited thereto, and can be performed using a method widely known in the art.

The present invention provides a composition, a kit, and a differential diagnosis method for simultaneous differential diagnosis of canine / feline parvovirus (CPV/FPV) and canine / feline coronavirus (CCoV/FCoV), and when the composition, kit, and method of the present invention are used, canine/feline parvovirus (CPV/FPV) and canine/feline coronavirus (CCoV/FCoV) can be detected with high sensitivity and specificity in a single PCR reaction, so that infection with canine/feline parvovirus and canine/feline coronavirus can be confirmed quickly and accurately.

Figure 1 shows the results of evaluating the sensitivity of the present invention by performing real-time RT-PCR: (a) differential diagnosis of canine parvovirus (CPV) and canine coronavirus (CCoV); (b) differential diagnosis of feline parvovirus (FPV) and feline coronavirus (FCoV).
Figure 2 shows the results of a comparative evaluation of the differential diagnosis of (a) canine parvovirus (CPV) and (b) canine coronavirus (CCoV) according to the diagnostic method of the present invention and the conventional RT-PCR diagnostic method.
Figure 3 shows the results of a comparative evaluation of the differential diagnosis of (a) feline parvovirus (FPV) and (b) feline coronavirus (FCoV) according to the diagnostic method of the present invention and the conventional RT-PCR diagnostic method.
Figure 4 shows the results of evaluating the effectiveness of the CPV/FPV and CCoV/FCoV real-time simultaneous differential genetic diagnostic method of the present invention for canine parvovirus (CPV) test samples.
Figure 5 shows the results of evaluating the effectiveness of the CPV/FPV and CCoV/FCoV real-time simultaneous differential genetic diagnostic method of the present invention for canine coronavirus (CCoV) test samples.
Figures 6a and 6b show the results of evaluating the effectiveness of the CPV/FPV and CCoV/FCoV real-time simultaneous differential genetic diagnostic method of the present invention for feline parvovirus (FPV) test samples.
Figures 7a and 7b show the results of evaluating the effectiveness of the CPV/FPV and CCoV/FCoV real-time simultaneous differential genetic diagnostic method of the present invention for feline coronavirus (FCoV) test samples.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

Example

Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and (volume/volume) % for liquid/liquid, unless otherwise noted.

Example 1. CPV/FPV( Canine / Feline parvovirus ) and CCoV/FCoV( Canine / Feline coronavirus ) Preparation of primers and probes for detection

The present inventors performed multiple sequence alignment of the base sequences of canine / feline parvovirus (CPV) and canine / feline coronavirus (CCoV) registered in the National Center for Biotechnology Information (NCBI) (Table 1), and then selected the most conserved regions to produce specific primers and probes capable of simultaneously distinguishing CPV/FPV and CCoV/FCoV.

Accession number of the virus strain used in the analysis for primer and probe production Genbank Accession No. CPV/FPV AY742932, AY742933, MT010564, M38245, MF134808, MH106698, M19296, MG013488, AY742935, KY403998, MW653251, MH476582, MH476583, JN867613, JN867615, EF011664, KX774252, MK388674, KU508407, KX434459, KF638400, D26079, MH165481, MH165482, MG764510, MN862744, MN862745, MN862746, MF069445, MF069447, MN862749, MN127779, KP280068, KX434461, KX434462, MN400979, KP769859, MN908257, MN400980, MH559110, KT899746, KT899745, KC713592 CCoV/FCoV LC190907, GQ477367, JQ404409, MT906864, JQ404410, AB781790, MF095854, MF095850, MF095852, MF095853, MF095847, MF095849, MF095848, MF095851, FJ938051, AB535528, KP849472, EU186072, KX722530, KU215421, DQ286389, GQ152141, FJ917522, MG893511, AB781789, MW030108, MW030110, KR061459, AB907624, FJ938059, MT239440, FJ917531, FJ917530, FJ917535, FJ917534, FJ917533, FJ917532, FJ917521, FJ917525, FJ917526, FJ917529, FJ917528, KC461237, KX900407, KX900406, NC_038861, DQ811788

Primer and probe sequences that specifically bind to CPV/FPV and CCoV/FCoV division target gene designation Primer and probe sequences Sequence number CPV/FPV NS1 K060F GCAATCCTCAGAGTCAAGACC 1 K063R GAGTACTCCACGGTTCCAGT 2 K062P CY5-ACTCCGGACGTAGTGGACCTTGC-BHQ2 3 CCoV/FCoV 3'-UTR K022F GGTCATCGCGCTG Y CTACTC 4 K023R ATCTAAACTTCCTAATATGCAATAG 5 K024P FAM-AGCACGTGTAATAGGAGGTACAAGC-BHQ1 6

Example 2. CPV/FPV according to the present invention Canine/Feline parvovirus ) and CCoV/FCoV( Canine / Feline coronavirus ) Evaluation of differential diagnosis efficacy

To evaluate the differential diagnostic efficacy of canine/feline parvovirus (CPV/FPV) and canine/feline coronavirus (CCoV/FCoV) according to the present invention, one-step real-time RT-PCR, in which reverse transcription (RT) and PCR occur sequentially in the same tube, was performed on the virus strains shown in Table 1 above. The conditions for real-time RT-PCR are as shown in Table 3 below.

Real-time RT-PCR conditions Step temperature (℃) hour Cycle (repetition count) reverse transcription (RT) 50 30 minutes 1 initial denaturation 95 15 minutes 1 denaturation 95 10 seconds 40 annealing % extension 60 60 seconds

Example 2.1. Sensitivity Evaluation

The detection limit of the real-time RT-PCR was investigated against standard viruses for each target virus to evaluate the sensitivity of the simultaneous differential diagnosis method for canine/feline parvovirus (CPV/FPV) and canine/feline coronavirus (CCoV/FCoV) of the present invention.

First, the results for canine parvovirus (CPV) and canine coronavirus (CCoV) are shown in Table 4 below and Figure 1 (a), and it was confirmed that CPV and CCoV could be detected up to 1 copy/㎕ for all virus strains.

Confirmation of the sensitivity evaluation of the genetic diagnostic method of the present invention for CPV and CCoV No. Copies/ul Ct value Cy5(CPV)
(Th=50) FAM(CCoV)
(Th=150) 1 10^7 13.87 14.17 2 10^6 17.50 17.55 3 10^5 20.98 21.26 4 10^4 24.25 24.69 5 10^3 27.80 28.05 6 10^2 30.39 31.26 7 10^1 33.53 33.83 8 10^0 35.92 35.19 9 Negative control - -

Next, the results for feline parvovirus (FPV) and feline coronavirus (FCoV) are shown in Table 5 below and (b) of Figure 1, and it was confirmed that FPV and FCoV could be detected up to 1 copy/㎕ for all virus strains.

Confirmation of the sensitivity evaluation of the developed diagnostic method of the present invention for FPV and FCoV No. Copies/ul Ct value Cy5(FPV)
(Th=50) FAM (FCoV)
(Th=150) 1 10^7 13.17 14.22 2 10^6 18.50 17.03 3 10^5 20.98 21.2 4 10^4 24.05 24.16 5 10^3 27.19 28.27 6 10^2 30.24 30.26 7 10^1 34.53 33.78 8 10^0 35.72 35.19 9 Negative control - -

Example 2.2. Specificity Evaluation

In addition, the specificity of the real-time simultaneous differential genetic diagnostic method for canine/feline parvovirus (CPV/FPV) and canine/feline coronavirus (CCoV/FCoV) according to the present invention was evaluated.

Specifically, since the development diagnostic method of the present invention is performed by detecting pathogens in genes extracted from dog and cat tissues, it was intended to confirm whether non-specific reactions exist in general organ tissues and bacterial causative agents of dogs and cats.

As a result, it was confirmed that no non-specific reaction was shown in the organ tissues of dogs and cats (brain, heart, kidney, brain, liver, spleen, intestines, etc.), and as a result of confirming with 15 types of bacterial pathogens, it was confirmed that no non-specific reaction was shown in the bacterial pathogens as shown in Table 6 below.

Evaluation of the specificity of the genetic diagnostic method of the present invention in 15 bacterial pathogens No. bacterial agents Cy5
(CPV) FAM
(CCoV) Cy5
(FPV) FAM
(FCoV) positive control group 1 B. bronchiseptica - - - - + 2 K. pneumoniae - - - - + 3 E. coli - - - - + 4 M. canis - - - - + 5 M. cynos - - - - + 6 Staphylococcus spp. - - - - + 7 Streptococcus spp. - - - - + 8 C. coli - - - - + 9 C. jejuni - - - - + 10 Giardia spp. - - - - + 11 C. perfringens - - - - + 12 Clostridioides difficile - - - - + 13 Salmonella spp. - - - - + 14 Cryptosporidium sp . - - - - + 15 Cystoisospora spp. - - - - +

Example 2.3. Comparative evaluation with existing diagnostic methods using standard viruses.

Next, the inventors of the present invention performed a comparative evaluation of the CPV/FPV and CCoV/FCoV real-time simultaneous differential genetic diagnostic method according to the present invention and existing diagnostic methods using standard viruses of each virus to be detected.

To perform a comparative evaluation of the differential diagnosis of CPV/FPV and CCoV/FCoV, currently commercially available RT-PCR kit products were used as the existing diagnostic methods in the comparison group. Specifically, for CPV, the commercially available product Lilif CPV PCR Kit (Cat. No. IP11074, iNtRON) was used, and for CCoV, the commercially available product Lilif CCoV RT-PCR Kit (Cat. No. IP12071, iNtRON) was used. However, since there is currently no commercially available diagnostic kit for FPV, we used the method described in Xiao, Xiangyu et al. "Two Multiplex PCR Methods for Detecting Several Pathogens Associated with Feline Respiratory and Intestinal Tracts." Veterinary sciences vol. 10,1 14. (2022).(doi:10.3390/vetsci10010014) was used as a reference for the diagnostic method, and FCoV was tested using the commercially available Lilif FCoV RT-PCR Kit (cat. No. IP12176, iNtRON). Each CPV/FPV and CCoV/FCoV standard virus was serially diluted and applied to this example.

The results are shown in Figures 2 and 3.

First, as shown in (a) of the following Figure 2, it was confirmed that the diagnostic method of the present invention exhibits sensitivity that is about 100 times higher than that of the existing commercially available CPV diagnostic kit, and as shown in (b) of the following Figure 2, it was confirmed that the diagnostic method of the present invention exhibits sensitivity that is about 100 times higher than that of the existing commercially available CCoV diagnostic kit.

In addition, as shown in (a) of the following Figure 3, it was confirmed that the diagnostic method of the present invention exhibits a sensitivity that is about 1000 times higher than that of the existing known FPV diagnostic method, and as shown in (b) of the following Figure 3, it was confirmed that the diagnostic method of the present invention exhibits a sensitivity that is about 100 times higher than that of the existing commercially available FCoV diagnostic kit.

Example 2.4. Validation using field samples

The present inventors evaluated the effectiveness of the developed diagnostic method of the present invention on canine/feline parvovirus (CPV/FPV) and canine/feline coronavirus (CCoV/FcoV) test samples that were evaluated as positive or negative samples by existing diagnostic methods, i.e., field samples for which diagnosis was completed.

2.4.1. CPV test sample

First, the inventors evaluated the developed diagnostic method of the present invention on a total of 75 CPV test samples, and the results are shown in Table 7 and Figure 4 below.

As shown in Table 7 below, it was confirmed that all 25 of the positive samples showed positive reactions, as in the previous diagnosis results, and 44 of the 50 negative samples showed negative reactions, as in the previous results, but 6 samples showed positive reactions.

[Table 7]

Evaluation of CPV positive samples (+) and negative samples (-) according to the developed diagnostic method of the present invention

Thus, as shown in Fig. 4 below, it was confirmed that some negative sample groups (6 samples) as well as samples (25 samples) confirmed to be positive by the existing diagnostic method showed positive reactions, thereby confirming that the real-time simultaneous differential genetic diagnosis method for CPV/FPV and CCoV/FCoV according to the present invention showed excellent diagnostic sensitivity compared to the existing diagnostic method.

2.4.2. CCoV test samples

In addition, the inventors of the present invention evaluated the developed diagnostic method of the present invention on a total of 75 CCoV test samples, and the results are shown in Table 8 and Figure 5 below.

As shown in Table 8 below, it was confirmed that the 25 positive samples showed a positive reaction, the same as the previous diagnosis results, and 46 of the 50 negative samples showed a negative reaction, the same as the previous results, but it was confirmed that 4 samples showed a positive reaction.

[Table 8]

Evaluation of CCoV positive samples (+) and negative samples (-) according to the developed diagnostic method of the present invention

Thus, as shown in Figure 5 below, it was confirmed that some negative sample groups (4) as well as samples (25) confirmed to be positive by the existing diagnostic method showed positive reactions, thereby confirming that the real-time simultaneous differential genetic diagnosis method for CPV/FPV and CCoV/FCoV according to the present invention showed excellent diagnostic sensitivity compared to the existing diagnostic method.

2.4.3 FPV test sample

Next, the inventors evaluated the developed diagnostic method of the present invention on a total of 75 CPV test samples, and the results are shown in Table 9 and Figures 6a and 6b below.

As shown in Table 9 below, it was confirmed that the 25 positive samples showed a positive reaction, the same as the previous diagnosis results, and 41 of the 50 negative samples showed a negative reaction, the same as the previous results, but it was confirmed that 9 samples showed a positive reaction.

[Table 9]

Evaluation of FPV positive samples (+) and negative samples (-) according to the developed diagnostic method of the present invention

Thus, as shown in Figures 6a and 6b below, it was confirmed that some negative sample groups (9 samples) as well as samples (25 samples) confirmed to be positive by the existing diagnostic method showed positive reactions, thereby confirming that the real-time simultaneous differential genetic diagnosis method for CPV/FPV and CCoV/FCoV according to the present invention showed excellent diagnostic sensitivity compared to the existing diagnostic method.

2.4.4. FCoV test samples

In addition, the inventors of the present invention evaluated the developed diagnostic method of the present invention on a total of 75 FCoV test samples, and the results are shown in Table 10 below and Figures 7a and 7b.

As shown in Table 10 below, it was confirmed that all 25 of the positive samples showed positive reactions as in the previous diagnosis results, and 39 of the 50 negative samples showed negative reactions as in the previous results, but it was confirmed that 11 samples showed positive reactions.

[Table 10]

Evaluation of FCoV positive samples (+) and negative samples (-) according to the developed diagnostic method of the present invention

Thus, as shown in Figures 7a and 7b below, it was confirmed that some negative sample groups (15 samples) as well as samples (25 samples) confirmed to be positive by existing diagnostic methods showed positive reactions, thereby confirming that the real-time simultaneous differential genetic diagnostic method for CPV/FPV and CCoV/FCoV according to the present invention showed excellent diagnostic sensitivity compared to existing diagnostic methods.

Therefore, from the above results, it was confirmed that the novel canine/feline parvovirus (CPV/FPV) and canine/feline coronavirus (CCoV/FCoV) simultaneous differential genetic diagnostic method of the present invention has higher sensitivity and specificity than existing diagnostic methods.

Attach an electronic file of the sequence list

Claims (13)

A composition for simultaneous differential diagnosis of canine and feline parvovirus (CPV) and feline parvovirus (FPV) and canine and feline coronavirus (CCoV) and feline parvovirus (FPV), comprising (i) a first primer set consisting of nucleotide sequences of SEQ ID NOs : 1 and 2; and (ii) a second primer set consisting of nucleotide sequences of SEQ ID NOs : 4 and 5.
In claim 1, the composition further comprises a CPV and FPV differential diagnosis probe comprising a nucleotide sequence of SEQ ID NO: 3; a CCoV and FCoV differential diagnosis probe comprising a nucleotide sequence of SEQ ID NO: 6; or a combination thereof.
In the second paragraph, a composition for simultaneous differential diagnosis, wherein the 5'-end of the nucleotide sequence of sequence number 3 and sequence number 6 is labeled with one different fluorophore selected from the group consisting of FAM, TET, HEX, JOE, ROX, TMR, Cy5, TxR, and Cal Red 610, and the 3'-end is modified with a quencher of BHQ-1, BHQ-2, or BHQ-3.
A kit for simultaneous differential diagnosis of canine and feline parvovirus and canine and feline coronavirus, comprising a composition of any one of claims 1 to 3.
In the fourth paragraph, the kit is a simultaneous differential diagnosis kit performed by gene amplification.
A simultaneous differential diagnosis kit in claim 5, wherein the gene amplification is performed by PCR (polymerase chain reaction).
A simultaneous differential diagnosis kit in claim 5, wherein the gene amplification is performed by real-time PCR.
A method for simultaneous differential diagnosis of canine and feline parvovirus and canine and feline coronavirus, including the following steps:
(a) a step of preparing nucleic acid from a specimen sample collected from a dog or cat or the environment;
(b) a step of amplifying the target nucleotide sequence in the specimen sample using a composition for simultaneous differential diagnosis of canine and feline parvovirus (CPV and FPV) and canine and feline coronavirus (CCoV and FCoV), comprising (i) a first primer set consisting of nucleotide sequences of SEQ ID NOs: 1 and 2; and (ii) a second primer set consisting of nucleotide sequences of SEQ ID NOs: 4 and 5, using the nucleic acid extracted in the step (a) as a template; and
(c) A step of confirming the above amplification results.
In paragraph 8,
A simultaneous differential diagnosis method, wherein the composition comprising the primer sets of (i) and (ii) in the above step (b) additionally comprises a CPV and FPV differential diagnosis probe comprising a nucleotide sequence of SEQ ID NO: 3, a CCoV and FCoV differential diagnosis probe comprising a nucleotide sequence of SEQ ID NO: 6, or a combination thereof.
A simultaneous differential diagnosis method in claim 8, wherein the amplification is performed by PCR (polymerase chain reaction).
A simultaneous differential diagnosis method in claim 8, wherein the amplification is performed by real-time PCR.
A simultaneous differential diagnosis method in claim 11, wherein the real-time PCR is performed using a TaqMan probe method.
A simultaneous differential diagnosis method in claim 12, wherein the probe is combined with a label, and the confirmation in step (C) is performed by detecting a signal generated from the probe.