Primer pair for detection of bovine viral diarrhea virus, probe for detection of bovine viral diarrhea virus type 1, probe for detection of bovine viral diarrhea virus type 2, detection kit for bovine viral diarrhea virus, and detection method for bovine viral diarrhea virus

Abstract

To provide a method for detecting bovine viral diarrhea virus, enabling quick examination of bovine viral diarrhea virus.SOLUTION: This detection method comprises reverse-transcribing RNA contained in a sample into cDNA via a reverse transcriptase without extracting the RNA from the sample, generating an amplification product by a polymerase chain reaction with the cDNA as a template by using a forward primer and a reverse primer, each having a specific sequence, and detecting bovine viral diarrhea virus type 1 and bovine viral diarrhea virus type 2 respectively via a type 1 detection probe and a type 2 detection probe, each having a specific sequence.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a primer pair for detecting bovine viral diarrhea virus, a probe for detecting bovine viral diarrhea virus type 1, a probe for detecting bovine viral diarrhea virus type 2, a bovine viral diarrhea virus detection kit, and a method for detecting bovine viral diarrhea virus. [Background technology]

[0002] Bovine viral diarrhea virus (BVD) (hereinafter referred to as "BVDV"), which significantly reduces the productivity of cattle, is spreading throughout Japan. BVD is also registered on the disease list established by the World Organization for Animal Health (WOAH). BVDV is highly contagious and can be transmitted vertically within the womb of a cow. Calves infected through vertical transmission become persistently infected (PI) cattle and shed large amounts of BVDV throughout their lives. PI cattle cannot be cured, and the disease suddenly becomes severe and can lead to death. The most important measure against BVD is to quickly identify, isolate, and cull PI cattle, which are the source of infection on farms.

[0003] PI cattle are often asymptomatic, making it difficult to suspect infection based on appearance alone. To prevent the spread of BVDV as quickly as possible, it is essential to actively promote testing. The internationally adopted BVDV testing method involves extracting RNA from cattle blood and performing quantitative reverse transcription polymerase chain reaction (PCR) (see Non-Patent Document 1). Non-Patent Document 2 also discloses that BVDV genotypes can be identified by quantitative reverse transcription PCR (RT-qPCR) using probes corresponding to each genotype. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] B. Hoffmann, et al., "A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses", 2006, Journal of Virological Methods, 136, 200-209 [Non-patent document 2] Viviana Mari, 8 others, "Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1,type 2 and HoBi-like pestivirus", 2016, Journal of Virological Methods, 229, 1-7 Summary of the Invention [Problem to be solved by the invention]

[0005] BVDV has an envelope with a diameter of 40 to 60 nm and contains positive-sense single-stranded RNA as the viral genome. Genotypes 1 to 3 of BVDV are known. The test method disclosed in Non-Patent Document 1 cannot distinguish genotypes and is known to have lower detection sensitivity for BVDV type 2 (BVDV2) than for BVDV type 1 (BVDV1). If it were possible to distinguish between BVDV1 and BVDV2, excluding at least type 3, which has not been reported in Japan, it would be possible to implement quarantine measures such as using a vaccine compatible with the epidemic strain to prevent the production of newly infected cattle.

[0006] The method disclosed in Non-Patent Document 2, like Non-Patent Document 1, requires RNA extraction from cattle samples and then performing reverse transcription PCR on a reference strain of BVDV to obtain standard RNA for absolute quantification. This makes the test complicated and costly, and it takes time for the test results to become clear. In particular, speed is required for BVDV testing of newborn calves and externally introduced cattle.

[0007] The present invention has been made in consideration of the above-mentioned circumstances, and aims to provide a primer pair for detecting bovine viral diarrhea virus, a probe for detecting bovine viral diarrhea virus type 1, a probe for detecting bovine viral diarrhea virus type 2, a bovine viral diarrhea virus detection kit, and a method for detecting bovine viral diarrhea virus, which can rapidly test for BVDV. [Means for solving the problem]

[0008] One-step PCR, in which RT-qPCR is performed directly on a sample without extracting RNA from the sample, is simpler and faster than two-step PCR, in which RNA is extracted from the sample and then RT-qPCR is performed. The inventors attempted one-step PCR using the primers and probes described in Non-Patent Document 2, but observed non-specific reactions and reduced probe reactivity. Therefore, the inventors conducted extensive research and discovered primer and probe base sequences and reaction conditions that do not cause non-specific reactions, thereby completing the present invention.

[0009] The primer pair for detecting bovine viral diarrhea virus according to the first aspect of the present invention comprises: a forward primer whose nucleotide sequence is shown in SEQ ID NO: 1; a reverse primer whose nucleotide sequence is shown in SEQ ID NO: 2; Equipped with.

[0010] A probe for detecting bovine viral diarrhea virus type 1 according to a second aspect of the present invention comprises: The base sequence is shown in SEQ ID NO:3.

[0011] A probe for detecting bovine viral diarrhea virus type 2 according to a third aspect of the present invention comprises: The base sequence is shown in SEQ ID NO:4.

[0012] A bovine viral diarrhea virus detection kit according to a fourth aspect of the present invention comprises: a primer pair for detecting bovine viral diarrhea virus according to the first aspect of the present invention; The probe for detecting bovine viral diarrhea virus type 1 according to the second aspect of the present invention; The probe for detecting bovine viral diarrhea virus type 2 according to the third aspect of the present invention; Equipped with.

[0013] A primer pair for detecting bovine viral diarrhea virus according to a fifth aspect of the present invention comprises: a forward primer whose nucleotide sequence is shown in SEQ ID NO: 5; a reverse primer whose nucleotide sequence is shown in SEQ ID NO: 6; Equipped with.

[0014] A probe for detecting bovine viral diarrhea virus type 1 according to a sixth aspect of the present invention comprises: The base sequence is shown in SEQ ID NO:7.

[0015] A bovine viral diarrhea virus detection kit according to a seventh aspect of the present invention comprises: A primer pair for detecting bovine viral diarrhea virus according to the fifth aspect of the present invention; The probe for detecting bovine viral diarrhea virus type 1 according to the sixth aspect of the present invention; The probe for detecting bovine viral diarrhea virus type 2 according to the third aspect of the present invention; Equipped with.

[0016] A method for detecting bovine viral diarrhea virus according to an eighth aspect of the present invention comprises: reverse transcribing RNA contained in a sample into cDNA using a reverse transcriptase without extracting it from the sample; generating an amplification product by polymerase chain reaction using the cDNA as a template and a forward primer whose nucleotide sequence is shown in SEQ ID NO: 1 and a reverse primer whose nucleotide sequence is shown in SEQ ID NO: 2; Bovine viral diarrhea virus type 1 and bovine viral diarrhea virus type 2 are detected using a probe for detecting bovine viral diarrhea virus type 1, whose base sequence is shown in SEQ ID NO: 3, and a probe for detecting bovine viral diarrhea virus type 2, whose base sequence is shown in SEQ ID NO: 4, respectively.

[0017] A method for detecting bovine viral diarrhea virus according to a ninth aspect of the present invention comprises: reverse transcribing RNA contained in a sample into cDNA using a reverse transcriptase without extracting it from the sample; generating an amplification product by polymerase chain reaction using the cDNA as a template and a forward primer whose nucleotide sequence is shown in SEQ ID NO: 5 and a reverse primer whose nucleotide sequence is shown in SEQ ID NO: 6; Bovine viral diarrhea virus type 1 and bovine viral diarrhea virus type 2 are detected using a probe for detecting bovine viral diarrhea virus type 1, whose base sequence is shown in SEQ ID NO: 7, and a probe for detecting bovine viral diarrhea virus type 2, whose base sequence is shown in SEQ ID NO: 4, respectively.

[0018] The sample is It is serum, This may also be the case. [Effects of the Invention]

[0019] According to the present invention, BVDV can be rapidly tested. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 shows an RT-qPCR amplification curve according to Example 1. [Figure 2] This figure shows the amplification curves of RT-qPCR using RNA (A) extracted from the serum of BVDV1-infected cattle, RNA (B) extracted from the serum of BVDV2-infected cattle, a mixed sample (C), and RNA (D) extracted from the serum of BVDV-uninfected cattle as templates in Example 2. [Figure 3] FIG. 1 shows the amplification curves of RT-qPCR using RNA extracted from the milk of BVDV-negative cows, BVDV1-11r, and BVDV2-25r as templates in Example 2. [Figure 4]This figure shows the amplification curves of RT-qPCR using the BVDV1-infected cell culture supernatant (A), the BVDV2-infected cell culture supernatant (B), a mixed sample of the BVDV1-infected cell culture supernatant and the BVDV2-infected cell culture supernatant (C) and serum from a BVDV-uninfected cow (D) as templates in Example 3. [Figure 5] FIG. 10 shows an RT-qPCR amplification curve according to Example 5. [Figure 6] FIG. 10 is a diagram showing the relationship between virus titer and Ct value in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0021] Embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "have," "include," or "contain" also include the meaning of "consisting of" or "consisting of."

[0022] (Embodiment 1: Primer pair for detecting BVDV) The primer pair for detecting BVDV according to this embodiment is, for example, a primer pair 10 for detecting BVDV. The primer pair 10 for detecting BVDV comprises a forward primer 11 whose nucleotide sequence is shown in SEQ ID NO: 1 and a reverse primer 12 whose nucleotide sequence is shown in SEQ ID NO: 2. The forward primer 11 is designed for the 5'-end untranslated region (5'UTR) of the BVDV genome. The reverse primer 12 is designed for the 5'UTR and nonstructural protein N of the BVDV genome. pro The PCR product obtained by PCR using the BVDV detection primer pair 10 and cDNA obtained by reverse transcription of the viral genome as a template is 80 bp.

[0023] The BVDV detection primer pair according to this embodiment may be BVDV detection primer pair 20. BVDV detection primer pair 20 comprises a forward primer 21 whose nucleotide sequence is set forth in SEQ ID NO: 5 and a reverse primer 22 whose nucleotide sequence is set forth in SEQ ID NO: 6. The nucleotide sequences of forward primer 21 and reverse primer 22 are each extended by two nucleotides from the nucleotide sequences of forward primer 11 and reverse primer 12 so that they can be used at a high annealing temperature to suppress non-specific reactions. The amplified product obtained by PCR using BVDV detection primer pair 20 and cDNA obtained by reverse transcription of the viral genome as a template is 82 bp.

[0024] The forward primer 11, reverse primer 12, forward primer 21 and reverse primer 22 can be chemically synthesized according to the base sequence by a known method, for example, using a commercially available automatic nucleic acid synthesizer.

[0025] As shown in the examples below, the primer pair for detecting BVDV according to this embodiment can amplify a region in the viral genome suitable for distinguishing between BVDV1 and BVDV2.

[0026] (Embodiment 2: Probes for detecting BVDV1 and BVDV2) The BVDV1 detection probe according to this embodiment is primarily used to detect an amplification product amplified with the BVDV detection primer pair according to the first embodiment. The BVDV1 detection probe has a nucleotide sequence complementary to at least a portion of the amplification product obtained from the BVDV1 viral genome and specifically hybridizes to at least a portion of the amplification product. The BVDV1 detection probe is, for example, BVDV1 detection probe 13, the nucleotide sequence of which is set forth in SEQ ID NO: 3. Alternatively, the BVDV1 detection probe may be, for example, BVDV1 detection probe 14, the nucleotide sequence of which is set forth in SEQ ID NO: 7. The nucleotide sequence of BVDV1 detection probe 14 is the same as that of BVDV1 detection probe 13, but with two bases deleted from the 5' end to further suppress non-specific hybridization.

[0027] The BVDV2 detection probe according to this embodiment is also used mainly to detect an amplification product amplified with the BVDV detection primer pair according to embodiment 1. The BVDV2 detection probe has a nucleotide sequence complementary to at least a portion of the amplification product obtained from the BVDV2 viral genome and specifically hybridizes to at least a portion of the amplification product. An example of the BVDV2 detection probe according to this embodiment is BVDV2 detection probe 15, the nucleotide sequence of which is set forth in SEQ ID NO: 4.

[0028] Preferably, the BVDV1 detection probe and the BVDV2 detection probe have a fluorescent dye and a quencher that suppresses the emission of the fluorescent dye. For example, the BVDV1 detection probe and the BVDV2 detection probe have a fluorescent dye at the 5' end and a quencher attached to the 3' end. Examples of the BVDV1 detection probe and the BVDV2 detection probe include hydrolysis probes, specifically TaqMan (trademark) probes.

[0029] When the hydrolysis probe hybridizes to DNA, the fluorescent dye and quencher are physically close to each other, resulting in fluorescence resonance energy transfer (FRET), which transfers the energy of the fluorescent dye to the quencher and suppresses fluorescence. As the extension reaction of the BVDV detection primer pair progresses, the hydrolysis probe is hydrolyzed by the 5'→3' exonuclease activity of the DNA polymerase, causing the fluorescent dye to dissociate from the quencher. As a result, FRET ceases to occur, and fluorescence is generated. Strong fluorescence is detected only when amplification is performed using DNA hybridized with the hydrolysis probe as a template. Therefore, strong fluorescence is not detected if nonspecific fragments are amplified by PCR or if primer dimers are formed.

[0030] If the hydrolysis probe does not completely hybridize to the base sequence to be detected, the intensity of the emitted fluorescence will decrease significantly. If one to several bases in the base sequence of the hydrolysis probe do not hybridize, the melting temperature (Tm) will decrease and the hydrolysis probe will be released from the base sequence to be detected. In this case, decomposition of the hydrolysis probe by 5'→3' exonuclease activity will not occur, and the fluorescent dye and quencher will remain attached to the 5' and 3' ends of the hydrolysis probe, respectively. Because the fluorescent dye will not separate from the quencher, the quencher will suppress the generation of fluorescence.

[0031] Examples of fluorescent dyes include 6-FAM, TET, HEX, JOE, Yakima Yellow, TAMRA, ATTO550, ATTO565, ATTO633, ATTO647, ROX, Texas Red-X, Cy3, and Cy5. A fluorescent dye having a detectable fluorescence wavelength can be selected depending on the PCR device used.

[0032] Examples of quenchers include TAMRA, BHQ™-1, BHQ™-2, BHQ™-3, Iowa Black™ RQ, Iowa Black™ FQ, Eclipse™, etc. Since each quencher can suppress different wavelengths of fluorescence, it is sufficient to select a quencher that can suppress the fluorescent dye to be used.

[0033] A minor groove binder (MGB) may be added to the quencher in the BVDV1 detection probe and the BVDV2 detection probe. The MGB fits into the minor groove of the double helix structure of DNA, further strengthening the double helix structure when the BVDV1 detection probe or the BVDV2 detection probe hybridizes. This allows a higher Tm to be obtained, enabling the annealing temperature to be set higher in PCR. As a result, the specificity of the BVDV1 detection probe and the BVDV2 detection probe can be further enhanced. The TaqMan (trademark) MGB probe is known as a probe with an MGB added.

[0034] Another example of a BVDV1 detection probe and a BVDV2 detection probe is a molecular beacon probe. A molecular beacon probe is a single-stranded oligonucleotide that has a base sequence complementary to a portion of the target base sequence and further has complementary base sequences on both sides. Similar to a hydrolysis probe, a molecular beacon probe has a fluorescent dye and a quencher at both ends. When a molecular beacon probe is not hybridized to the target base sequence, it forms a hairpin-like stem-loop structure in which the complementary base sequences at both ends of the molecular beacon probe form a stem structure and the base sequence complementary to the target base sequence forms a loop structure. In the stem-loop structure, the fluorescent dye and quencher at both ends are in close proximity, suppressing fluorescence.

[0035] Unlike the hydrolysis probes mentioned above, the suppression of fluorescence in molecular beacon probes is due to collisional quenching caused by overlapping electron orbitals between the fluorescent dye and quencher. Thermal denaturation of molecular beacon probes opens the stem structure, making them linear, and in this state, they hybridize to the target base sequence, increasing the physical distance between the fluorescent dye and quencher. This increase in physical distance between the fluorescent dye and quencher relieves the suppression of fluorescence by the quencher. The use of molecular beacon probes enables analysis with low background levels and high specificity.

[0036] The probes for detecting BVDV1 and BVDV2 can be chemically synthesized according to the base sequences by known methods, for example, using a commercially available automatic nucleic acid synthesizer.

[0037] As shown in the examples below, the BVDV1 detection probe and the BVDV2 detection probe according to this embodiment can detect and distinguish between BVDV1 and BVDV2 while minimizing non-specific reactions.

[0038] (Embodiment 3: BVDV detection kit and BVDV detection method) The BVDV detection kit according to this embodiment comprises a BVDV detection primer pair 10, a BVDV1 detection probe 13, and a BVDV2 detection probe 15. The BVDV detection kit according to this embodiment may also comprise a BVDV detection primer pair 20, a BVDV1 detection probe 14, and a BVDV2 detection probe 15.

[0039] The BVDV detection kit may include buffers necessary for PCR, particularly RT-qPCR, and reagents other than those mentioned above.

[0040] Here, the BVDV detection method according to this embodiment will be described using as an example a BVDV detection kit including a BVDV detection primer pair 20, a BVDV1 detection probe 14, and a BVDV2 detection probe 15. The BVDV detection method includes a reverse transcription step, a PCR step, and a detection step.

[0041] In the reverse transcription step, RNA contained in the sample is reverse transcribed into cDNA using reverse transcriptase without extraction from the sample. Samples are collected from any animal that can be infected with BVDV, such as cattle, buffalo, goats, sheep, pigs, and deer. Cattle are not particularly limited as long as they belong to the genus Bos, but examples include domestic cattle (Bos taurus) and Zebu cattle (Bos indicus). Samples include, for example, blood, serum, saliva, tissue, etc., and preferably serum. Tissues include respiratory tissue, etc., and may be tissue from an aborted fetus.

[0042] The reverse transcriptase is not particularly limited, and commercially available reverse transcriptases may be used. Preferably, the reverse transcriptase is a reverse transcriptase used in RT-qPCR. The reverse transcription reaction solution contains known reagents such as a buffer solution, dNTPs, DTT, an RNase inhibitor, and RNase-free water in addition to the template RNA and the reverse transcriptase. The conditions for the reverse transcription reaction are appropriately set depending on the reverse transcriptase used. The temperature for the reverse transcription reaction is, for example, 45 to 58°C, 48 to 55°C, or 50 to 52°C. The reaction time for the reverse transcription reaction is, for example, 3 to 20 minutes, 4 to 18 minutes, or 5 to 16 minutes.

[0043] In the PCR step, an amplification product is generated by PCR using a forward primer 21 and a reverse primer 22 with cDNA as a template. In PCR, a deoxynucleotide triphosphate complementary to the template cDNA is added to the 3' end of the forward primer 21 and the reverse primer 22 by DNA polymerase, and the forward primer 21 and the reverse primer 22 are extended in the direction from the 5' end to the 3' end. In PCR, the double-stranded template DNA is unwound (denatured) into single strands by changing the reaction temperature, the primers are annealed to the DNA, an extension reaction is carried out by DNA polymerase, and the extended primers and DNA are unwound. By repeating this process, the amplification product can be amplified.

[0044] The DNA polymerase is not particularly limited, and commercially available DNA polymerases may be used. The PCR reaction solution contains known reagents such as a buffer solution, dNTPs, and the like, in addition to the cDNA template and DNA polymerase. PCR conditions are appropriately set depending on the DNA polymerase used. The initial denaturation and denaturation temperatures are, for example, 93 to 98°C, 94 to 97°C, or 95°C. The initial denaturation time is, for example, 10 seconds to 4 minutes, 10 seconds to 3 minutes, 10 seconds, or 3 minutes. The denaturation time is, for example, 10 to 20 seconds, 12 to 18 seconds, 13 to 16 seconds, or 15 seconds. The annealing temperature is, for example, 60 to 65°C, 61 to 63°C, 61°C, or 62°C. The annealing time is, for example, 18 to 35 seconds, 19 to 33 seconds, 20 to 30 seconds, 20 seconds, or 30 seconds. The number of cycles of denaturation and annealing is, for example, 33 to 45, 34 to 42, 35 to 40, 35, or 40.

[0045] In the case of PCR using forward primer 21 and reverse primer 22, the preferred cycles are initial denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 62°C for 20 seconds, and 40 cycles. In the case of PCR using forward primer 11 and reverse primer 12, the preferred cycles are initial denaturation at 95°C for 10 seconds, denaturation at 95°C for 15 seconds, annealing at 61°C for 30 seconds, and 35 cycles.

[0046] In the detection step, BVDV1 is detected using the BVDV1 detection probe 14, and BVDV2 is detected using the BVDV2 detection probe 15. The detection step is performed under conditions under which the BVDV1 detection probe 14 and the BVDV2 detection probe 15 hybridize to amplification products derived from the BVDV1 viral genome and amplification products derived from the BVDV2 viral genome, respectively. The hybridization conditions are, for example, stringent conditions under which the BVDV1 detection probe and the BVDV2 detection probe hybridize to amplification products with complementary nucleotide sequences but do not hybridize to amplification products with non-complementary nucleotide sequences. Stringent conditions can be determined appropriately based on, for example, Molecular Cloning: A Laboratory Manual, Third Edition (2001), and are, for example, 0.2×SSC, 0.1% SDS, and incubation at 65°C.

[0047] When hydrolysis probes are used as the BVDV1 detection probe 14 and the BVDV2 detection probe 15, the detection step detects the emission of a fluorescent dye whose inhibition by a quencher has been released upon hybridization of the BVDV1 detection probe 14 to an amplification product derived from the BVDV1 viral genome, and the detection step detects the emission of a fluorescent dye whose inhibition by a quencher has been released upon hybridization of the BVDV2 detection probe 15 to an amplification product derived from the BVDV2 viral genome. BVDV1 and BVDV2 can be detected by using fluorescent dyes with different fluorescence wavelengths for the BVDV1 detection probe 14 and the BVDV2 detection probe 15. The emission can be detected by measuring fluorescence intensity, etc., using a commercially available device.

[0048] The reverse transcription step, PCR step, and detection step may be performed in a single reaction solution. In this embodiment, the BVDV detection method has been described using as an example a BVDV detection kit comprising BVDV detection primer pair 20, BVDV1 detection probe 14, and BVDV2 detection probe 15. However, the above BVDV detection method can also be applied to a BVDV detection kit comprising BVDV detection primer pair 10, BVDV1 detection probe 13, and BVDV2 detection probe 15.

[0049] In the reverse transcription step of the BVDV detection method according to this embodiment, RNA contained in a sample is reverse transcribed into cDNA using reverse transcriptase without extraction from the sample. As shown in Examples 1 and 7 below, by using the BVDV detection primer pair, BVDV1 detection probe, and BVDV2 detection probe described above, BVDV can be specifically and accurately detected by directly performing the BVDV detection method on serum as a sample, even if RNA is not extracted from serum in advance. Therefore, the presence or absence of BVDV infection and the BVDV genotype can be rapidly tested. Performing the reverse transcription step, PCR step, and detection step in a single reaction solution, i.e., direct one-step RT-qPCR, enables simple and rapid on-site testing.

[0050] Furthermore, by using the BVDV detection primer pair 20, the BVDV1 detection probe 14, and the BVDV2 detection probe 15, mutant strains can also be specifically detected, as shown in Example 2 below.

[0051] Furthermore, the BVDV detection method according to this embodiment allows for the discrimination of genotypes, making it possible to identify prevalent strains and select vaccines that take into consideration cross-reactivity.

[0052] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples. [Example]

[0053] Example 1: Study of one-step PCR Sera isolated from blood collected from a BVDV1-infected cow (#11) and a BVDV2-infected cow (#25) by standard methods were designated samples BVDV1-11s and BVDV2-25s, respectively. RNA was extracted from each serum sample using a MagMAX CORE Nucleic Acid Purification Kit (Thermo Fisher Scientific, #A32700) and an automated nucleic acid extractor, the KingFisher Duo Prime (Thermo Fisher Scientific). The extracted RNA samples were designated samples BVDV1-11r and BVDV2-25r, respectively. Furthermore, a mixed sample of equal amounts of BVDV1-11s and BVDV2-25s was designated sample 11-25s (mix), and a mixed sample of equal amounts of BVDV1-11r and BVDV2-25r was designated sample 11-25r (mix).

[0054] The RT-qPCR enzyme used was One Step PrimeScript III RT-qPCR Mix (Takara Bio, #RR600A). The nucleotide sequences of the forward primer BVDV_317-335F (50 μM) and the reverse primer BVDV_376-396R (50 μM) are shown in SEQ ID NOs: 1 and 2, respectively. The nucleotide sequences of the probe BVDV1_357-373P (10 μM) designed for the BVDV1-derived amplification product and the probe BVDV2_336-353P (10 μM) designed for the BVDV2-derived amplification product are shown in SEQ ID NOs: 3 and 4, respectively. The 5' end of BVDV1_357-373P is labeled with FAM and the 3' end has an MGB-eclipse. The 5' end of BVDV2_336-353P is labeled with HEX and the 3' end has an MGB-eclipse. ROX Reference (Invitrogen, 12223012) was used as an internal standard, and Nuclease-Free Water (Invitrogen, AM9930) was used as water.

[0055] Using the above sample as a template, a reaction solution with the composition shown in Table 1 below was prepared, and RT-qPCR was performed using a real-time PCR instrument, Quant Studio 3 (Applied Biosystems). Instead of a template, both PI bovine serum and RNA were used as positive controls, and water was added as a negative control.

[0056] [Table 1]

[0057] The reaction conditions for RT-qPCR in Example 1 are shown in Table 2. The cutoff value was determined manually or by automatic analysis within a fluorescence intensity (ΔRn) range of 0.1 to 0.2. Samples whose ΔRn exceeded the cutoff value within 35 cycles were considered PCR-positive.

[0058] [Table 2]

[0059] (result) The RT-qPCR amplification curves are shown in Figure 1. No nonspecific reactions were observed, and amplification was confirmed in serum, extracted RNA, and the mixed sample.

[0060] (Example 2: Examination of application to mutant strains) Genetic analysis of BVDV isolates in Miyazaki Prefecture revealed that approximately half of the isolates had mutations in the target region of BVDV1_357-373P used to detect BVDV1 in Example 1. Therefore, the nucleotide sequence of the probe used to detect BVDV1 was modified to correspond to the mutation. Furthermore, the nucleotide sequence of the primer was extended to withstand high annealing, and the nucleotide sequence of the probe was shortened to suppress nonspecific hybridization of the probe. The nucleotide sequences of the forward primer BVDV_315-335F (50 μM) and the reverse primer BVDV_374-396R (50 μM) are shown in SEQ ID NOs: 5 and 6, respectively. The nucleotide sequence of the probe BVDV1_359-373P (10 μM) designed for the BVDV1-derived amplification product is shown in SEQ ID NO: 7. The same probe BVDV2_336-353P (10 μM) as in Example 1 was used for the BVDV2-derived amplification product. The 5' end of BVDV1_359-373P is labeled with FAM and the 3' end has an MGB-Eclipse.

[0061] The enzyme used for RT-qPCR was PrimeTime™ One-Step 4× Broad-Range Master Mix (Integrated DNA Technologies, #10011744). The internal standard and water were the same as in Example 1.

[0062] For the samples, serum was separated by standard methods from blood collected from BVDV1-infected cattle, BVDV2-infected cattle, and BVDV-uninfected cattle, and RNA was extracted from each serum in the same manner as in Example 1. In addition, a mixed sample was prepared by mixing equal amounts of RNA extracted from BVDV1-infected cattle and BVDV2-infected cattle.

[0063] Using the above samples as templates, reaction solutions with the compositions shown in Table 3 below were prepared, and RT-qPCR was performed using Quant Studio 3. Instead of templates, PI cattle RNA and water were added to the positive and negative controls, respectively.

[0064] [Table 3]

[0065] The reaction conditions for RT-qPCR according to Example 2 are shown in Table 4. The cutoff value was determined manually or by automatic analysis within a fluorescence intensity (ΔRn) range of 0.1 to 0.2. Samples whose ΔRn exceeded the cutoff value within 40 cycles were considered PCR-positive.

[0066] [Table 4]

[0067] (result) Figures 2A, 2B, 2C, and 2D show the RT-qPCR amplification curves using RNA extracted from the serum of BVDV1-infected cattle, RNA extracted from the serum of BVDV2-infected cattle, and RNA extracted from the mixed sample and the serum of BVDV-uninfected cattle, respectively. No nonspecific reactions were observed, and the genotypes were successfully identified.

[0068] The RT-qPCR amplification curves using RNA (24 species) extracted from the milk of BVDV-negative cows, BVDV1-11r, and BVDV2-25r as templates are shown in Figure 3. BVDV1 and BVDV2 were detected and differentiated, while the negative control and RNA extracted from milk were negative.

[0069] Example 3: Detection of BVDV from BVDV-infected cell culture supernatant We investigated the feasibility of detecting BVDV and distinguishing its genotype from the culture supernatant of MDBK-HS cells infected with BVDV. The culture medium was a mixture of 500 mL of Eagle's minimum essential medium (L-Glu-free) (Merck, #M2279-500 mL), 5 mL of penicillin-streptomycin (Merck, #P4333-100 mL), 5 mL of 200 mM L-glutamine (Thermo Fisher Scientific, #25030081), 25 mL of heat-inactivated horse serum (Merck, #H1138-100 mL), HEPES solution (Merck, #H3537-100 mL), and 1 mL of TPB solution. The TPB solution was prepared by dissolving 45 g of tryptose phosphate broth (Merck, #T8782-500G) in 30 mL of Milli Q, followed by filter sterilization.

[0070] The cells were passaged according to the following procedure. 1. Aspirate all of the culture supernatant and wash the cell sheet once with sterilized 1x phosphate-buffered saline (PBS). 2. Aspirate the 1x PBS, add 2-4 mL of Trypsin / EDTA Solution (Merck, #R-001-100), mix well, and incubate at 37°C for 10-20 minutes. 3. Once the cells are digested, add 10 mL of culture medium and pipette thoroughly to disperse the cell clumps. 4. Collect in a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. 5. Aspirate the supernatant and resuspend in 10 mL of culture medium. 6. Mix 13 mL of culture medium and 2 mL of the above resuspended material in a new flask. 7. Gently tilt the flask and incubate at 37°C for 2-3 days. 8. Once a full sheet is formed, repeat the passage from step 1 to maintain the MDBK-HS cells.

[0071] 2 μL to 50 μL of serum from a BVDV-uninfected cow or BVDV fluid was inoculated into a 6-well plate. After inoculation, 2 mL of the above resuspension diluted 5-fold with culture medium was inoculated. The plate was gently tilted and incubated at 37°C for 3 to 7 days. The cell supernatant was collected and stored at -80°C. This supernatant was used as the virus-infected cell culture supernatant.

[0072] RT-qPCR was carried out as in Example 2 above, using the virus-infected cell culture supernatant as a template.

[0073] (result) Figures 4A, 4B, 4C, and 4D show the amplification curves of RT-qPCR using BVDV1-infected cell culture supernatant, BVDV2-infected cell culture supernatant, a mixture of BVDV1-infected cell culture supernatant and BVDV2-infected cell culture supernatant, and serum from a BVDV-uninfected cow as templates, respectively. BVDV was detected directly from the cell culture supernatant and its genotype was identified.

[0074] Example 4: BVDV gene lineage analysis RNA was extracted from the serum of a BVDV-PI cattle in the same manner as in Example 1. The 5'UTR gene was amplified by one-step conventional PCR. The nucleotide sequences of the forward primer 324T (10 μM) and reverse primer 326 (10 μM) used in one-step conventional PCR are shown in SEQ ID NOs: 8 and 9, respectively.

[0075] The enzyme used for one-step conventional PCR was the OneStep RT-PCR Kit (QIAGEN, #210212). Using the extracted RNA as a template, a reaction solution with the composition shown in Table 5 below was prepared, and PCR was performed using a MiniAmp (Thermo Fisher Scientific). The reaction conditions for one-step conventional PCR are shown in Table 6.

[0076] [Table 5]

[0077] [Table 6]

[0078] The reaction mixture was electrophoresed on a 2% agarose gel. After electrophoresis, the band containing the PCR product (approximately 280 bp) was excised from the gel using an LED Transilluminator Gel Mier (Fujifilm Wako Pure Chemical Industries, Ltd.) and collected in a 2 mL Eppendorf tube. The PCR product was purified using a QIAquick Gel Extraction Kit (QIAGEN, #28704).

[0079] Dye terminator reactions were performed using the SupreDye v3.1 Cycle Sequencing Kit (EdgeBioSystems) according to the manufacturer's instructions. The forward and reverse primers for the dye terminator reactions were 324T and 326, respectively. Base calling of the dye terminator reaction products was performed using a SeqStudio Genetic Analyzer (Thermo Fisher Scientific) or an Applied Biosystems 3130 Genetic Analyzer (Thermo Fisher Scientific). The decoded data was used to confirm the base sequence using GENETYX Ver. 15 software (GENETYX).

[0080] The confirmed nucleotide sequences were combined with the reference strains of each BVDV genetic subtype listed below to create a genetic phylogenetic tree using MEGA-X Software (Kumar, S., et al., "MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Molecular biology and evolution", 2018, 35(6), 1547-1549), and the genetic subtypes were identified by confirming the clusters of the isolated strains and the reference strains. BVDV1a subtype: Nose strain (Accession No. AB019670) BVDV1b subtype: IS27CP / 01 strain (Accession No. AB359924) BVDV2a subtype: KZ-91-CP strain (Accession No. LC006970) BVDV2b subtype: Hokudai-Lab / 09 strain (Accession No. AB567658)

[0081] The sera for which the genetic subtype of the infecting BVDV was determined were subjected to RT-qPCR as described in Example 2 above.

[0082] (result) The genotypes determined by gene phylogenetic tree analysis and the genotypes detected by RT-qPCR are shown in Table 7. The genotypes detected by RT-qPCR were consistent with the genotypes determined by gene phylogenetic tree analysis.

[0083] [Table 7]

[0084] Example 5: Specificity Test RT-qPCR was performed as described in Example 2 above using RNA extracted from the serum of BVDV-PI cattle and other pathogens such as bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus 3, Akabane virus, bovine herpesvirus, bovine immunodeficiency virus, or bovine infectious lymphoma virus (BLV) as templates.

[0085] For bovine respiratory syncytial virus, bovine parainfluenza virus 3, Akabane virus, bovine herpesvirus, and BLV, RNA was extracted from TSV3 (Zoetis), a vaccine containing the BRSV / 375 strain; TSV3, a vaccine containing the RLB103 strain; Akabane disease live vaccine (Nisseiken), a vaccine containing the TS-C2 strain; and TSV3, a vaccine containing the RLB106 strain, using MagDEA™ Dx SV RNA reagent (Precision System Science) and a nucleic acid extraction system magLEAD 12gc (Precision System Science).

[0086] DNA was extracted from the whole blood of BLV-infected cattle using MagDEA (trademark) Dx SV RNA reagent (Precision System Science) with a nucleic acid extraction system magLEAD 12gc (Precision System Science).

[0087] For bovine coronavirus, RNA was extracted from the culture supernatant of the CS5 strain using the MagMAX CORE Nucleic Acid Purification Kit and Kingfisher Duo Prime. For bovine immunodeficiency virus, a plasmid encoding the full-length nucleotide sequence of the virus was used.

[0088] (result) 5 shows the RT-qPCR amplification curves according to Example 5. BVDV1 and BVDV2 were positive, and pathogens other than BVDV were negative.

[0089] (Example 6: Examination of detection limit) RNA was extracted from serum of PI cattle infected with the M5-3 / G1B / Cattle / Japan / 2018 strain (accession number: OR674901) for BVDV1 and the M2-2 / G2A / Cattle / Japan / 2018 strain (accession number: OR674907) using the MagMAX CORE Nucleic Acid Purification Kit and Kingfisher Duo Prime. The extracted RNA was serially diluted 10-fold using nuclease-free water. RT-qPCR was performed as described in Example 2 above using the resulting dilution series as a template. For comparison, the dilution series was also subjected to the Hoffmann method disclosed in Non-Patent Document 1.

[0090] The nucleotide sequence of the forward primer BVDV 190F (20 μM) used in the Hoffmann method is shown in SEQ ID NO: 10. The reverse primer used was 326 (20 μM) from Example 4 above. The nucleotide sequence of the probe TQ-pesti (10 μM) is shown in SEQ ID NO: 11. The 5' end of TQ-pesti is labeled with FAM, and the 3' end has TAMRA.

[0091] A reaction solution with the composition shown in Table 8 below was prepared, and RT-qPCR was performed using Quant Studio 3. The reaction conditions for PCR using the Hoffmann method are shown in Table 9.

[0092] [Table 8]

[0093] [Table 9]

[0094] (result) Table 10 shows the number of positive detections in triplicate (n=3) for each dilution. The RT-qPCR in Example 2 had the same detection sensitivity as the Hoffmann method for BVDV1. On the other hand, the RT-qPCR in Example 2 had a higher detection sensitivity than the Hoffmann method for BVDV2.

[0095] [Table 10]

[0096] (Example 7: Investigation of the detection limit of direct virus detection from serum) 1. Quantification of viral titer in PI bovine serum Serum from a PI cow infected with the M6-1 / G1B / Cattle / Japan / 2018 strain (accession number: OR674904) was used as a BVDV1 specimen. Serum from a PI cow infected with the M2-1 / G2A / Cattle / Japan / 2016 strain (accession number: OR674894) was used as a BVDV2 specimen. MDBK-HS cells were cultured as in Example 3, and the above-mentioned diluted suspension of MDBK-HS cells was prepared. The serum from the PI cow was serially diluted 10-fold with the above-mentioned culture medium. 50 μL of the diluted serum was inoculated into a 96-well plate for each dilution step (n=4). 100 μL of the diluted suspension was simultaneously inoculated into each well and incubated for 3 to 7 days.

[0097] BVDV-infected cells were stained by immunostaining, and wells containing even one BVDV-infected cell were scored. For immunostaining, the supernatant from each well was decanted, and the plate was air-dried overnight. The primary antibody (JCU / BVD / CF10 (TropBio)) was then diluted 1:1000 in antibody diluent (1% BSA PBST). The antibody diluent (1% BSA PBST) contained 500 mL of 1x PBS, 250 μL of Tween 20 (Merck, #P7949-100ML), and 5.25 g of bovine serum albumin (Merck, #A7030). 100 μL of diluted primary antibody was added to each well. The plate was incubated for 1 hour and washed three times with 1x PBS.

[0098] The secondary antibody (Goat Anti-Mouse IgG H&L (Abcam, #ab6708) was diluted 1:1000 in antibody diluent. 100 μL of the diluted secondary antibody was added to each well. The plate was incubated for 1 hour and washed three times with 1×PBS. 100 μL of colorimetric substrate solution was added to each well. The colorimetric substrate solution contained 10 mL of 50 mM acetate buffer, 500 μL of AEC solution, and 25 μL of 30% H2O. The acetate buffer was prepared by mixing 3.4 g of sodium acetate trihydrate with 500 mL of H2O, adjusting the pH to 5.0 with acetic acid, and autoclaving. The AEC solution was prepared by mixing three tablets of 3-amino-9-ethyl-carbazole (Sigma, Cat. No. A-6926) and 7.5 mL of N,N-dimethylformamide. The mixture was mixed with 1 mL of PBS, protected from light, and stored at 4°C.

[0099] Each well was washed once with tap water, and the TCID was determined using the Reed-Muench method (L.J. Reed and 1 other researchers, American Journal of Epidemiology, 1938, Volume 27, Issue 3, pp. 493-497). 50 / 50μL was calculated

[0100] 2. Evaluation of the detection limit of virus isolation and propagation culture Based on the determined virus titer, the PI bovine serum was diluted with cell culture medium and diluted to 10 3 , 10 2 , 10 1 and 10 0 TCID 50 The serum with adjusted viral titer was inoculated onto MDBK-HS cells (n=3) for each viral titer, and immunostaining was performed. Wells containing even one BVDV-infected cell were considered positive.

[0101] 3. Evaluation of the detection limit of RT-qPCR from PI bovine serum Based on the virus titer determined as described above, the PI bovine serum was diluted with the serum of a BVDV-uninfected bovine that was confirmed negative by the Hoffmann method, and 10 3, 10 2 , 10 1 , 10 0 The serum with adjusted virus titer was subjected to RT-qPCR in Example 2 above (n=3) for each virus titer.

[0102] (result) Table 11 shows each TCID 50 The figure shows the number of times that a positive result was detected in three determinations per 1 / mL sample. The RT-qPCR in Example 2 had the same detection sensitivity for BVDV2 as that of virus isolation.

[0103] [Table 11]

[0104] Example 8: Correlation analysis between virus titer and RT-qPCR Ct value Serum from a PI cow infected with the M6-1 / G1B / Cattle / Japan / 2018 strain (accession number: OR674904) was used as a BVDV1 sample, and serum from a PI cow infected with the M2-1 / G2A / Cattle / Japan / 2016 strain (accession number: OR674894) was used as a BVDV2 sample.

[0105] The virus in the sample was amplified through virus isolation and propagation culture in the same manner as in Example 3. The virus titer of the recovered virus-infected cultured cell supernatant was quantified in the same manner as in Example 7. Based on the virus titer, the virus-infected cultured cell supernatant was diluted with serum from a BVDV-uninfected cow that had been confirmed negative by the Hoffmann method, and 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0The virus titer was adjusted to TCID50 / mL. The virus-infected culture cell supernatants with adjusted virus titers were detected by RT-qPCR as described in Example 2 for each virus titer (n=3), and the Ct values ​​were recorded. The correlation between the virus titer and the Ct value was analyzed using the statistical software R.

[0106] (result) As shown in Figure 6, a correlation was observed between the virus titer and the Ct value. This demonstrated that the virus titer of infected cattle can be estimated from the Ct value of RT-qPCR in Example 2.

[0107] The above-described embodiments are intended to explain the present invention and are not intended to limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not by the embodiments. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention. [Industrial Applicability]

[0108] The present invention is suitable for detecting BVDV. [Explanation of symbols]

[0109] 10,20 Primer pair, 11,21 Forward primer, 12,22 Reverse primer, 13,14 BVDV1 detection probe, 15 BVDV2 detection probe

Claims

1. a forward primer whose base sequence is shown in SEQ ID NO: 1; a reverse primer whose base sequence is shown in SEQ ID NO: 2; A primer pair for detecting bovine viral diarrhea virus, comprising:

2. The base sequence is shown in SEQ ID NO:

3. Probe for detecting bovine viral diarrhea virus type 1.

3. The base sequence is shown in SEQ ID NO:

4. Probe for detecting bovine viral diarrhea virus type 2.

4. The primer pair for detecting bovine viral diarrhea virus according to claim 1; The probe for detecting bovine viral diarrhea virus type 1 according to claim 2; The probe for detecting bovine viral diarrhea virus type 2 according to claim 3; A bovine viral diarrhea virus detection kit comprising:

5. a forward primer whose base sequence is shown in SEQ ID NO: 5; A reverse primer whose base sequence is shown in SEQ ID NO: 6; A primer pair for detecting bovine viral diarrhea virus, comprising:

6. The base sequence is shown in SEQ ID NO:

7. Probe for detecting bovine viral diarrhea virus type 1.

7. The primer pair for detecting bovine viral diarrhea virus according to claim 5; The probe for detecting bovine viral diarrhea virus type 1 according to claim 6; The probe for detecting bovine viral diarrhea virus type 2 according to claim 3; A bovine viral diarrhea virus detection kit comprising:

8. reverse transcribing RNA contained in a sample into cDNA using a reverse transcriptase without extracting it from the sample; generating an amplification product by polymerase chain reaction using the cDNA as a template and a forward primer whose nucleotide sequence is shown in SEQ ID NO: 1 and a reverse primer whose nucleotide sequence is shown in SEQ ID NO: 2; The bovine viral diarrhea virus type 1 and the bovine viral diarrhea virus type 2 are detected by a probe for detecting bovine viral diarrhea virus type 1 whose base sequence is shown in SEQ ID NO: 3 and a probe for detecting bovine viral diarrhea virus type 2 whose base sequence is shown in SEQ ID NO: 4, respectively. Methods for detecting bovine viral diarrhea virus.

9. reverse transcribing RNA contained in a sample into cDNA using a reverse transcriptase without extracting it from the sample; generating an amplification product by polymerase chain reaction using the cDNA as a template and a forward primer whose nucleotide sequence is shown in SEQ ID NO:5 and a reverse primer whose nucleotide sequence is shown in SEQ ID NO:6; The bovine viral diarrhea virus type 1 and the bovine viral diarrhea virus type 2 are detected by a probe for detecting bovine viral diarrhea virus type 1 whose base sequence is shown in SEQ ID NO: 7 and a probe for detecting bovine viral diarrhea virus type 2 whose base sequence is shown in SEQ ID NO: 4, respectively. Methods for detecting bovine viral diarrhea virus.

10. The sample is It is serum, The method for detecting a bovine viral diarrhea virus according to claim 8 or 9.

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