Method for detecting resistance alleles to bovine lymphoma and kit for detecting resistance alleles to bovine lymphoma

Abstract

The allele detection method comprises a reaction step in which a polymerase chain reaction is carried out using a reaction solution comprising: a nucleic acid containing an allele base sequence that forms a template; primers for base sequence amplification by a polymerase chain reaction; DNA polymerase that performs primer elongation when the 1 base at the 3'-terminal of primer hybridized to the base sequence is complementary to the base at the position in the base sequence corresponding to the position of the 1 base; a probe that hybridizes to at least a portion of the base sequence that is amplified by the DNA polymerase; and a labeling substance that indicates that the probe has hybridized to the base sequence.

Description

Technical Field

[0001] The present invention relates to Resistance to bovine lymphoma a method for detecting an allele and Resistance to bovine lymphoma allele detection for to a kit.

Background Art

[0002] Genes that occupy the same locus and have different genetic information are each called alleles. Diploid organisms have two alleles derived from each parent, and these alleles are greatly involved in diverse traits of organisms. In particular, for genes included in the major histocompatibility complex, some genes are known to have tens to thousands of alleles, and their association with various diseases including infectious diseases has been reported.

[0003] Livestock infectious diseases such as African swine fever, foot-and-mouth disease and avian influenza not only cause enormous economic losses to the livestock industry, but also represent a very important issue in terms of ensuring a stable food supply for humanity. Among these, the number of cases of chronic intractable infectious diseases such as enzootic bovine leukosis (EBL) and Johne's disease is increasing year by year, which has become an international problem. Animals affected with these infectious diseases remain lifelong infected with the pathogen, and there are no effective treatments or vaccines. Therefore, an epidemic prevention strategy that prevents exposure of livestock to pathogens is important.

[0004] As a measure to prevent pathogen transmission within farms, animals that exhibit resistance to pathogens have attracted attention. These animals have characteristics such as, for example, not transmitting the virus even if they are infected with the virus, or the disease does not progress.

[0005] In livestock, the genotype of the major histocompatibility complex (BoLA)-DRB3 gene is strongly associated with susceptibility or resistance to pathogens. This gene is highly polymorphic, and currently, 357 different alleles are registered in the database for cattle. Non-patent document 1 identifies cattle possessing the allele BoLA-DRB3*009:02 as being resistant to bovine lymphoma virus (BLV) infection.

[0006] Non-patent document 2 describes that BLV-resistant cattle do not spread the virus. Therefore, in disease control strategies to prevent BLV infection, it is crucial to identify cattle that possess the allele BoLA-DRB3*009:02.

[0007] For allele identification, sequencing, PCR-RFLP (restriction fragment length polymorphism) methods, and real-time PCR using SYBR Green have been employed. Sequencing is a method for deciphering the base sequence of alleles. While highly accurate, it is costly and time-consuming, making it unsuitable for testing large volumes of samples.

[0008] PCR-RFLP is a method for identifying alleles by treating allele fragments amplified by polymerase chain reaction (PCR) with restriction enzymes and then examining the band patterns by electrophoresis. While PCR-RFLP is inexpensive and simple, interpreting the results is difficult. Furthermore, it cannot distinguish alleles when multiple alleles exhibit the same cleavage pattern. BoLA-DRB3*009:01 and BoLA-DRB3*009:03 are known alleles that exhibit a cleavage pattern similar to BoLA-DRB3*009:02.

[0009] The method using real-time PCR with SYBR Green, disclosed in Non-Patent Document 3, is simple and suitable for testing multiple samples. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Takumi HAYASHI, 7 others, "Cattle with the BoLA class II DRB3*0902 allele have significantly lower bovine leukemia proviral loads", 2017, Journal of Veterinary Medical Science, 79(9), 1552-1555 [Non-Patent Document 2] Marcela A. Juliarena and 3 others, “Hot topic: Bovine leukemia virus (BLV)-infected cows with low proviral load are not a source of infection for BLV-free cattle”, 2016, Journal of Dairy Science, 99, 4586-4589 [Non-Patent Document 3] A. Forletti and 5 others, “Identification of cattle carrying alleles associated with resistance and susceptibility to the Bovine Leukemia Virus progression by real-time PCR”, 2013, Research in Veterinary Science, 95, 991-995 [Overview of the project] [Problems that the invention aims to solve]

[0011] In the method using SYBR Green, any allele having a base sequence to which a primer can hybridize, other than the target allele to be detected, will also be amplified, and all amplified double-stranded DNAs are detected. Therefore, it is difficult to use this method for genes for which a very large number of alleles have been identified. Non-Patent Document 3 reports that false negatives were confirmed when this method was used to detect BoLA-DRB3*009:02.

[0012] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for detecting an allele that can simply and highly accurately detect a specific allele, Resistance to bovine lymphoma an allele detection method and Resistance to bovine lymphoma allele detection for a kit. [Means for Solving the Problem]

[0013] The allele detection method according to the first aspect of the present invention Resistance to bovine lymphoma comprises: a nucleic acid comprising the base sequence of Resistance to bovine lymphoma an allele BoLA-DRB3*009:02 serving as a template, a primer for amplifying said base sequence by polymerase chain reaction, a DNA polymerase, forward a probe that hybridizes to at least a part of said base sequence amplified by said DNA polymerase, and reverse primer and HiDi (registered trademark) a labeling substance that indicates that said probe has hybridized to said base sequence; and the method includes a reaction step of performing a polymerase chain reaction with a reaction solution containing the above components HiDi (registered trademark) . fruit, The forward primer is Having the base sequence shown in Sequence ID No. 2, The aforementioned reverse primer is Having the base sequence shown in Sequence ID No. 3, The aforementioned probe It has the base sequence shown in Sequence ID No. 4. . ​​

[0014] In this case, the HiDi (registered trademark) DNA polymerase has 5'→3' exonuclease activity, the probe is a hydrolysis probe comprising a fluorescent dye as the labeling substance and a quencher that suppresses light emission of the fluorescent dye, wherein the resistance hydrolysis probe hybridized to the base sequence of the allele is degraded by the HiDi (registered trademark) DNA polymerase, whereby suppression of light emission of the fluorescent dye by the quencher is released, this may also be adopted.

[0018] Further, the temperature of the annealing and extension reaction in the polymerase chain reaction is 65°C, this may also be adopted.

[0019] According to a second aspect of the present invention, the Resistance to bovine lymphoma allele detection for detection kit comprises: a primer for amplifying the base sequence of an allele serving as a template in a polymerase chain reaction, Resistance to bovine lymphoma the allele BoLA-DRB3*009:02 for amplifying the base sequence of forward the primer and reverse primer , HiDi (registered trademark) a DNA polymerase, the HiDi (registered trademark) a probe that hybridizes to at least a part of the base sequence amplified by the DNA polymerase, a labeling substance that indicates that the probe has hybridized to the base sequence, comprising 、 The forward primer is Having the base sequence shown in Sequence ID No. 2, The aforementioned reverse primer is Having the base sequence shown in Sequence ID No. 3, The aforementioned probe It has the base sequence shown in Sequence ID No. 4. . Effects of the Invention

[0020] According to the present invention, specific alleles can be detected simply and with high accuracy. [Brief explanation of the drawing]

[0021] [Figure 1] This figure shows the nucleotide sequence of BoLA-DRB3*009:02. [Figure 2] This figure shows the results of agarose gel electrophoresis according to Example 1. [Figure 3] This figure shows the fluorescence intensity for each cycle of real-time PCR according to Example 2. (A) and (B) show the results when the annealing and extension reaction temperatures were 60°C and 64°C, respectively. [Figure 4] This figure shows the fluorescence intensity for each cycle of real-time PCR according to Example 2. (A) and (B) show the results when the annealing and extension reaction temperatures were 64.5°C and 65°C, respectively. [Figure 5] This figure shows the fluorescence intensity for each cycle of real-time PCR performed at a temperature of 66°C for the annealing and extension reactions according to Example 2. [Figure 6] This figure shows the fluorescence intensity for each cycle of real-time PCR according to Example 3. [Figure 7] This figure shows the fluorescence intensity for each cycle of real-time PCR used to examine the amount of template genomic DNA in Example 4. [Figure 8] This figure shows the fluorescence intensity for each cycle of real-time PCR performed in Example 4, where the template genomic DNA amount was 200 ng. [Figure 9] This figure shows the fluorescence intensity for each cycle of real-time PCR according to Example 5. [Figure 10] This figure shows the fluorescence intensity for each cycle of real-time PCR using the conventional method according to Example 7. [Figure 11] This figure shows the fluorescence intensity for each cycle of real-time PCR according to Example 8. [Modes for carrying out the invention]

[0022] The allele detection method according to this embodiment is useful for detecting or determining the genotype of an allele. The organisms possessing the alleles are not particularly limited, but preferably include animals, including humans, and plants. The alleles are not particularly limited, but include, for example, alleles related to the prevention, progression, and cure of various diseases. Preferably, the alleles are resistance alleles or susceptibility alleles to diseases in livestock.

[0023] Livestock includes farm animals, companion animals, and laboratory animals, and in particular farm animals. Specifically, livestock include cattle, buffalo, sheep, goats, pigs, horses, dogs, cats, rabbits, camels, llamas, alpacas, reindeer, donkeys, minks, ferrets, hamsters, mice, rats, guinea pigs, chickens, pigeons, turkeys, quail, guinea fowl, ducks, geese, carp, goldfish, silkworms, and honeybees. Preferably, livestock are cattle, pigs, or chickens.

[0024] The following describes the detection of BoLA-DRB3*009:02 in individuals resistant to BLV infection and EBL resulting from BLV infection, as an example of applying allele detection methods. The number of EBL cases in Japan is on the rise. BLV mainly infects B cells and is incorporated into the host's DNA as a provirus, resulting in persistent infection. As the disease progresses, 2-3% of BLV-infected cows develop B-cell lymphoma, i.e., EBL. There is no vaccine for BLV or treatment for EBL, and cows that develop EBL are discarded. BLV-infected cows suffer from reduced milk yield and decreased immunity.

[0025] The allele detection method according to this embodiment includes a reaction step of performing PCR, particularly real-time PCR, using a reaction solution containing a nucleic acid containing the base sequence of a template allele, a primer, DNA polymerase, a probe, and a labeling substance. The nucleic acid is not particularly limited as long as it contains the base sequence of a template allele. Preferably, the nucleic acid is the genomic DNA of the subject. Genomic DNA can be extracted and purified from blood or the like by known methods.

[0026] PCR is a method of amplifying DNA fragments with a predetermined base sequence using DNA polymerase. Real-time PCR, a type of PCR, is also called quantitative PCR (qPCR). Real-time PCR allows for real-time monitoring and analysis of DNA fragment amplification by measuring the fluorescence signal generated during the amplification of the DNA fragment.

[0027] Primers are extended from the 5' end to the 3' end by DNA polymerase, with a deoxynucleotide triphosphate complementary to the template DNA being added to its 3' end. In PCR, changes in reaction temperature unwind the double-stranded template DNA into single strands, the primers anneal to the DNA, and the DNA polymerase extension reaction occurs, unwinding the extended primers and DNA. By repeating this process, the amplification product (amplicon) can be amplified. Primers consist of a forward primer and a reverse primer, which hybridize to each strand of the double-stranded DNA and extend from the 5' end to the 3' end. The forward primer has a portion of the nucleotide sequence upstream of the region containing the allele's nucleotide sequence. The reverse primer has a nucleotide sequence complementary to a portion of the nucleotide sequence downstream of the region containing the allele's nucleotide sequence.

[0028] Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) of BoLA-DRB3*009:02. For example, the forward primer and reverse primer are designed for the nucleotide sequences F and R shown in Figure 1, respectively. In this case, the nucleotide sequences of the forward primer shown in SEQ ID NO: 2 and the reverse primer shown in SEQ ID NO: 3 are the nucleotide sequences F and R shown in Figure 1, respectively.

[0029] The DNA polymerase used in PCR in this embodiment extends the primer when the single base at the 3' end of the primer hybridized to the allele's base sequence is complementary to the base at the position corresponding to that single base in the base sequence (hereinafter also referred to as "base X"). Some DNA polymerases allow the extension reaction to proceed even if there are several bases that are not complementary between the primer's base sequence and the template's base sequence. In contrast, the DNA polymerase according to this embodiment extends the primer only when at least one base at the 3' end of the primer is complementary to base X. In the case of BoLA-DRB3*009:02 shown in Figure 1, the underlined "G" in base sequence F and the underlined "T" in base sequence R correspond to base X. Preferably, a DNA polymerase modified so that the amplification efficiency is significantly reduced when at least one base at the 3' end of the primer is not complementary to base X is preferred. Examples of such DNA polymerases include HiDi DNA polymerase and HiDi Taq DNA polymerase (myPOLS Biotec).

[0030] Due to the characteristics of DNA polymerase described above, the extension reaction will not proceed if at least one base at the 3' end of at least one of the forward primer and reverse primer is not complementary to base X. Therefore, primers are designed so that at least one base at the 3' end of at least one of the forward primer and reverse primer is as non-complementary as possible to base X in alleles other than the allele being detected. Preferably, primers are designed so that one base at the 3' end of the forward primer or reverse primer is not complementary to base X in 95% or more, more preferably 97% or more, and more preferably 98% or more of alleles other than the allele being detected. Most preferably, primers are designed so that one base at the 3' end of the forward primer or reverse primer is not complementary to base X in all alleles other than the allele being detected. Preferably, primers are designed so that less than 3% of alleles have base X that is complementary to the one base at the 3' end of both the forward primer and the reverse primer.

[0031] Furthermore, as long as the primer hybridizes to the allele's base sequence and DNA polymerase functions, there may be 1 to 5 or 1 to 3 bases in the base sequence other than the single base at the 3' end that are not complementary to the allele's base sequence. Preferably, all bases of the primer except the single base at the 3' end are complementary to the bases at their respective positions in the allele.

[0032] In this embodiment, PCR uses a hybridization method. The hybridization method uses a probe that has a base sequence complementary to a portion of the base sequence amplified by DNA polymerase. With the hybridization method, a strong signal can be detected only when a DNA fragment is amplified using the DNA hybridized with the probe as a template.

[0033] The probe is an oligonucleotide of approximately 10-40 mers. The probe specifically hybridizes to at least a portion of the base sequence of the amplification product amplified by DNA polymerase. More specifically, the probe has a base sequence complementary to at least a portion of the base sequence amplified by DNA polymerase. The region to which the primer hybridizes is located between the region to which the forward primer hybridizes and the region to which the reverse primer hybridizes. Therefore, the region to which the primer hybridizes and the region to which the probe hybridizes do not overlap. Preferably, the base sequence of the probe is a base sequence that does not hybridize to the base sequence amplified in alleles other than the allele to be detected, as much as possible. In the case of BoLA-DRB3*009:02 shown in Figure 1, base sequence P is exemplified as the region to which the primer hybridizes.

[0034] In BoLA-DRB3, which has 357 reported alleles, when the forward primer whose sequence is shown in SEQ ID NO: 2 and the reverse primer whose sequence is shown in SEQ ID NO: 3 are used to detect BoLA-DRB3*009:02, at least one base at the 3' end of the forward primer is not complementary to base X of the 333 alleles, and at least one base at the 3' end of the reverse primer is not complementary to base X of 16 of the remaining 24 alleles.

[0035] Comparing the eight alleles that can be amplified using the forward primer shown in SEQ ID NO: 2 and the reverse primer shown in SEQ ID NO: 3, a probe with a nucleotide sequence that is not completely complementary to alleles other than BoLA-DRB3*009:02 is preferred as much as possible. The nucleotide sequence of the probe used to detect BoLA-DRB3*009:02 is, for example, the nucleotide sequence shown in SEQ ID NO: 4. The probe whose nucleotide sequence is shown in SEQ ID NO: 4 is not completely complementary to the nucleotide sequences of six of the eight alleles. The remaining two alleles are BoLA-DRB3*163:01 and the target allele, BoLA-DRB3*009:02. BoLA-DRB3*163:01 is an allele specific to humped cattle (Bos Indicus) and, although it is in the database, it is not an allele that is present when targeting domestic cattle (Bos Taurus). Therefore, cattle samples that exhibit specific amplification by PCR using the forward primer shown in SEQ ID NO: 2, the reverse primer shown in SEQ ID NO: 3, and the probe shown in SEQ ID NO: 4 are samples containing BoLA-DRB3*009:02.

[0036] The hybridization conditions are stringent, meaning that the probe or primer hybridizes with nucleic acids with complementary base sequences but not with nucleic acids with non-complementary base sequences. Stringent conditions can be determined appropriately based on, for example, the Molecular Cloning: A Laboratory Manual, 3rd Edition (2001), and include conditions such as 0.2×SSC, 0.1% SDS, and incubation at 65°C.

[0037] The labeling substance is not particularly limited as long as it indicates that the probe has hybridized to the base sequence amplified by DNA polymerase. Preferably, the labeling substance is attached to the probe and emits a signal due to hybridization with the base sequence of the allele. An example of such a probe is a hydrolysis probe having a fluorescent dye and a quencher that suppresses the emission of the fluorescent dye.

[0038] When a hydrolysis probe hybridized to the amplified allele's base sequence is degraded by the 5'→3' exonuclease activity of DNA polymerase, fluorescence is released as the suppression of fluorescent dye emission by the quencher is released. Preferably, the fluorescent dye is attached to the 5' end of the hydrolysis probe and the quencher to the 3' end. An example of a hydrolysis probe is the TaqMan® probe. As a DNA polymerase having 5'→3' exonuclease activity, for example, the above-mentioned HiDi Taq DNA polymerase (manufactured by myPOLS Biotec) is preferred.

[0039] The hydrolysis probe hybridizes to the base sequence of the allele. In this state, because the physical distance between the fluorescent dye and the quencher is small, fluorescence resonance energy transfer (FRET) occurs, transferring energy from the fluorescent dye to the quencher and suppressing fluorescence generation. FRET occurs when the distance between the fluorescent dye and the quencher is 1-10 nm. As the extension reaction progresses, the hydrolysis probe is hydrolyzed by the 5'→3' exonuclease activity of DNA polymerase, and the fluorescent dye dissociates from the quencher. As a result, FRET no longer occurs, and a strong fluorescence signal is generated. Therefore, if nonspecific fragment amplification occurs by PCR or primer dimers are formed, a strong fluorescence signal will not be detected.

[0040] Furthermore, if the hydrolysis probe does not completely hybridize with the allele's base sequence, the resulting fluorescence signal is significantly reduced. If one to several bases of the hydrolysis probe's base sequence do not hybridize, the melting temperature (Tm) decreases, and the hydrolysis probe is released from the allele's base sequence. In this case, degradation of the probe by 5'→3' exonuclease activity does not occur, and the fluorescent dye and quencher remain attached to the 5' and 3' ends of the hydrolysis probe, respectively. Since the fluorescent dye does not detach from the quencher, the quencher suppresses the generation of the fluorescence signal. Therefore, by making the hydrolysis probe's base sequence not completely complementary to the base sequences of alleles other than the allele being detected, the specificity of fluorescence generation can be increased. For example, even if there are many alleles and it is not possible to design primers in a position that suppresses the amplification of all alleles other than the allele being detected, the allele being detected can be detected with high accuracy by making the hydrolysis probe's base sequence not completely complementary to the base sequences of alleles other than the allele being detected.

[0041] Examples of fluorescent dyes used in hydrolysis probes include 6-FAM, TET, HEX, JOE, Yakima Yellow, TAMRA, ATTO550, ATTO565, ATTO633, ATTO647, ROX, Texas Red-X, Cy3, and Cy5. A fluorescent dye with a detectable fluorescence wavelength should be selected depending on the real-time PCR instrument used.

[0042] Examples of quenchers used in hydrolysis probes include TAMRA, BHQ(trademark)-1, BHQ(trademark)-2, BHQ(trademark)-3, Iowa Black(trademark)RQ, Iowa Black(trademark)FQ, and Eclipse(trademark). Since each quencher can suppress different wavelengths of fluorescence, one should select a quencher that can suppress the fluorescent dye being used.

[0043] Alternatively, a hydrolysis probe with a minor groove binder (MGB) added to the quencher may be used. MGB enters the minor grooves of the DNA double helix structure, further strengthening the double helix structure when the hydrolysis probe hybridizes. This allows for a higher Tm (transfer temperature), enabling a higher annealing temperature in PCR. As a result, the specificity of the probe can be further enhanced. TaqMan® MGB probes are a well-known example of probes with added MGB.

[0044] Alternatively, a double-quencher probe may be used, which has another type of quenching agent added to the probe. In a double-quencher probe, an internal quencher such as ZEN® quencher or TAO® quencher is added between the base sequences, which enhances the quenching effect within the probe and reduces the background level.

[0045] Another example of a probe that can be used in PCR in this embodiment 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 allele to be detected, and further has complementary base sequences on both sides. Like hydrolysis probes, molecular beacon probes have a fluorescent dye and a quencher at both ends. When the molecular beacon probe is not hybridized to the allele to be detected, the molecular beacon probe takes the form of a hairpin-shaped stem-loop structure, where the complementary base sequences at both ends form a stem structure and the base sequence complementary to the allele forms a loop structure. In the stem-loop structure, the fluorescent dye and quencher at both ends are in close proximity, and fluorescence is suppressed.

[0046] Unlike the hydrolysis probes mentioned earlier, fluorescence suppression in molecular beacon probes is due to collision quenching caused by the overlap of electron orbitals between the fluorescent dye and the quencher. Collision quenching occurs when the distance between the fluorescent dye and the quencher is 0.3 to 1 nm. In molecular beacon probes, thermal denaturation causes the stem structure to open and become linear, and in this state, hybridizes to the target sequence, increasing the physical distance between the fluorescent dye and the quencher. This increased physical distance releases the fluorescence suppression by the quencher. The use of molecular beacon probes enables analysis with low background levels and high specificity.

[0047] Another example of a probe that can be used in PCR in this embodiment is a dual hybridization probe. As a dual hybridization probe, two probes having a base sequence complementary to a portion of the allele to be detected are used. An acceptor fluorescent dye is attached to the end of the first probe, and a donor fluorescent dye is attached to the end of the second probe. The first and second probes are designed so that the acceptor fluorescent dye and the donor fluorescent dye are in close proximity when both hybridize to the allele. The energy of the donor fluorescent dye, excited by excitation light, is transferred to the acceptor fluorescent dye, generating a fluorescent signal. An example of a dual hybridization probe is the Light Cycler® probe.

[0048] Another example of a probe that can be used in PCR in this embodiment is a cycling probe. A cycling probe is a chimeric probe containing DNA and RNA having a base sequence complementary to a portion of the allele to be detected, with a reporter fluorescent dye attached to one end and a quencher to the other. When PCR is performed with the addition of RNaseH, the probe hybridizes to the allele, and the RNA portion of the probe is cleaved by the RNaseH, generating strong fluorescence. If there is a base near the RNA that is not complementary to the corresponding base of the allele to be detected, cleavage of the RNA by RNaseH does not occur, and fluorescence remains suppressed. Therefore, cycling probes are effective when detection is based on a single base difference in the allele to be detected.

[0049] The primers and probes according to this embodiment can be chemically synthesized, for example, using a commercially available automated nucleic acid synthesizer. The optimal reaction conditions in PCR, such as the amount of template DNA, the amount of deoxynucleoside triphosphate (dNTP), the annealing temperature, the extension reaction time, and the number of cycles, are appropriately set according to the sequence and length of the primers used, the type and base sequence of the probes used, the length of the sequence to be amplified, and the type of real-time PCR instrument used. PCR may also be performed in two steps: a thermal denaturation step and an annealing and extension reaction step, or in three steps: a thermal denaturation step, an annealing step, and an extension reaction step.

[0050] The annealing temperature in PCR using the forward primer whose base sequence is shown in SEQ ID NO: 2, the reverse primer shown in SEQ ID NO: 3, and the probe shown in SEQ ID NO: 4 is, for example, 64°C to 65°C, preferably 65°C. The dNTP concentration is, for example, less than 280 μM, preferably 200 to 280 μM, more preferably 200 to 240 μM, and even more preferably 200 μM. The primer concentration is, for example, 0.08 to 0.8 μM, preferably 0.4 to 0.8 μM, and more preferably 0.6 μM. The probe concentration is, for example, 0.08 to 0.5 μM, preferably 0.1 to 0.5 μM, and more preferably 0.3 μM. The amount of template DNA is, for example, 1.56 to 200 ng, preferably 10 to 100 ng, and more preferably 50 ng.

[0051] The allele detection method according to this embodiment allows for the simple and highly accurate detection of a specific allele from among a large number of alleles. By applying this detection method to livestock samples, for example, individuals possessing disease-resistant or susceptible alleles in livestock can be identified simply, accurately, and inexpensively. Individuals possessing susceptible alleles are more susceptible to disease, making it possible to take measures such as isolating and raising such individuals. Furthermore, breeding strategies utilizing disease-resistant animals enable the maintenance and production of resistant animals. This detection method can serve as the basis for livestock infectious disease control strategies utilizing resistant animals.

[0052] Furthermore, the allele detection method according to this embodiment can be used to detect any gene having multiple alleles, or single nucleotide polymorphisms (SNPs), in any organism, including animals and plants.

[0053] In another embodiment, the allele detection method may include a determination step based on a labeling substance to determine whether or not a probe has hybridized to the base sequence amplified by DNA polymerase. Based on the determination result, a specific allele can be detected, or the genotype of the allele can be determined.

[0054] In another embodiment, an allele detection kit is provided. The allele detection kit comprises the primers described above, DNA polymerase, a probe, and a labeling substance. This detection kit can easily and accurately detect a specific allele or determine the genotype of an allele in the genomic DNA of an acquired sample. The allele detection kit may further include various reagents such as buffers necessary for PCR.

[0055] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples. [Examples]

[0056] (Example 1: Design of a primer for detecting BoLA-DRB3*009:02) We investigated the nucleotide sequences of primers for detecting BoLA-DRB3*009:02 by real-time PCR. All alleles of BoLA-DRB3 registered in the IPD-MHC database (https: / / www.ebi.ac.uk / ipd / mhc / ) as of December 21, 2020, were aligned, and the 3' end bases of the primers were determined based on the following conditions: 1) BoLA-DRB3*009:02 has different bases from the majority of other alleles, 2) primers with appropriate Tm values ​​and GC content can be designed, and 3) appropriate probes can be created between the primers. From the determined bases, the primer sets shown in Table 1 were designed to have appropriate lengths, Tm values, and GC content.

[0057] [Table 1]

[0058] Conventional PCR was performed to confirm the effectiveness of each primer set. The following four types of test samples, S1 to S4, were used. Genome of a heterozygous cattle with S1:DRB3*009:02 and DRB3*015:01 (both the forward and reverse primer sequences are perfectly complementary to the DRB3*009:02 sequence) Plasmid DNA containing the S2:DRB3*009:01 nucleotide sequence (both the forward and reverse primer sequences are perfectly complementary to the DRB3*009:02 nucleotide sequence) Genome of a heterozygous cattle with S3:DRB3*034:01 and DRB3*005:03 (forward primer sequences are perfectly complementary to the sequences of both alleles, but reverse primer sequences are not perfectly complementary to the sequences of either allele). Four types of genomes were used from heterozygous cattle with S4:DRB3*001:0 and DRB3*014:01:01 alleles (where the sequences of both the forward and reverse primers were not perfectly complementary to the sequences of either allele).

[0059] Bovine genomic DNA was isolated and purified from bovine blood according to standard procedures. PCR reaction solutions, as shown in Table 2, were prepared in PCR tubes or 96-well plates. Bovine genome was prepared to 100 ng / μL and used as the test sample. Plasmid DNA was prepared to 1 ng / μL.

[0060] [Table 2]

[0061] The PCR conditions consisted of holding at 95°C for 2 minutes, followed by 15 seconds at 95°C, 30 seconds at the annealing temperature, and 30 seconds at 72°C. This cycle was repeated 27 times. The annealing temperatures were 62°C for primer set A, 57°C for primer set B, and 54°C for primer sets C and D. A MiniAmp (Applied Biosystems) was used for the reaction of primer set A, and a 2720 Thermal Cycler (Applied Biosystems) was used for the reactions of primer sets B, C, and D.

[0062] (result) The PCR products were confirmed by agarose gel electrophoresis. As shown in Figure 2, it was confirmed that all primer sets accurately amplified DNA fragments of approximately 220 base pairs in S1 and S2. Note that sample S5 in Figure 2 is distilled water.

[0063] (Example 2: Investigation of annealing temperature and extension reaction temperature) The annealing and extension reaction temperatures in real-time PCR for detecting BoLA-DRB3*009:02 were investigated. Sample 1 was the genome of a heterozygous cattle carrying DRB3*009:02 and DRB3*015:01, and Sample 2 was a plasmid DNA containing the DRB3*009:01 sequence. The cattle genome was prepared to 100 ng / μL and used as the test sample. The plasmid DNA concentration was 160 pg / μL. Primer set A from Example 1 (forward primer with the sequence shown in SEQ ID NO: 2 and reverse primer with the sequence shown in SEQ ID NO: 3) was used. The nucleotide sequences of the probes used are shown in SEQ ID NO: 4. A PCR reaction solution containing the following reagents was prepared in a PCR tube or 96-well plate as shown in Table 3.

[0064] The reagents used were HiDi Taq DNA polymerase (myPOLS Biotec, 9201S), 10×HiDi reaction buffer, dNTPs Mixture (2mM each of A, C, G, and T, Toyobo Co., Ltd., NTP-201), forward primer (Eurofins), reverse primer (Eurofins), TaqMan® MGB probe (Eurofins) with FAM® attached to the 5' end and MGB and Eclipse® attached to the 3' end, ROX Reference (invitrogen, 12223012), and Nuclease-Free Water (invitrogen, AM9930).

[0065] [Table 3]

[0066] PCR was performed by setting tubes or plates in a Quant Studio3 (Applied Biosystems), holding at 95°C for 2 minutes, followed by a denaturation step of 10 seconds at 95°C, and then repeating the annealing and extension reaction step of 1 minute at 60°C, 64°C, 64.5°C, 65°C, or 66°C for 40 cycles. Fluorescence intensity was measured after each cycle of the annealing and extension reaction step.

[0067] (result) The results at each annealing temperature are shown in Figures 3, 4, and 5. As shown in Figure 3(A), at 60°C, an increase in fluorescence intensity was observed in both Sample 1 and Sample 2, and DRB3*009:02 could not be identified. As shown in Figures 3(B) and 4(A), a difference in fluorescence intensity was observed between Sample 1 and Sample 2 at 64°C and 64.5°C. On the other hand, as shown in Figure 4(B), a specific increase in fluorescence intensity was observed in Sample 1 at 65°C. The fluorescence intensity of Sample 2 was suppressed to the background level, and the non-smooth amplification curve is also characteristic of non-specific amplification. As shown in Figure 5, at 66°C, the amplification curves collapsed in both samples, and no specific increase in fluorescence intensity was observed. This is thought to be because the Tm value of the reverse primer was 66.7°C, preventing the probe from hybridizing.

[0068] The results above indicate that the optimal annealing and extension temperature for real-time PCR to detect BoLA-DRB3*009:02 is 65°C. Furthermore, plasmid DNA containing the DRB3*009:01 sequence did not show a clear increase in fluorescence intensity despite containing an excess amount of template compared to the genome. At 65°C, it was shown that the genomes of DRB3*009:02 and heterozygous cattle can be distinguished.

[0069] (Example 3: Investigation of primer concentration) The primer concentrations for real-time PCR to detect BoLA-DRB3*009:02 were investigated. As test samples, heterozygous cattle genomes of DRB3*009:02 and 015:01, prepared at 25 ng / μL, were used. The primer concentrations were set to 0.8 μM, 0.6 μM, 0.4 μM, or 0.16 μM, and the PCR reaction solutions shown in Table 4 were prepared in PCR tubes or 96-well plates. PCR was performed under the same reaction conditions as in Example 2, and the fluorescence intensity was measured. Note that in the tests with primer concentrations of 0.8 μM, 0.6 μM, 0.4 μM, and 0.16 μM, the values ​​of x in Table 4 were 2, 1.5, 1, and 0.4, respectively.

[0070] [Table 4]

[0071] (result) As shown in Figure 6, accurate amplification of fluorescence intensity was observed at all primer concentrations, but the amplification curve rose with the fewest cycles at 0.6 μM. These results indicate that 0.6 μM is the optimal primer concentration for real-time PCR to detect BoLA-DRB3*009:02.

[0072] (Example 4: Examination of template genomic DNA quantity) The amount of template genomic DNA used in real-time PCR to detect BoLA-DRB3*009:02 was investigated. In addition to Sample 1 described above, the genome of a DRB3*010:01 and 015:01 heterozygous cattle was used as Sample 3. PCR reaction solutions were prepared in PCR tubes or 96-well plates with template genomic DNA amounts of 1.56 ng, 3.13 ng, 6.25 ng, 12.5 ng, 25 ng, 50 ng, 100 ng, or 200 ng, according to the compositions shown in Table 5. PCR was performed under the same reaction conditions as in Example 2, and the fluorescence intensity was measured. For Sample 3, the composition of the PCR reaction solution was changed by replacing 0.05 μL of Nuclease-free water with 0.05 μL of ROX Reference as shown in Table 3 above.

[0073] [Table 5]

[0074] (result) As shown in Figure 7, sample 1 showed a precise increase in fluorescence intensity at amounts ranging from 1.56 to 200 ng. Figure 8 shows the fluorescence intensity of samples 1 and 3 when each was 200 ng. Sample 3, which does not contain DRB3*009:02, also showed an increase in fluorescence intensity. However, when sample 3 was prepared to 50 ng and retested, no increase in fluorescence intensity was observed. Considering the rapid rise of the amplification curve and the elimination of false positives, 50 ng of template genomic DNA was shown to be the optimal amount for real-time PCR to detect BoLA-DRB3*009:02.

[0075] (Example 5: Detection of BoLA-DRB3*009:02 by real-time PCR) Based on the real-time PCR conditions optimized in Examples 1-4, detection of BoLA-DRB3*009:02 was performed by real-time PCR. Genomics collected from 64 cattle were prepared at 25 ng / μL each as test samples. Of these 64 cattle, 13 contained BoLA-DRB3*009:02. PCR reaction solutions were prepared in PCR tubes or 96-well plates with the same composition as in Example 4, except that genomic DNA was used instead of test samples.

[0076] PCR was performed by setting tubes or plates in a Quant Studio3 (Applied Biosystems), holding at 95°C for 2 minutes, and then repeating a denaturation step at 95°C for 10 seconds followed by an annealing and extension step at 65°C for 1 minute for 40 cycles. Fluorescence intensity was measured after each cycle of the annealing and extension step. PCR was completed in approximately 1 hour and 10 minutes.

[0077] (result) As is clear from Figure 9, only the samples from 13 cattle containing BoLA-DRB3*009:02 showed amplification curves, clearly distinguishing them from the samples from 51 cattle not containing BoLA-DRB3*009:02. This demonstrates that BoLADRB3*009:02 can be detected by this real-time PCR.

[0078] (Example 6: Evaluation of detection sensitivity and detection specificity using field samples) To confirm whether real-time PCR can correctly identify samples prepared from individuals possessing BoLA-DRB3*009:02 as positive and samples prepared from individuals lacking BoLADRB3*009:02 as negative, it was compared with conventional PCR-RFLP and sequencing methods. Field samples were prepared by collecting and purifying genomic DNA from 150 cattle and adjusting the concentration to 25 ng / μL. Real-time PCR was performed following the same procedure as in Example 5.

[0079] The PCR-RFLP method was performed using TaKaRa Ex Taq® Hot Start Version (RR006A, manufactured by Takara Bio Inc.) as follows. The restriction enzymes used (all from New England BioLabs) were Rsa I (R0167S), Hae III (R0108S), and BstY I (R0523S), and the reaction mixtures used were NEB Buffer ver 2.1 and CutSmart (both from New England BioLabs). The sample DNA was derived from bovine blood (100-200 ng / μL).

[0080] First, semi-nested PCR targeting BoLA-DRB3 exon 2 was performed. The reaction mix for the first round of PCR consisted of 14.8 μL of nuclease-free water and 10× buffer (20 mM Mg). 2+The following preparations were used: 2.0 μL of (additional) dNTP Mixture (2.5 mM each), 1.6 μL of primer HL030 (10 μM), 0.2 μL of primer HL031 (10 μM), and 0.2 μL of Takara Ex Taq HS (5 U / μL). The nucleotide sequences of primer HL030 and primer HL031 are shown in SEQ ID NOs. 10 and 11, respectively.

[0081] 1 μL of sample DNA was added to the above reaction mix, and PCR was performed. The reaction conditions were as follows: holding at 98°C for 2 minutes, followed by 10 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 72°C for 30 seconds, followed by holding at 72°C for 7 minutes, and finally settling at 10°C.

[0082] The reaction mix for the second round of PCR consists of 30.2 μL of nuclease-free water and 10× buffer (20 mM Mg). 2+ The following preparations were used: 4.0 μL of dNTP Mixture (2.5 mM each), 3.2 μL of primer HL030 (10 μM), 0.2 μL of primer HL032 (10 μM), and 0.2 μL of Takara Ex Taq HS (5 U / μL). The nucleotide sequences of primer HL032 are shown in SEQ ID NO: 12.

[0083] 2 μL of the PCR product from the first round was added to the above reaction mix, and PCR was performed. The reaction conditions were as follows: holding at 98°C for 2 minutes, followed by 35 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 72°C for 30 seconds, followed by holding at 72°C for 7 minutes, and then set to 10°C.

[0084] Next, the PCR products obtained in the second round of PCR were cleaved with BstY I, Rsa I, and Hae III. The reaction mix for the BstY I reaction consisted of 3.0 μL of distilled water, 1.5 μL of 10×NEB Buffer, and 0.5 μL of BstY I (10 U / μL). 10 μL of the PCR product was added to this reaction mix and incubated at 60°C for 5 hours.

[0085] The reaction mix for Rsa I and Hae III consisted of 3.0 μL of distilled water, 1.5 μL of 10×CutSmart Buffer, and 0.5 μL of Rsa I or Hae III (10 U / μL). 10 μL of the PCR product was added to the reaction mix and incubated at 37°C for 6 hours.

[0086] The polyacrylamide gel was composed of 16.3 mL of 1× Tris-Borate-EDTA Buffer (TBE buffer, T9121, Takara Bio Inc.), 3.5 mL of acrylamide solution (40 w / v% acrylamide / bis-compound (19:1), 06140-45, Nacalai Tesque Inc.), 200 μL of 10% APS (ammonium persulfate, 1610700, Biorad Inc.), and 14 μL of TEMED (tetramethylethylenediamine, 1610800, Biorad Inc.). After fixing the polyacrylamide gel in the electrophoresis apparatus, 1× TBE buffer was added to the apparatus, and 15 μL of the sample mixed with 6× sample loading buffer was added to each well. Electrophoresis was performed at 140 V for 28 minutes. The gel was added to a mixture of 40 mL of distilled water and 2 μL of GelRed (41002, Cosmo Bio Co., Ltd.) and stirred for 20 minutes. The gel was photographed under ultraviolet light, and bovine MHC class II allyls were typed by band pattern analysis.

[0087] Since the PCR-RFLP method cannot distinguish between BoLA-DRB3*009:01, BoLA-DRB3*009:02, and BoLA-DRB3*009:03, if the cleavage pattern indicated BoLA-DRB3*009:01, BoLA-DRB3*009:02, or BoLA-DRB3*009:03, the DNA sequence was determined by sequencing to determine the genotype of the allele.

[0088] (result) Based on PCR-RFLP and sequencing methods, 17 cows were found to possess BoLA-DRB3*009:02. All samples that showed amplification by real-time PCR were derived from cows possessing BoLA-DRB3*009:02. Therefore, in this example, real-time PCR successfully identified samples prepared from individuals possessing BoLA-DRB3*009:02 as positive. Furthermore, it demonstrated the ability to correctly identify samples prepared from individuals lacking BoLA-DRB3*009:02 as negative. Additionally, 23 PCR-RFLP patterns were obtained from field samples. This indicates that the alleles in field samples were not biased towards a specific allele, but rather contained a diverse range of alleles.

[0089] (Example 7: Comparison with real-time PCR using SYBR Green) Using genomic DNA collected and purified from 150 cattle used in Example 6, real-time PCR according to the present invention (Example A) and real-time PCR using SYBR Green (Comparative Example) were compared. Test samples were prepared at 25 ng / μL. Example A was performed using the same procedure as the real-time PCR in Example 5.

[0090] In the comparative example, the PCR reaction mixtures shown in Table 6 were prepared in PCR tubes or 96-well plates. The reagents used were FastStart Universal SYBR Green Master (Rox) (Roche, part number 04913850001 201S), forward primers with the nucleotide sequence shown in SEQ ID NO: 13 (Eurofins), reverse primers with the nucleotide sequence shown in SEQ ID NO: 14 (Eurofins), and Nuclease-Free Water (invitrogen, AM9930). For the positive control, instead of the test sample, genomic DNA prepared at 25 ng / μL from cattle containing BoLA-DRB3*009:02 prepared in Example 1 was used.

[0091] [Table 6]

[0092] PCR was performed by setting tubes or plates in a Quant Studio3 (Applied Biosystems), holding them at 50°C for 2 minutes and 95°C for 10 minutes, followed by a thermal denaturation step of 15 seconds at 95°C and an annealing and extension reaction step of 1 minute at 60°C, repeated for 40 cycles. Fluorescence intensity was measured after each cycle of the annealing and extension reaction step. Subsequently, to perform melting curve analysis, the temperature was changed to 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds, and fluorescence intensity was measured during the temperature increase from 60°C to 95°C.

[0093] (result) Figure 10 shows the fluorescence intensity in the comparative example. It was found that the amplification curve of the sample containing BoLA-DRB3*009:02 rose quickly, while the amplification curve of the sample without BoLA-DRB3*009:02 also rose at a large number of cycles. According to the comparative example, BoLA-DRB3*009:02 can be distinguished by the difference in the number of cycles, but it cannot be distinguished solely by the rise of the amplification curve, so there is a risk of misidentifying a false positive.

[0094] Table 7 also shows the number of cycles (Threshold Cycle (Ct) values) required for fluorescence intensity to reach the detection threshold in Example A and the Comparative Example. Note that the Tm values ​​for the Comparative Example in Table 7 represent the results of melting curve analysis.

[0095] [Table 7]

[0096] As shown in Table 7, sample 10 showed a high Ct value in the second test and could not be distinguished from a negative sample. Although not shown in Table 7, sample 14 also tested negative in the preliminary test. Furthermore, since samples lacking BoLA-DRB3*009:02 also show a peak Tm value around 85°C, it is difficult to distinguish between false negatives and true negatives using melting curve analysis.

[0097] These results revealed that the method using SYBR Green carries risks of false positives and false negatives, and also has problems with the reproducibility of results. On the other hand, the detection of BoLA-DRB3*009:02 using the method according to the present invention avoids the risks of false positives and false negatives, and furthermore, it was shown that BoLA-DRB3*009:02 can be accurately detected simply by whether or not the amplification curve rises.

[0098] (Example 8: Detection of BLV resistance gene in mixed samples) DNA pool 1, containing DNA collected from BoLA-DRB3*009:02-possessing cattle and DNA collected from 29 other allele-type cattle that do not possess BoLA-DRB3*009:02, or DNA pool 2, containing DNA collected from 29 other allele-type cattle that do not possess BoLA-DRB3*009:02, were used as template genomes. A PCR reaction solution was prepared with the same composition as in Table 5 of Example 4, except that a template genome was used instead of a test sample. PCR was performed under the same reaction conditions as in Example 5, and the fluorescence intensity was measured. For DNA pool 1 and DNA pool 2, the PCR reaction solution was prepared so that the amount of template genome in the PCR reaction solution was 500 ng, 250 ng, 100 ng, 50 ng, 10 ng, or 1 ng. For DNA pool 1, the amounts of BoLA-DRB3*009:02 DNA in the PCR reaction solutions with template genome amounts of 500 ng, 250 ng, 100 ng, 50 ng, 10 ng, and 1 ng were 16.7 ng, 8.3 ng, 3.3 ng, 1.7 ng, 333 pg, and 33.3 pg, respectively.

[0099] (result) As shown in Figure 11, when a fluorescence intensity (ΔRn) of 0.15 or higher was judged as positive, DNA pool 1, which contained more than 10 ng of template genome, could be detected as positive with high sensitivity. DNA pool 2, which did not contain BoLA-DRB3*009:02 DNA, did not become positive even when the amount of template genome increased. The fact that BoLA-DRB3*009:02 DNA in mixed DNA pool samples can also be detected with high sensitivity indicates that it is possible to screen for the presence of BoLA-DRB3*009:02-carrying cattle by mixing bovine blood, extracting DNA, and performing PCR testing on each herd. BoLA-DRB3*009:02-carrying cattle can be identified by further testing the individuals that make up the herd in which positive results were detected.

[0100] The embodiments described above are for illustrative purposes only and do not limit the scope of the present invention. That is, the scope of the present invention is defined not by the embodiments, but by the claims. Various modifications made within the scope of the claims and equivalent inventive meaning are considered to be within the scope of the present invention.

[0101] This application is based on Japanese Patent Application No. 2021-66098, filed on 8 April 2021. The entire specification, claims, and drawings of Japanese Patent Application No. 2021-66098 are incorporated herein by reference. [Industrial applicability]

[0102] This invention can be used in diagnostic testing technologies in the fields of livestock farming and veterinary medicine, or in the breeding of animals and plants, including livestock.

Claims

1. A nucleic acid containing the base sequence of BoLA-DRB3*009:02, which is a resistance allele to bovine lymphoma that serves as a template, A forward primer and a reverse primer for amplifying the aforementioned base sequence by polymerase chain reaction, HiDi® DNA polymerase and A probe that hybridizes to at least a portion of the base sequence amplified by the HiDi® DNA polymerase, A labeling substance indicating that the probe has hybridized to the base sequence, The reaction step includes carrying out a polymerase chain reaction in a reaction solution containing the following: The forward primer is Having the base sequence shown in Sequence ID No. 2, The aforementioned reverse primer is Having the base sequence shown in Sequence ID No. 3, The aforementioned probe Having the base sequence shown in Sequence ID No. 4, A method for detecting resistance alleles to bovine lymphoma.

2. The aforementioned HiDi® DNA polymerase is It has 5'→3' exonuclease activity, The aforementioned probe A hydrolysis probe comprising a fluorescent dye as a labeling substance and a quencher that suppresses the emission of light from the fluorescent dye, When the hydrolysis probe hybridized to the base sequence of the resistance allele is degraded by the HiDi® DNA polymerase, the suppression of the luminescence of the fluorescent dye by the quencher is released. A method for detecting resistance alleles to bovine lymphoma according to claim 1.

3. The temperatures for the annealing and extension reactions in the polymerase chain reaction are: It is 65°C. A method for detecting resistance alleles to bovine lymphoma according to claim 1 or 2.

4. Forward primers and reverse primers for amplifying the nucleotide sequence of BoLA-DRB3*009:02, a resistance allele to bovine lymphoma that serves as a template in polymerase chain reaction, HiDi® DNA polymerase and A probe that hybridizes to at least a portion of the base sequence amplified by the HiDi® DNA polymerase, A labeling substance indicating that the probe has hybridized to the base sequence, Equipped with, The forward primer is Having the base sequence shown in Sequence ID No. 2, The aforementioned reverse primer is Having the base sequence shown in Sequence ID No. 3, The aforementioned probe Having the base sequence shown in Sequence ID No. 4, A kit for detecting resistance alleles to bovine lymphoma.

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