WO2017212904A1 - Method for rapid detection of african swine fever virus using lamp method in which multiple primer sets are combined - Google Patents

Method for rapid detection of african swine fever virus using lamp method in which multiple primer sets are combined Download PDF

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WO2017212904A1
WO2017212904A1 PCT/JP2017/019084 JP2017019084W WO2017212904A1 WO 2017212904 A1 WO2017212904 A1 WO 2017212904A1 JP 2017019084 W JP2017019084 W JP 2017019084W WO 2017212904 A1 WO2017212904 A1 WO 2017212904A1
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seq
base sequence
primer
african swine
swine fever
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PCT/JP2017/019084
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French (fr)
Japanese (ja)
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渉 山崎
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国立大学法人 宮崎大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the present invention relates to an oligonucleotide primer useful for detection of African swine fever virus. More specifically, the present invention relates to oligonucleotide primers used in the LAMP method, which is a simple, highly sensitive and rapid gene detection method, a primer set combining these, and a rapid detection method for African swine fever using the primer set. .
  • African swine fever virus (African swine fever virus) is a causative virus of African swine fever virus, and is a large double-stranded DNA virus classified as Asfairidae. African swine fever is highly transmissible to pigs, and the affected pigs mainly suffer from fever, loss of appetite, conjunctivitis, lymphadenopathy, respiratory distress, constipation, diarrhea, postparalysis, ataxia, and difficulty standing. Cause up to 100% mortality.
  • African swine fever is classified into these five types depending on the strength of the pathogenicity of the virus: hyperacute, acute, subacute, chronic and inapparent.
  • affected animals are slaughtered as soon as they are found to prevent the spread of infection to other livestock. Since the economic damage caused by this slaughtering procedure is enormous, African swine fever is designated as a legally contagious disease by the Livestock Infectious Disease Prevention Law in Japan. Under these circumstances, prevention of epidemics of African swine cholera and rapid diagnosis and detection of intrusions from overseas are among the most important issues in Japan.
  • Diagnosis of African swine fever is performed from materials such as blood of infected individuals by PCR, which is a microbiological test, antibody measurement, and virus isolation using cultured cells.
  • PCR a microbiological test, antibody measurement, and virus isolation using cultured cells.
  • virus isolation using cultured cells is not a useful diagnostic method from the viewpoint of rapid diagnosis because it requires days for culture.
  • the PCR method can obtain the result most quickly among the three test methods, but it still has a problem that it requires at least about 3 hours from obtaining the sample.
  • Non-Patent Documents 1 and 2 the LAMP method is a simpler and quicker test method than the PCR method, and many primer sets for detecting various pathogens such as foot-and-mouth disease have been reported.
  • the LAMP method for detecting African swine fever virus has been developed by a group in the UK (James et al.) (Non-patent Document 3).
  • Non-Patent Document 3 in the case of African swine fever virus strains isolated mainly in Africa, it takes 40 minutes or more to make a positive test, even though a sufficient amount of virus is used for diagnosis. Virus strains (hereinafter referred to as “positive-determination delayed strains”) were found in 2 strains, 5.2% of the 38 African swine fever virus strains used.
  • the complete base sequence information of African swine fever virus has 232 data registered in GenBank, a base sequence database. These base sequence information has been registered for many years based on base sequence information derived from African swine fever virus strains isolated mainly from Africa. In general, it is common knowledge that a mismatch is present in a 5 base region (hereinafter referred to as “reaction important region”) from the reaction site side of each primer used in the LAMP method, thereby inhibiting the reaction and reducing the amplification efficiency. Known as. The inventor verified the specificity of the primer of the LAMP method reported by James et al. Using the above-mentioned 232 African swine fever virus base sequence information. Confirmed that there was a mismatch. These mismatches are presumed to be the cause of the positive-delayed strains found in James et al.
  • an object of the present invention is to develop a detection method that can easily and quickly detect the presence or absence of African swine fever cholera and can cover the diversity of swine cholera viruses.
  • the inventor found a site that is highly conserved as a sequence in African swine fever virus and was useful for detection, and confirmed the usefulness of the primer set designed based on this, The invention has been completed. Furthermore, the inventor has confirmed that it is possible to detect African swine fever virus more sensitively and rapidly by performing detection by the LAMP method using a combination of primer sets, and has completed the invention. is there.
  • the present invention has the following configuration.
  • the first configuration of the present invention is an oligonucleotide primer set comprising the following base sequences (i) to (iv) for the purpose of detecting African swine fever virus.
  • Inner primer F (a) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 3)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 4) -3' Or
  • Outer primer F (c) SEQ ID NO: 2 or (d) SEQ ID NO: 12
  • Inner primer B (e) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 5)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 6) -3' Or (f)
  • the oligonucleotide primers (i), (ii), (iii), and (iv) are represented by the nucleotide sequences of SEQ ID NO: 8, SEQ ID NO: 2, SEQ ID NO: 9, and SEQ ID NO: 7, respectively.
  • the oligonucleotide primer set according to the first configuration characterized by comprising:
  • the oligonucleotide primers (i), (ii), (iii), and (iv) are represented by the nucleotide sequences of SEQ ID NO: 18, SEQ ID NO: 12, SEQ ID NO: 19, and SEQ ID NO: 17, respectively.
  • the oligonucleotide primer set according to the first configuration characterized by comprising:
  • a fourth configuration of the present invention is an oligonucleotide primer comprising the above SEQ ID NOs: 2 to 9 and 12 to 19, or a complementary base sequence thereof, and includes at least 15 consecutive bases identical to these base sequences
  • the oligonucleotide primer set according to the first configuration characterized by comprising oligonucleotide primers of a base sequence.
  • a fifth configuration of the present invention is characterized in that an amplification reaction of a target nucleic acid region of African swine fever virus is performed by using the oligonucleotide primer set described in the first to fourth configurations alone or in combination.
  • the sixth configuration of the present invention is the method for detecting African swine fever virus according to the fifth configuration, wherein the amplification reaction of the target nucleic acid region of African swine fever virus is the LAMP method.
  • the seventh configuration of the present invention is the primer set 1 of the oligonucleotide primer set described in the second configuration and the combination of the oligonucleotide primers of SEQ ID NO: 10 and SEQ ID NO: 11, and the oligonucleotide primer set described in the third configuration And the detection of African swine fever virus by combining the primer sets 1 and 2 with the oligonucleotide primers of SEQ ID NO: 20 and SEQ ID NO: 21 as primer set 2 Virus detection method.
  • a detection kit comprising the oligonucleotide primer set according to any one of the first to fourth aspects in a method for detecting African swine fever virus infection.
  • an oligonucleotide primer refers to a nucleic acid fragment having a plurality of base sequences, unless otherwise specified, and can be used for DNA synthesis by a DNA polymerase.
  • a primer set is not only a combination of a plurality of primers, but also a primer set including a plurality of such primer combinations.
  • the LAMP method (Loop-Mediated Isothermal Amplification) is a method of annealing the 3 'end of the template nucleotide itself as the starting point for complementary strand synthesis, and combining the primer that anneals with the loop formed at this time.
  • it is defined as a nucleic acid amplification method that enables an isothermal complementary strand synthesis reaction.
  • a detection method that can easily and quickly detect the presence or absence of African swine fever cholera and can cover the diversity of African swine fever virus. That is, by using the target nucleic acid region in the present invention, a primer useful for detection of African swine fever virus can be prepared. In addition, detection of African swine fever virus can be performed easily and quickly by detecting the LAMP method using the prepared primer.
  • the oligonucleotide primer etc. of this invention are demonstrated.
  • the present invention relates to nucleotide sequences 1503 to 2072 (hereinafter referred to as “target nucleic acid region”) of the nucleotide sequence of the African swine fever virus gene (GenBank accession No. AY578689) as a nucleotide sequence region useful for detection of African swine fever virus. ) Based on the discovery of the inventor. In addition, the inventor can perform high-sensitivity and rapid amplification using the oligonucleotide primer shown below (hereinafter simply referred to as “primer”) using the base sequence (SEQ ID NO: 22) imitating the target nucleic acid region as a positive control.
  • primer the oligonucleotide primer shown below (hereinafter simply referred to as “primer”) using the base sequence (SEQ ID NO: 22) imitating the target nucleic acid region as a positive control.
  • ASF-IPF-1 5'-CGTTTACACGCTTGTGGATCGTACGCATTCTTTGTGCCG-3 '(SEQ ID NO: 8)
  • ASF-OPF-1 5'-ACTCGCAGTAGTAAACCAA-3 '(SEQ ID NO: 2)
  • ASF-IPB-1 5'-TCCGTTGCGAGGAAACGTTTGATGCTCCGATTCAGGGC-3 '(SEQ ID NO: 9)
  • ASF-OPB-1 5'-TGTCATTCGTCCTGGCA-3 '(SEQ ID NO: 7)
  • ASF-LPF-1 5'-CTTTTGGGAGACCCATTGT-3 '(SEQ ID NO: 10)
  • Primer set 2 ASF-IPF-2: 5'-TTGAGTCAAATCGAAGAAACACAT
  • the target nucleic acid region was found as a highly conserved portion in 232 African swine fever viruses, and is a nucleic acid region that can cover and amplify the diversity of African swine fever virus.
  • the primer in the present invention is not particularly limited as long as such a target nucleic acid region can be designed as an amplification target, and can be designed as a primer for use in a nucleic acid amplification method such as a PCR method or a LAMP method.
  • the method for detecting African swine fever virus of the present invention is not particularly limited as long as the target nucleic acid region can be amplified, and various detection methods using nucleic acid amplification can be used. Typically, PCR methods and LAMPs are used. Can be used. Hereinafter, the LAMP method, which is a particularly suitable nucleic acid amplification method in the present invention, will be described as an example.
  • LAMP is a new nucleic acid amplification method developed by Natomi et al. That does not require temperature control, which is essential for PCR, and is a loop-mediated isothermal amplification method (Patent Publication WO 00/28082). This is a method referred to as an abbreviation for “No.
  • the LAMP method anneals its 3 'end to the template nucleotide to serve as a starting point for complementary strand synthesis, and by combining the primer that anneals with the loop formed at this time, isothermal complementary strand synthesis reaction is performed. Make it possible.
  • the 3 'end of the primer is always annealed to the region derived from the sample, and the check mechanism based on complementary binding of the base sequence functions repeatedly, resulting in high sensitivity and specificity.
  • a highly nucleic acid amplification reaction is possible.
  • primers corresponding to a total of 6 regions of the base sequence of the template nucleic acid are required for nucleic acid amplification. That is, the LAMP method requires at least four types of primers that recognize the F3c, F2c, and F1c regions from the 3 ′ end, the B3, B2, and B1 regions from the 5 ′ end, and these six base sequences. These primers are called inner primer F and B and outer primer F and B, respectively.
  • base sequences complementary to F1c, F2c and F3c are called F1, F2 and F3.
  • base sequences complementary to B1, B2, and B3 are called B1c, B2c, and B3c.
  • F in each primer is a primer display that binds complementarily to the sense strand of the target base sequence and provides a starting point for synthesis.
  • B is a primer display that complementarily binds to the antisense strand of the target base sequence and provides a starting point for synthesis.
  • An inner primer is a nucleic acid synthesis reaction product that recognizes a “specific nucleotide sequence region” on a target base sequence and has a base sequence that gives a synthesis starting point at the 3 ′ end, and at the same time starts from this primer.
  • primers containing “a base sequence selected from F2” and “a base sequence selected from F1c” are used as inner primer F (hereinafter “IPF”), and “a base sequence selected from B2” and “from B1c A primer containing the “selected base sequence” is referred to as an inner primer B (hereinafter “IPB”).
  • the base sequence may be the F2 region (or B2 region) or F1c region (or B1c region) from the 3 ′ end.
  • a non-specific base sequence region (linker) not involved in amplification may be present between the F2 region and the F1c region, and this non-specific region is included. It may be an array.
  • the sequence length of the non-specific region may be a sequence length that does not affect amplification, and typically 50 sequences or less.
  • an outer primer is an oligonucleotide having a base sequence that recognizes "a specific nucleotide sequence region existing on the 3 'end side of a" specific nucleotide sequence region "on the target base sequence and provides a starting point for synthesis. It is.
  • a primer containing “a base sequence selected from F3” is designated as outer primer F (hereinafter “OPF”)
  • a primer containing “a base sequence selected from B3” is designated as outer primer B (hereinafter “OPB”).
  • a loop primer in addition to the inner primer and the outer primer, another primer, that is, a loop primer can be used.
  • the loop primer is a primer having a base sequence complementary to the base sequence of the single-stranded part of the loop structure on the 5 ′ end side of the dumbbell structure.
  • the base sequence of the loop primer may be selected from the base sequence of the target gene or its complementary strand as long as it is complementary to the base sequence of the single-stranded portion of the loop structure on the 5 ′ end side of the dumbbell structure described above. Other base sequences may be used. Further, one or two types of loop primers may be used.
  • the target nucleic acid region is used as an amplification target, the F3c, F2c, F1c base sequence region from the 3 ′ end side, the B3, B2, B1 base sequence region from the 5 ′ end side, and the respective base sequence regions.
  • F3, F2, and F1 base sequence regions, B3c, B2c, and B1c base sequence regions that are complementary to these selected base sequence regions, and their respective base sequence regions are selected.
  • the following oligonucleotide primers are prepared.
  • a base sequence having an F3 region A base sequence having a B2 region, a non-specific base sequence region, and a B1c region from the 3 ′ end side.
  • IV A base sequence having a B3 region.
  • the length of the base sequence prepared as a primer can be adjusted as appropriate in consideration of the sensitivity and specificity for each region or the stability as a primer, but it falls under (II) to (IV) above.
  • the oligonucleotide primer to be prepared is preferably 10 bases or more, more preferably 15 bases or more.
  • the oligonucleotide primer prepared according to the above (I) to (III) is preferably 20 bases or more, more preferably 40 bases or more.
  • the base sequence of (I) corresponds to IPF
  • the base sequence of (II) corresponds to OPF
  • the base sequence of (III) corresponds to IPB
  • the base sequence of (IV) corresponds to OPB.
  • the oligonucleotide primer set of the present invention comprises the following primers (i) to (iv) for the purpose of detecting African swine fever virus.
  • Inner primer F (a) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 3)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 4) -3' Or (b) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 13)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 14) -3' (ii) Outer primer F (c) SEQ ID NO: 2 or (d) SEQ ID NO: 12 (iii) Inner primer B (e) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 5)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 6) -3' Or (f) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 15)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 16) -3' (iv) Outer primer B (g) S
  • the sequences (a) and (b) which are IPFs are designed by combining the sequence corresponding to F2 and the sequence corresponding to F1c from the 3 'side. It is also known as technical common knowledge that a non-specific base sequence (linker) that does not participate in amplification may be present between the base sequence corresponding to F2 and the base sequence corresponding to F1c.
  • the length of the linker need not be particularly limited, but can typically be 50 or less.
  • IPB sequences (e) to (f) are designed by combining the sequence corresponding to B2 and the sequence corresponding to B1c from the 3 'side.
  • IPF it is known as technical common knowledge that IPB may have a linker between the base sequence corresponding to B2 and the base sequence corresponding to B1c.
  • the number of bases can be 50 or less.
  • the primers (i), (ii), (iii), and (iv) are SEQ ID NO: 8, SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 7, or SEQ ID NO: 18, SEQ ID NO: 12, respectively.
  • SEQ ID NO: 19 and SEQ ID NO: 17 can be designed as the nucleotide sequence.
  • the primers (i), (ii), (iii), and (iv) may be designed as complementary nucleotide sequences to these nucleotide sequences.
  • the primers (i), (ii), (iii), and (iv) can amplify the target nucleic acid region even when they contain a continuous base sequence of these base sequences or complementary base sequences. It can be designed as an oligonucleotide primer containing at least 15 consecutive bases.
  • IPF, OPF, IPB, and OPB can be used as one set of oligonucleotide primers for use in the LAMP method, and in addition to these, loop primers may be used in combination.
  • loop primer an oligonucleotide primer having a base sequence described in SEQ ID NO: 10 or 20 as LPF and SEQ ID NO: 11 or 21 as LPB can be used.
  • a nucleic acid amplification operation can be performed using a single primer set, or a plurality of nucleic acid amplification operations can be performed in combination.
  • nucleic acid amplification operation by combining multiple primer sets, the diversity of African swine fever virus can be covered, and the detection sensitivity, specificity, and speed can be improved. Examples of these combinations include the detection of African swine fever virus by combining primer set 1 and primer set 2 described above.
  • Samples are required for detection of African swine fever virus by the LAMP method.
  • any specimen specimen that is usually used can be used. Examples thereof include viruses, cultured cells containing viruses, and specimens derived from animals such as culture fluids, pigs, rabbits, and lobsters.
  • an animal-derived specimen as the specimen sample.
  • blood, serum, body fluid, tissue extract, or tissue wiping solution and tissue wiping specimen can be used as the specimen sample. This has the effect of enabling rapid detection of African swine fever virus.
  • Specimen samples for detecting African swine fever virus can be used as samples for use in the LAMP method described below after pretreatment that is normally used.
  • samples for use in the LAMP method described below after degrading tissue cell-derived protein with proteolytic enzymes, etc., nucleic acid extraction and purification using phenol and chloroform, or using a commercially available extraction kit (for example, RNeasy Mini Mini Kit from Qiagen)
  • nucleic acid is extracted and used as a specimen.
  • the specimen sample When the specimen sample is ready, perform the procedure for nucleic acid amplification using the primer set described above.
  • the primer set described above As an operation for this nucleic acid amplification, for example, at least 4 types of primers (IPF, IPB, OPF, OPB), template-dependent nucleic acid synthase, deoxynucleotide triphosphate, etc. in the reaction buffer are used in 60 to 65. Incubate at 15 ° C for 15 minutes to 1 hour. Further, as described above, a loop primer can be further used.
  • the enzyme used in nucleic acid synthesis is not particularly limited as long as it is a template-dependent nucleic acid synthase having strand displacement activity.
  • Such enzymes include Bst DNA polymerase (large fragment), Bca (exo-) DNA polymerase, Csa DNA polymerase, Gsp DNA polymerase (large fragment), GspSSD DNA polymerase (large fragment), Tin DNA polymerase (large fragment) , Klenow fragment of E. coli DNA polymerase I and the like, preferably Bst DNA polymerase (large fragment).
  • the nucleic acid amplification product is detected.
  • Any commonly used detection method can be used to detect the nucleic acid amplification product. For example, detection using a labeled oligonucleotide that specifically recognizes the amplified base sequence, detection by a fluorescent intercalator method, agarose gel electrophoresis, and the like. In the agarose gel electrophoresis method, a large number of bands with different base lengths are detected in a ladder form in the LAMP amplification product. In addition, the LAMP method may allow visual detection, which is preferable when rapid detection of African swine fever virus is required.
  • a large amount of substrate is consumed by nucleic acid synthesis, and pyrophosphate, a by-product, reacts with coexisting magnesium to become magnesium pyrophosphate, and the reaction solution becomes cloudy to the extent that it can be visually confirmed.
  • Detection of swine cholera virus is possible.
  • reagents necessary for detection of nucleic acid amplification using the primer of the present invention can be packaged in advance to form a kit.
  • various oligonucleotides necessary as a primer or loop primer of the present invention, four types of dNTPs serving as a substrate for nucleic acid synthesis, a template-dependent nucleic acid synthase having a strand displacement activity, and conditions suitable for enzymatic reaction Provided buffers and salts, protecting agents for stabilizing enzymes and templates, and reagents necessary for detection of reaction products as necessary are provided as kits.
  • reaction reagents were prepared so that each reagent concentration in 25 ⁇ L of the final reaction solution was as follows.
  • ⁇ LAMP reaction solution composition 15.0 ⁇ L Isothermal Master Mix for GenieIII (manufactured by Optigene) 5.0 ⁇ L purified water 1.6 ⁇ M IPF (SEQ ID NO: 8 or SEQ ID NO: 18) 1.6 ⁇ M IPB (SEQ ID NO: 9 or 19) 0.2 ⁇ M OPF (SEQ ID NO: 2 or SEQ ID NO: 12) 0.2 ⁇ M OPB (SEQ ID NO: 7 or SEQ ID NO: 17) 0.8 ⁇ M LPF (SEQ ID NO: 10 or SEQ ID NO: 20) 0.8 ⁇ M LPB (SEQ ID NO: 11 or SEQ ID NO: 21) 3.
  • reaction reagent 5.0 ⁇ L of each concentration of sample solution containing template DNA was added to make 25.0 ⁇ L of the final reaction solution, and each LAMP reaction was performed twice. 4).
  • the reaction was detected in real time using a real-time fluorescence analyzer GenieIII (manufactured by Optigene) at 65 ° C for 60 minutes in a dedicated tube.
  • the results of the reaction were visually confirmed in real time using a graph on the monitor built in Genie III, a real-time fluorescence measurement device. 5).
  • a specimen having a fluorescence ratio of amplification rate within a predetermined time exceeding 0.02 was determined to be positive.
  • PCR method 1.
  • the method disclosed in Non-Patent Document 4 was used as a reference. That is, a combination of PPA-1 (SEQ ID NO: 23) and PPA-2 (SEQ ID NO: 24) disclosed in Non-Patent Document 4 was used as a primer used in the PCR method.
  • reaction reagents were prepared so that the concentration of each reagent in 25 ⁇ L of the final reaction solution was as follows.
  • PCR reaction solution composition PCR buffer ⁇ 10 (Qiagen) 2.5 ⁇ L 2 mM dNTPs (Qiagen) 2 ⁇ L dDW (sterilized ultrapure water) 20.98 ⁇ L 100pmol / ⁇ L OPF (SEQ ID NO: 23) 0.16 ⁇ L 100pmol / ⁇ L OPB (SEQ ID NO: 24) 0.16 ⁇ L 5U / ⁇ L Taq Polymerase (Qiagen) 0.1 ⁇ L 3.
  • each sample solution containing the template DNA was added to make 25.0 ⁇ L of the final reaction solution, and each PCR reaction was performed twice.
  • the temperature cycle conditions for the PCR reaction were 95 ° C for 2 minutes, heat denaturation at 95 ° C for 15 seconds, annealing at 62 ° C for 30 seconds, and polymerase extension reaction at 72 ° C for 30 seconds. After 30 cycles, the reaction was terminated after standing at 72 ° C for 7 minutes. The time required was about 2 hours.
  • 3 ⁇ L of the reaction solution after completion of the reaction was electrophoresed on a 2% agarose gel and stained with ethidium bromide.
  • FIG. 1 to FIG. 3 show the experimental results when using the LAMP method.
  • the horizontal axis represents the time (minutes) during which the nucleic acid amplification reaction was performed
  • the vertical axis represents the fluorescence intensity ratio. The more the nucleic acid is amplified, the higher the fluorescence intensity ratio in the reaction solution. As a result, the target virus can be detected.
  • the larger the number of copies of the artificial base sequence in the reaction tube the easier the nucleic acid amplification reaction proceeds, and the faster the fluorescence intensity ratio can be detected. Conversely, the smaller the copy number, the more difficult the nucleic acid amplification reaction proceeds, so the increase in the fluorescence intensity ratio becomes slower and the detection takes longer.
  • FIG. 1 shows the results when primer set 1 was used. (1) The fluorescence intensity exceeded 0.02 in the reaction tubes of 1378.0 copies (well 1 and well 2) and 137.8 copies (well 3 and well 4). (2) In the 13.8 copies of the reaction tube, those exceeding 0.02 (well 6) and those not exceeding (well 5) were observed. (3) The 1.4 copy sample did not exceed 0.02, and no increase in fluorescence intensity was observed (well 7 and well 8). 3.
  • FIG. 2 shows the results when Primer Set 2 was used. The result was the same as in FIG. 4).
  • FIG. 3 shows the results when primer set 1 and primer set 2 are used in combination.
  • the LAMP method using the primer set of the present invention could be detected earlier than real-time PCR. 3.
  • detection using a combination of primer sets was generally capable of faster detection than detection alone.
  • Table 3 shows a simple comparison between the experimental results in the present application and the experimental results disclosed in Prior Art 3. 2.
  • the positive average time in real-time PCR is 25.35 cycles for the present application and 21.42 cycles for the prior art. In consideration of normal real-time PCR, this application is about 4 cycles higher. It was thought that there were generally few. 3. However, in the results using the primer set, the average positive time required for the present application was 19 to 22 minutes shorter than the prior art. 4). From this comparison, it was considered that the technology of the present application could be detected more quickly than the prior art.
  • Table 4 to Table 6 show the results of examining mismatches with the target sequences in the primer set of the present invention and the primer set in Prior Art Document 3.
  • the base in which the underline of the sequence in each table is written is the target mismatch site. 6).
  • primer set 1 although there were a total of 10 mismatches, there was no mismatch in the reaction critical region. 7).
  • primer set 2 the total number of mismatches was 11, and there were 3 mismatches in the reaction critical region, but the sequence was found only in one sequence of LPB. 8).
  • the mismatch in this reaction critical region extended to three sequences, F2, LF, and LB. 9. From these, the primer set of the present invention has no or few mismatches in the reaction important region as compared with the prior art, and this point is considered to be related to the above-mentioned rapid detection time. It was.

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Abstract

[Problem] To develop a method for detecting the presence/absence of African swine fever virus infection easily and rapidly. [Solution] The present invention provides: an oligonucleotide primer designed on the basis of an optional base sequence, or a base sequence complimentary thereto, the optional base sequence being selected from base sequence Nos. 1503 to 2072 (hereinafter "target nucleic acid") of the base sequence of African swine cholera virus genome (GenBank accession No. AY578689) depicted in SEQ ID NO: 1; and a method for amplifying nucleic acids, such as the LAMP method in which such oligonucleotide primers are used.

Description

複数のプライマーセットを組み合わせたＬＡＭＰ法を用いたアフリカ豚コレラウイルスの迅速検出法Rapid detection method of African swine fever virus using LAMP method combining multiple primer sets
　本発明は，アフリカ豚コレラウイルスの検出に有用なオリゴヌクレオチドプライマー等に関する。さらに詳しくいうと本発明は，簡易・高感度かつ迅速な遺伝子検出法であるLAMP法に用いられるオリゴヌクレオチドプライマー，これを組み合わせたプライマーセット，ならびにプライマーセットを用いたアフリカ豚コレラの迅速検出法に関する。 The present invention relates to an oligonucleotide primer useful for detection of African swine fever virus. More specifically, the present invention relates to oligonucleotide primers used in the LAMP method, which is a simple, highly sensitive and rapid gene detection method, a primer set combining these, and a rapid detection method for African swine fever using the primer set. .
　アフリカ豚コレラウイルス（African swine fever virus）は，アフリカ豚コレラの原因ウイルスで，アスファウイルス科（Asfaviridae）に分類される大型の２本鎖DNAウイルスである。
　アフリカ豚コレラは，豚に高い伝播性を持つとともに，罹患した豚に，発熱，食欲不振，結膜炎，リンパ節腫脹，呼吸障害，便秘，下痢，後躯麻痺や運動失調，起立困難などを主とした症状を引き起こし，最大で100％の致死率を示す。 African swine fever virus (African swine fever virus) is a causative virus of African swine fever virus, and is a large double-stranded DNA virus classified as Asfairidae.
African swine fever is highly transmissible to pigs, and the affected pigs mainly suffer from fever, loss of appetite, conjunctivitis, lymphadenopathy, respiratory distress, constipation, diarrhea, postparalysis, ataxia, and difficulty standing. Cause up to 100% mortality.
　アフリカ豚コレラは，ウイルスの病原性の強さにより，甚急性，急性，亜急性，慢性および不顕性，これら５種類に分類される。
　日本をはじめとするアフリカ豚コレラ清浄国において感染が確認された場合，他の家畜への感染拡大を防ぐために，罹患した患畜は，発見され次第，殺処分される。この殺処分措置による経済的被害は甚大なものとなることから，日本においてアフリカ豚コレラは，家畜伝染病予防法により法定伝染病に指定されている。
　これらの事情から，アフリカ豚コレラの防疫，および海外からの侵入時の迅速診断ならびに摘発淘汰は，我が国においても最重要課題の一つとなっている。 African swine fever is classified into these five types depending on the strength of the pathogenicity of the virus: hyperacute, acute, subacute, chronic and inapparent.
When infection is confirmed in Japan and other African swine fever cholera countries, affected animals are slaughtered as soon as they are found to prevent the spread of infection to other livestock. Since the economic damage caused by this slaughtering procedure is enormous, African swine fever is designated as a legally contagious disease by the Livestock Infectious Disease Prevention Law in Japan.
Under these circumstances, prevention of epidemics of African swine cholera and rapid diagnosis and detection of intrusions from overseas are among the most important issues in Japan.
　アフリカ豚コレラの被害拡大を抑制するためには，初発例を確実に診断するとともに，臨床症状を示す前の感染初期の個体を，迅速に診断し摘発淘汰することが極めて重要である。
　アフリカ豚コレラの診断は，感染個体の血液等の材料から，微生物学的試験法であるPCR法，抗体測定，培養細胞を用いたウイルス分離などにより行われている。
　これらの診断手法のうち，抗体測定では，感染初期の個体を摘発することは原理的に困難である。また，培養細胞を用いたウイルス分離は，培養のための日数を必要とするため，迅速診断という観点からは有用な診断方法とはいえない。
　これらと比較してPCR法は，３つの検査法の中では最も迅速に結果を得ることが出来るが，それでも検体入手から，少なくとも約３時間を必要とすることが問題となっている。 In order to control the spread of damage caused by African swine fever, it is extremely important to diagnose the first case and to quickly diagnose and detect the early stage of infection before clinical manifestations.
Diagnosis of African swine fever is performed from materials such as blood of infected individuals by PCR, which is a microbiological test, antibody measurement, and virus isolation using cultured cells.
Of these diagnostic methods, it is in principle difficult to detect individuals in the early stages of infection in antibody measurement. Moreover, virus isolation using cultured cells is not a useful diagnostic method from the viewpoint of rapid diagnosis because it requires days for culture.
Compared with these, the PCR method can obtain the result most quickly among the three test methods, but it still has a problem that it requires at least about 3 hours from obtaining the sample.
　一方，LAMP法はPCR法よりも簡易迅速な検査法であり，口蹄疫等の様々な病原体を検出するプライマーセットが多数論文報告されている（非特許文献１，２）。また，アフリカ豚コレラウイルスを検出するためのLAMP法は，英国の１グループ（Jamesら）によって開発されている（非特許文献３）。 On the other hand, the LAMP method is a simpler and quicker test method than the PCR method, and many primer sets for detecting various pathogens such as foot-and-mouth disease have been reported (Non-Patent Documents 1 and 2). In addition, the LAMP method for detecting African swine fever virus has been developed by a group in the UK (James et al.) (Non-patent Document 3).
　非特許文献３の報告によると，主にアフリカで分離されたアフリカ豚コレラウイルス株では，診断を行うのに充分なウイルス量を使用しているにも関わらず，陽性判定に40分以上を要するウイルス株（以下，「陽性判定遅延株」という。）が，使用したアフリカ豚コレラウイルス38株中5.2%にあたる2株で認められた。 According to the report of Non-Patent Document 3, in the case of African swine fever virus strains isolated mainly in Africa, it takes 40 minutes or more to make a positive test, even though a sufficient amount of virus is used for diagnosis. Virus strains (hereinafter referred to as “positive-determination delayed strains”) were found in 2 strains, 5.2% of the 38 African swine fever virus strains used.
　アフリカ豚コレラウイルスの全塩基配列情報は，塩基配列データベースであるGenBankに232データが登録されている。これらの塩基配列情報は，長年に亘って，主にアフリカから分離されたアフリカ豚コレラウイルス株に由来した塩基配列情報に基づき登録されている。
　一般的に，LAMP法で使用する各プライマーの反応拠点側から５塩基の領域（以下，「反応重要領域」という。）にミスマッチが存在すると反応が阻害され，増幅効率が低下することが技術常識として知られている。
　発明者は，前述の232のアフリカ豚コレラウイルスの塩基配列情報を用いて，Jamesらの報告しているLAMP法のプライマーの特異性を検証したところ，反応重要領域ならびにそれ以外の領域に多くのミスマッチがあることを確認した。これらのミスマッチがJamesらの報告において陽性判定遅延株が認められた原因と推測される。 The complete base sequence information of African swine fever virus has 232 data registered in GenBank, a base sequence database. These base sequence information has been registered for many years based on base sequence information derived from African swine fever virus strains isolated mainly from Africa.
In general, it is common knowledge that a mismatch is present in a 5 base region (hereinafter referred to as “reaction important region”) from the reaction site side of each primer used in the LAMP method, thereby inhibiting the reaction and reducing the amplification efficiency. Known as.
The inventor verified the specificity of the primer of the LAMP method reported by James et al. Using the above-mentioned 232 African swine fever virus base sequence information. Confirmed that there was a mismatch. These mismatches are presumed to be the cause of the positive-delayed strains found in James et al.
　上記事情を背景として本発明では，アフリカ豚コレラの感染有無を簡易かつ迅速に検出しうるとともに，アフリカ豚コレラウイルスの多様性をカバーしうる検出方法の開発を課題とする。 In view of the above circumstances, an object of the present invention is to develop a detection method that can easily and quickly detect the presence or absence of African swine fever cholera and can cover the diversity of swine cholera viruses.
　発明者は，鋭意研究の結果，アフリカ豚コレラウイルスにおいて配列としての保存性が高く，検出に有用な部位を発見するとともに，これを基に設計されたプライマーセットの有用性を確認し，プライマーに関する発明を完成させたものである。
　さらに発明者は，プライマーセットを組み合わせて使用したLAMP法による検出を行うことにより，アフリカ豚コレラウイルスの，より高感度かつ迅速な検出が可能であることを確認し，発明を完成させたものである。 As a result of earnest research, the inventor found a site that is highly conserved as a sequence in African swine fever virus and was useful for detection, and confirmed the usefulness of the primer set designed based on this, The invention has been completed.
Furthermore, the inventor has confirmed that it is possible to detect African swine fever virus more sensitively and rapidly by performing detection by the LAMP method using a combination of primer sets, and has completed the invention. is there.
　本発明は，以下の構成からなる。
　本発明の第一の構成は，アフリカ豚コレラウイルス検出を目的とした下記(i)から(iv)の塩基配列からなることを特徴とするオリゴヌクレオチドプライマーセットである。
(i)　インナープライマーF
　(a)　5’－（配列番号３の塩基配列に相補的な塩基配列）－（塩基数０～５０の任意の塩基配列）－（配列番号４の塩基配列）－3’
又は，
　(b)　5’－（配列番号１３の塩基配列に相補的な塩基配列）－（塩基数０～５０の任意の塩基配列）－（配列番号１４の塩基配列）－3’
(ii)　アウタープライマーF
　(c)　配列番号２，又は，(d)　配列番号１２
(iii)　インナープライマーB
　(e)　5’－（配列番号５の塩基配列に相補的な塩基配列）－（塩基数０～５０の任意の塩基配列）－（配列番号６の塩基配列）－3’
又は，
　(f)　5’－（配列番号１５の塩基配列に相補的な塩基配列）－（塩基数０～５０の任意の塩基配列）－（配列番号１６の塩基配列）－3’
(iv)　アウタープライマーB
　(g)　配列番号７，又は，(h)　配列番号１７ The present invention has the following configuration.
The first configuration of the present invention is an oligonucleotide primer set comprising the following base sequences (i) to (iv) for the purpose of detecting African swine fever virus.
(i) Inner primer F
(a) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 3)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 4) -3'
Or
(b) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 13)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 14) -3'
(ii) Outer primer F
(c) SEQ ID NO: 2 or (d) SEQ ID NO: 12
(iii) Inner primer B
(e) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 5)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 6) -3'
Or
(f) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 15)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 16) -3'
(iv) Outer primer B
(g) SEQ ID NO: 7 or (h) SEQ ID NO: 17
　本発明の第二の構成は，前記(i)，(ii)，(iii)，(iv)のオリゴヌクレオチドプライマーが，それぞれ配列番号８，配列番号２，配列番号９，配列番号７の塩基配列からなることを特徴とする第一の構成に記載のオリゴヌクレオチドプライマーセットである。
　本発明の第三の構成は，前記(i)，(ii)，(iii)，(iv)のオリゴヌクレオチドプライマーが，それぞれ配列番号１８，配列番号１２，配列番号１９，配列番号１７の塩基配列からなることを特徴とする第一の構成に記載のオリゴヌクレオチドプライマーセットである。
　本発明の第四の構成は，前記配列番号２から９および１２から１９，もしくはこれらと相補的な塩基配列からなるオリゴヌクレオチドプライマーであって，これら塩基配列と同一の少なくとも連続した15塩基を含む塩基配列のオリゴヌクレオチドプライマーからなることを特徴とする第一の構成に記載のオリゴヌクレオチドプライマーセットである。 In the second configuration of the present invention, the oligonucleotide primers (i), (ii), (iii), and (iv) are represented by the nucleotide sequences of SEQ ID NO: 8, SEQ ID NO: 2, SEQ ID NO: 9, and SEQ ID NO: 7, respectively. The oligonucleotide primer set according to the first configuration, characterized by comprising:
In the third configuration of the present invention, the oligonucleotide primers (i), (ii), (iii), and (iv) are represented by the nucleotide sequences of SEQ ID NO: 18, SEQ ID NO: 12, SEQ ID NO: 19, and SEQ ID NO: 17, respectively. The oligonucleotide primer set according to the first configuration, characterized by comprising:
A fourth configuration of the present invention is an oligonucleotide primer comprising the above SEQ ID NOs: 2 to 9 and 12 to 19, or a complementary base sequence thereof, and includes at least 15 consecutive bases identical to these base sequences The oligonucleotide primer set according to the first configuration, characterized by comprising oligonucleotide primers of a base sequence.
　本発明の第五の構成は，第一から第四の構成に記載のオリゴヌクレオチドプライマーセットを，単独もしくは組み合わせて用いることにより，アフリカ豚コレラウイルスの標的核酸領域の増幅反応を行うことを特徴とするアフリカ豚コレラウイルスの検出方法である。
　本発明の第六の構成は，アフリカ豚コレラウイルスの標的核酸領域の増幅反応が，LAMP法であることを特徴とする第五の構成に記載のアフリカ豚コレラウイルスの検出方法である。
　本発明の第七の構成は，第二の構成に記載のオリゴヌクレオチドプライマーセットおよび配列番号１０，配列番号１１のオリゴヌクレオチドプライマーの組み合わせをプライマーセット１，第三の構成に記載のオリゴヌクレオチドプライマーセットおよび配列番号２０，配列番号２１のオリゴヌクレオチドプライマーをプライマーセット２として，これらプライマーセット１と２を組み合わせてアフリカ豚コレラウイルスの検出を行うことを特徴とする第六の構成に記載のアフリカ豚コレラウイルスの検出方法である。
　本発明の第八の構成は，アフリカ豚コレラウイルス感染症の検出方法において，第一から第四の構成に記載のオリゴヌクレオチドプライマーセットを含むことを特徴とする検出キットである。 A fifth configuration of the present invention is characterized in that an amplification reaction of a target nucleic acid region of African swine fever virus is performed by using the oligonucleotide primer set described in the first to fourth configurations alone or in combination. A method for detecting African swine fever virus.
The sixth configuration of the present invention is the method for detecting African swine fever virus according to the fifth configuration, wherein the amplification reaction of the target nucleic acid region of African swine fever virus is the LAMP method.
The seventh configuration of the present invention is the primer set 1 of the oligonucleotide primer set described in the second configuration and the combination of the oligonucleotide primers of SEQ ID NO: 10 and SEQ ID NO: 11, and the oligonucleotide primer set described in the third configuration And the detection of African swine fever virus by combining the primer sets 1 and 2 with the oligonucleotide primers of SEQ ID NO: 20 and SEQ ID NO: 21 as primer set 2 Virus detection method.
According to an eighth aspect of the present invention, there is provided a detection kit comprising the oligonucleotide primer set according to any one of the first to fourth aspects in a method for detecting African swine fever virus infection.
　本発明において，オリゴヌクレオチドプライマー（もしくは，プライマー）とは，特に限定しない限り，複数の塩基配列を有する核酸断片をいい，DNAポリメラーゼによるDNA合成の際，用いることができるものをいう。
　また，プライマーセットとは，複数個のプライマーの組合せのみならず，そのようなプライマーの組合せを複数個含むものも合わせてプライマーセットというものとする。
　さらに，LAMP法（Loop-Mediated Isothermal Amplification）とは，鋳型となるヌクレオチドに自身の3’末端をアニールさせて相補鎖合成の起点とするとともに，このとき形成されるループにアニールするプライマーを組み合わせることにより，等温での相補鎖合成反応を可能とした核酸増幅法として定義される。 In the present invention, an oligonucleotide primer (or primer) refers to a nucleic acid fragment having a plurality of base sequences, unless otherwise specified, and can be used for DNA synthesis by a DNA polymerase.
A primer set is not only a combination of a plurality of primers, but also a primer set including a plurality of such primer combinations.
Furthermore, the LAMP method (Loop-Mediated Isothermal Amplification) is a method of annealing the 3 'end of the template nucleotide itself as the starting point for complementary strand synthesis, and combining the primer that anneals with the loop formed at this time. Thus, it is defined as a nucleic acid amplification method that enables an isothermal complementary strand synthesis reaction.
　本発明により，アフリカ豚コレラの感染有無を簡易かつ迅速に検出しうるとともに，アフリカ豚コレラウイルスの多様性をカバーしうる検出方法の提供が可能となった。
　すなわち，本発明における標的核酸領域を用いることにより，アフリカ豚コレラウイルスの検出に有用なプライマーを作製することが可能となる。加えて，作製されたプライマーを用いてLAMP法による検出を行うことにより，簡易かつ迅速に，アフリカ豚コレラウイルスの検出を可能とするものである。 According to the present invention, it has become possible to provide a detection method that can easily and quickly detect the presence or absence of African swine fever cholera and can cover the diversity of African swine fever virus.
That is, by using the target nucleic acid region in the present invention, a primer useful for detection of African swine fever virus can be prepared. In addition, detection of African swine fever virus can be performed easily and quickly by detecting the LAMP method using the prepared primer.
LAMP法にて，プライマーセット１を用いた場合の蛍光強度の変化を示した図Diagram showing changes in fluorescence intensity when primer set 1 is used in the LAMP method LAMP法にて，プライマーセット２を用いた場合の蛍光強度の変化を示した図Diagram showing changes in fluorescence intensity when primer set 2 is used in the LAMP method LAMP法にて，プライマーセット１ならびにプライマーセット２を組み合わせて用いた場合の蛍光強度の変化を示した図Diagram showing changes in fluorescence intensity when primer set 1 and primer set 2 are used in combination with the LAMP method
　本発明のオリゴヌクレオチドプライマー等について，説明を行う。
　本発明は，アフリカ豚コレラウイルスの検出に有用な塩基配列領域として，アフリカ豚コレラウイルス遺伝子の塩基配列（GenBank accession No.AY578689）の1503番～2072番の塩基配列（以下，「標的核酸領域」）を発明者が発見したことに基づくものである。また，発明者は，かかる標的核酸領域に模した塩基配列（配列番号２２）を陽性対照として，下記に示すオリゴヌクレオチドプライマー（以下，単に「プライマー」という）により，高感度かつ迅速な増幅が可能であることを確認し，アフリカ豚コレラウイルスの検出に有用なプライマーおよび検出方法等にかかる発明を完成させたものである。
(1)　プライマーセット１
ASF-IPF-1:5'-CGTTTACACGCTTGTGGATCGTACGCATTCTTTGTGCCG-3' （配列番号8)
ASF-OPF-1:5'-ACTCGCAGTAGTAAACCAA-3' （配列番号2）
ASF-IPB-1:5'-TCCGTTGCGAGGAAACGTTTGATGCTCCGATTCAGGGC-3' （配列番号9）
ASF-OPB-1:5'-TGTCATTCGTCCTGGCA-3' （配列番号７）
ASF-LPF-1:5'-CTTTTGGGAGACCCATTGT-3' （配列番号１０）
ASF-LPB-1:5'-CCCCCATCTGGGATGA-3' （配列番号１１）
(2)　プライマーセット２
ASF-IPF-2:5'-TTGAGTCAAATCGAAGAAACACATACACTTTATTGTATTCAAACCCTA-3' （配列番号18）
ASF-OPF-2:5'-GGAAAAAGTCTCCGTACTG-3' （配列番号１２）
ASF-IPB-2:5'-GCAAGTCTTGGGCCAAGATACTTTTTGTCTTATTGCTAACGATGG-3' （配列番号19）
ASF-OPB-2:5'-ATATGGCATCAGGAGGAG-3' （配列番号１７）
ASF-LPF-2:5'-GCATTTTAATGCACATTTTAAGCCTT-3' （配列番号２０）
ASF-LPB-2:5'-AATCTTGTCGGCCTTCC-3' （配列番号２１）
　なお，詳細は後述するが，IPFはインナープライマーF，OPFはアウタープライマーF，IPBがインナープライマーB，OPBがアウタープライマーB，LPFがループプライマーF，LPBがループプライマーBの略である。 The oligonucleotide primer etc. of this invention are demonstrated.
The present invention relates to nucleotide sequences 1503 to 2072 (hereinafter referred to as “target nucleic acid region”) of the nucleotide sequence of the African swine fever virus gene (GenBank accession No. AY578689) as a nucleotide sequence region useful for detection of African swine fever virus. ) Based on the discovery of the inventor. In addition, the inventor can perform high-sensitivity and rapid amplification using the oligonucleotide primer shown below (hereinafter simply referred to as “primer”) using the base sequence (SEQ ID NO: 22) imitating the target nucleic acid region as a positive control. It was confirmed that the present invention was completed for primers and detection methods useful for detection of African swine fever virus.
(1) Primer set 1
ASF-IPF-1: 5'-CGTTTACACGCTTGTGGATCGTACGCATTCTTTGTGCCG-3 '(SEQ ID NO: 8)
ASF-OPF-1: 5'-ACTCGCAGTAGTAAACCAA-3 '(SEQ ID NO: 2)
ASF-IPB-1: 5'-TCCGTTGCGAGGAAACGTTTGATGCTCCGATTCAGGGC-3 '(SEQ ID NO: 9)
ASF-OPB-1: 5'-TGTCATTCGTCCTGGCA-3 '(SEQ ID NO: 7)
ASF-LPF-1: 5'-CTTTTGGGAGACCCATTGT-3 '(SEQ ID NO: 10)
ASF-LPB-1: 5'-CCCCCATCTGGGATGA-3 '(SEQ ID NO: 11)
(2) Primer set 2
ASF-IPF-2: 5'-TTGAGTCAAATCGAAGAAACACATACACTTTATTGTATTCAAACCCTA-3 '(SEQ ID NO: 18)
ASF-OPF-2: 5'-GGAAAAAGTCTCCGTACTG-3 '(SEQ ID NO: 12)
ASF-IPB-2: 5'-GCAAGTCTTGGGCCAAGATACTTTTTGTCTTATTGCTAACGATGG-3 '(SEQ ID NO: 19)
ASF-OPB-2: 5'-ATATGGCATCAGGAGGAG-3 '(SEQ ID NO: 17)
ASF-LPF-2: 5'-GCATTTTAATGCACATTTTAAGCCTT-3 '(SEQ ID NO: 20)
ASF-LPB-2: 5'-AATCTTGTCGGCCTTCC-3 '(SEQ ID NO: 21)
Although details will be described later, IPF stands for inner primer F, OPF stands for outer primer F, IPB stands for inner primer B, OPB stands for outer primer B, LPF stands for loop primer F, and LPB stands for loop primer B.
　標的核酸領域は，232種のアフリカ豚コレラウイルスにおける保存性が高い部分として見出されたものであり，アフリカ豚コレラウイルスの多様性をカバーし増幅しうる核酸領域である。
　本発明におけるプライマーは，かかる標的核酸領域を増幅対象として設計しうる限り特に限定する必要はなく，PCR法やLAMP法などの核酸増幅方法に用いる際のプライマーとして設計することができる。 The target nucleic acid region was found as a highly conserved portion in 232 African swine fever viruses, and is a nucleic acid region that can cover and amplify the diversity of African swine fever virus.
The primer in the present invention is not particularly limited as long as such a target nucleic acid region can be designed as an amplification target, and can be designed as a primer for use in a nucleic acid amplification method such as a PCR method or a LAMP method.
　また，本発明のアフリカ豚コレラウイルス検出方法は，標的核酸領域を増幅しうる限り特に限定する必要はなく，種々の核酸増幅による検出方法を用いることができ，典型的には，PCR法やLAMP法を用いることができる。
　以下では，本発明において特に好適な核酸増幅方法であるLAMP法を例にとり説明を行う。 The method for detecting African swine fever virus of the present invention is not particularly limited as long as the target nucleic acid region can be amplified, and various detection methods using nucleic acid amplification can be used. Typically, PCR methods and LAMPs are used. Can be used.
Hereinafter, the LAMP method, which is a particularly suitable nucleic acid amplification method in the present invention, will be described as an example.
　LAMP法は，納富らが開発した，PCR法で不可欠とされる温度制御が不要な新しい核酸増幅法であり，ループ媒介等温増幅法（Loop-mediated isothermal amplification method，特許公報国際公開第00/28082号パンフレット）の略称として称される方法である。
　LAMP法は，鋳型となるヌクレオチドに自身の3’末端をアニールさせて相補鎖合成の起点とするとともに，このとき形成されるループにアニールするプライマーを組み合わせることにより，等温での相補鎖合成反応を可能とする。また，LAMP法では，プライマーの3’末端が，常に試料に由来する領域に対してアニールしており，塩基配列の相補的結合によるチェック機構が繰り返し機能することとなり，結果として，高感度かつ特異性の高い核酸増幅反応を可能とするものである。 LAMP is a new nucleic acid amplification method developed by Natomi et al. That does not require temperature control, which is essential for PCR, and is a loop-mediated isothermal amplification method (Patent Publication WO 00/28082). This is a method referred to as an abbreviation for “No.
The LAMP method anneals its 3 'end to the template nucleotide to serve as a starting point for complementary strand synthesis, and by combining the primer that anneals with the loop formed at this time, isothermal complementary strand synthesis reaction is performed. Make it possible. In the LAMP method, the 3 'end of the primer is always annealed to the region derived from the sample, and the check mechanism based on complementary binding of the base sequence functions repeatedly, resulting in high sensitivity and specificity. A highly nucleic acid amplification reaction is possible.
　LAMP法においては，核酸増幅の際，鋳型核酸の塩基配列の計6領域に対応したプライマーを必要とする。
　すなわち，LAMP法では，3’末端側からF3c，F2c，F1cという領域，5’末端側からB3，B2，B1という領域，これら6つの塩基配列を認識する少なくとも４種類のプライマーを必要とする。これらのプライマーはそれぞれ，インナープライマーF及びB，アウタープライマーF及びBと呼ばれる。
　また，F1c，F2c，F3cと相補的な塩基配列は，F1，F2，F3と呼ばれる。一方，B1，B2，B3と相補的な塩基配列は，B1c，B2c，B3cと呼ばれる。
　なお，各プライマーにおけるFとは，標的塩基配列のセンス鎖と相補的に結合し，合成起点を提供するプライマー表示である。一方，Bとは，標的塩基配列のアンチセンス鎖と相補的に結合し，合成起点を提供するプライマー表示である。 In the LAMP method, primers corresponding to a total of 6 regions of the base sequence of the template nucleic acid are required for nucleic acid amplification.
That is, the LAMP method requires at least four types of primers that recognize the F3c, F2c, and F1c regions from the 3 ′ end, the B3, B2, and B1 regions from the 5 ′ end, and these six base sequences. These primers are called inner primer F and B and outer primer F and B, respectively.
In addition, base sequences complementary to F1c, F2c and F3c are called F1, F2 and F3. On the other hand, base sequences complementary to B1, B2, and B3 are called B1c, B2c, and B3c.
In addition, F in each primer is a primer display that binds complementarily to the sense strand of the target base sequence and provides a starting point for synthesis. On the other hand, B is a primer display that complementarily binds to the antisense strand of the target base sequence and provides a starting point for synthesis.
　インナープライマーとは，標的塩基配列上の「ある特定のヌクレオチド配列領域」を認識し，かつ合成起点を与える塩基配列を3’末端に有し，同時にこのプライマーを起点とする核酸合成反応生成物の任意の領域に対して相補的な塩基配列を5’末端に有するオリゴヌクレオチドである。
　また，「F2より選ばれた塩基配列」及び「F1cより選ばれた塩基配列」を含むプライマーをインナープライマーF（以下，「IPF」），そして「B2より選ばれた塩基配列」と「B1cより選ばれた塩基配列」を含むプライマーをインナープライマーB（以下，「IPB」）と呼ぶ。
　IPFならびにIPBともに，典型的には，その3'末端側から，F2領域（又はB2領域），F1c領域（又はB1c領域）とした塩基配列とすればよい。また，F2領域とF1c領域の間には，増幅に関与しない非特異的塩基配列領域（リンカー）を有していてもよいことが技術常識として知られており，この非特異的領域を含んだ配列としてもよい。非特異的領域の配列の長さについては，増幅に影響を及ぼさない配列長とすればよく，典型的には50配列以下とすればよい。 An inner primer is a nucleic acid synthesis reaction product that recognizes a “specific nucleotide sequence region” on a target base sequence and has a base sequence that gives a synthesis starting point at the 3 ′ end, and at the same time starts from this primer. An oligonucleotide having a base sequence complementary to an arbitrary region at the 5 ′ end.
In addition, primers containing “a base sequence selected from F2” and “a base sequence selected from F1c” are used as inner primer F (hereinafter “IPF”), and “a base sequence selected from B2” and “from B1c A primer containing the “selected base sequence” is referred to as an inner primer B (hereinafter “IPB”).
For both IPF and IPB, typically, the base sequence may be the F2 region (or B2 region) or F1c region (or B1c region) from the 3 ′ end. In addition, it is known as technical common sense that a non-specific base sequence region (linker) not involved in amplification may be present between the F2 region and the F1c region, and this non-specific region is included. It may be an array. The sequence length of the non-specific region may be a sequence length that does not affect amplification, and typically 50 sequences or less.
　一方，アウタープライマーとは，標的塩基配列上の「『ある特定のヌクレオチド配列領域』の3’末端側に存在する，ある特定のヌクレオチド配列領域」を認識かつ合成起点を与える塩基配列を有するオリゴヌクレオチドである。
　また，「F3より選ばれた塩基配列」を含むプライマーをアウタープライマーF（以下，「OPF」），「B3より選ばれた塩基配列」を含むプライマーをアウタープライマーB（以下，「OPB」）と呼ぶ。 On the other hand, an outer primer is an oligonucleotide having a base sequence that recognizes "a specific nucleotide sequence region existing on the 3 'end side of a" specific nucleotide sequence region "on the target base sequence and provides a starting point for synthesis. It is.
In addition, a primer containing “a base sequence selected from F3” is designated as outer primer F (hereinafter “OPF”), and a primer containing “a base sequence selected from B3” is designated as outer primer B (hereinafter “OPB”). Call.
　LAMP法の核酸増幅においては，インナープライマーとアウタープライマーに加え，さらにこれとは別のプライマー，すなわちループプライマーを用いることができる。
　ループプライマー（Loop Primer）は，ダンベル構造の5’末端側のループ構造の一本鎖部分の塩基配列に相補的な塩基配列を持つプライマーである。このプライマーを用いると，核酸合成の起点が増加し，反応時間の短縮と検出感度の上昇が可能となる（特許文献国際公開第02/24902号パンフレット）。
　ループプライマーの塩基配列は，上述のダンベル構造の5’末端側のループ構造の一本鎖部分の塩基配列に相補的であれば，標的遺伝子の塩基配列あるいはその相補鎖から選ばれても良く，他の塩基配列でも良い。また，ループプライマーは１種類でも２種類でも良い。 In the nucleic acid amplification of the LAMP method, in addition to the inner primer and the outer primer, another primer, that is, a loop primer can be used.
The loop primer is a primer having a base sequence complementary to the base sequence of the single-stranded part of the loop structure on the 5 ′ end side of the dumbbell structure. When this primer is used, the starting point of nucleic acid synthesis is increased, and the reaction time can be shortened and the detection sensitivity can be increased (Patent Document WO 02/24902 pamphlet).
The base sequence of the loop primer may be selected from the base sequence of the target gene or its complementary strand as long as it is complementary to the base sequence of the single-stranded portion of the loop structure on the 5 ′ end side of the dumbbell structure described above. Other base sequences may be used. Further, one or two types of loop primers may be used.
　本発明において，標的核酸領域を増幅のターゲットとして，その3’末端側からF3c，F2c，F1cの塩基配列領域，5’末端側からB3，B2，B1の塩基配列領域，これらそれぞれの塩基配列領域を選択する。また，これら選択された塩基配列領域と相補的な，F3，F2，F1の塩基配列領域，B3c，B2c，B1cの塩基配列領域，これらそれぞれの塩基配列領域を選択する。これら塩基配列領域を基に，下記のオリゴヌクレオチドプライマーが作製される。
　(I)　3’末端側から，F2領域，非特異的塩基配列領域，F1c領域を有する塩基配列。
　(II)　F3領域を有する塩基配列。
　(III)　3’末端側から，B2領域，非特異的塩基配列領域，B1c領域を有する塩基配列。
　(IV)　B3領域を有する塩基配列。 In the present invention, the target nucleic acid region is used as an amplification target, the F3c, F2c, F1c base sequence region from the 3 ′ end side, the B3, B2, B1 base sequence region from the 5 ′ end side, and the respective base sequence regions. Select. In addition, F3, F2, and F1 base sequence regions, B3c, B2c, and B1c base sequence regions that are complementary to these selected base sequence regions, and their respective base sequence regions are selected. Based on these base sequence regions, the following oligonucleotide primers are prepared.
(I) A base sequence having an F2 region, a non-specific base sequence region, and an F1c region from the 3 ′ end side.
(II) A base sequence having an F3 region.
(III) A base sequence having a B2 region, a non-specific base sequence region, and a B1c region from the 3 ′ end side.
(IV) A base sequence having a B3 region.
　なお，プライマーとして作製される塩基配列の長さは，各領域に対する感度や特異度ないしはプライマーとしての安定性を考慮し適宜調整することができるが，上記(II)ないし(IV)に該当して作製するオリゴヌクレオチドプライマーは，10塩基以上とすることが好ましく，さらに好ましくは15塩基以上とすることができる。また，上記(I)ないし(III)に該当して作製するオリゴヌクレオチドプライマーは，20塩基以上とすることが好ましく，さらに好ましくは40塩基以上とすることができる。 The length of the base sequence prepared as a primer can be adjusted as appropriate in consideration of the sensitivity and specificity for each region or the stability as a primer, but it falls under (II) to (IV) above. The oligonucleotide primer to be prepared is preferably 10 bases or more, more preferably 15 bases or more. In addition, the oligonucleotide primer prepared according to the above (I) to (III) is preferably 20 bases or more, more preferably 40 bases or more.
　本発明において，(I)の塩基配列はIPF，(II)の塩基配列はOPF，(III)の塩基配列はIPB，(IV)の塩基配列はOPBに該当するものである。
　すなわち，本発明のオリゴヌクレオチドプライマーセットは，アフリカ豚コレラウイルス検出を目的とした下記(i)から(iv)のプライマーからなることを特徴とするオリゴヌクレオチドプライマーセットである。
(i)　インナープライマーF
　(a)　5’－（配列番号３の塩基配列に相補的な塩基配列）－（塩基数０～５０の任意の塩基配列）－（配列番号４の塩基配列）－3’
又は，
　(b)　5’－（配列番号１３の塩基配列に相補的な塩基配列）－（塩基数０～５０の任意の塩基配列）－（配列番号１４の塩基配列）－3’
(ii)　アウタープライマーF
　(c)　配列番号２，又は，(d)　配列番号１２
(iii)　インナープライマーB
　(e)　5’－（配列番号５の塩基配列に相補的な塩基配列）－（塩基数０～５０の任意の塩基配列）－（配列番号６の塩基配列）－3’
又は，
　(f)　5’－（配列番号１５の塩基配列に相補的な塩基配列）－（塩基数０～５０の任意の塩基配列）－（配列番号１６の塩基配列）－3’
(iv)　アウタープライマーB
　(g)　配列番号７，又は，(h)　配列番号１７ In the present invention, the base sequence of (I) corresponds to IPF, the base sequence of (II) corresponds to OPF, the base sequence of (III) corresponds to IPB, and the base sequence of (IV) corresponds to OPB.
That is, the oligonucleotide primer set of the present invention comprises the following primers (i) to (iv) for the purpose of detecting African swine fever virus.
(i) Inner primer F
(a) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 3)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 4) -3'
Or
(b) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 13)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 14) -3'
(ii) Outer primer F
(c) SEQ ID NO: 2 or (d) SEQ ID NO: 12
(iii) Inner primer B
(e) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 5)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 6) -3'
Or
(f) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 15)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 16) -3'
(iv) Outer primer B
(g) SEQ ID NO: 7 or (h) SEQ ID NO: 17
　IPFである配列(a)ないし(b)については，3’側からF2に相当する配列と，F1cに相当する配列を組み合わせて設計されている。また，F2に相当する塩基配列と，F1cに相当する塩基配列の間には，増幅に関与しない非特異的な塩基配列（リンカー）を有していてもよいことが技術常識として知られており，そのリンカーの長さとして特に限定する必要はないが，典型的には，塩基数50以下とすることができる。 The sequences (a) and (b) which are IPFs are designed by combining the sequence corresponding to F2 and the sequence corresponding to F1c from the 3 'side. It is also known as technical common knowledge that a non-specific base sequence (linker) that does not participate in amplification may be present between the base sequence corresponding to F2 and the base sequence corresponding to F1c. The length of the linker need not be particularly limited, but can typically be 50 or less.
　同様に，IPBである配列(e)ないし(f)については，3’側からB2に相当する配列と，B1cに相当する配列を組み合わせて設計されている。また，IPF同様，IPBについても，B2に相当する塩基配列と，B1cに相当する塩基配列の間には，リンカーを有していてもよいことが技術常識として知られており，そのリンカーの長さとして特に限定する必要はないが，典型的には，塩基数50以下とすることができる。 Similarly, the IPB sequences (e) to (f) are designed by combining the sequence corresponding to B2 and the sequence corresponding to B1c from the 3 'side. Similarly to IPF, it is known as technical common knowledge that IPB may have a linker between the base sequence corresponding to B2 and the base sequence corresponding to B1c. Although there is no particular limitation, typically, the number of bases can be 50 or less.
　本発明において，前記(i)，(ii)，(iii)，(iv)のプライマーは，それぞれ配列番号８，配列番号２，配列番号９，配列番号７，もしくは，配列番号１８，配列番号１２，配列番号１９，配列番号１７に記載の塩基配列として設計することが可能である。また，当然のことながら，前記(i)，(ii)，(iii)，(iv)のプライマーは，これらの塩基配列と相補的な塩基配列として設計してもよい。さらに前記(i)，(ii)，(iii)，(iv)のプライマーは，これらの塩基配列もしくは相補的な塩基配列における連続した塩基配列を含む場合であっても標的核酸領域の増幅しうるものであり，少なくとも連続する15塩基を含む塩基を含むオリゴヌクレオチドプライマーとして設計することができる。 In the present invention, the primers (i), (ii), (iii), and (iv) are SEQ ID NO: 8, SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 7, or SEQ ID NO: 18, SEQ ID NO: 12, respectively. , SEQ ID NO: 19 and SEQ ID NO: 17 can be designed as the nucleotide sequence. Naturally, the primers (i), (ii), (iii), and (iv) may be designed as complementary nucleotide sequences to these nucleotide sequences. Furthermore, the primers (i), (ii), (iii), and (iv) can amplify the target nucleic acid region even when they contain a continuous base sequence of these base sequences or complementary base sequences. It can be designed as an oligonucleotide primer containing at least 15 consecutive bases.
　本発明において，IPF，OPF，IPB，OPBを，LAMP法に用いる際のオリゴヌクレオチドプライマーの一つのセットとして用いることができ，これらに加え，ループプライマーを合わせて用いてもよい。これにより，核酸合成の起点が増加することとなり，反応時間の短縮と検出感度の上昇が可能となる効果を有する。
　ループプライマーは，LPFとして配列番号１０もしくは配列番号２０，LPBとして配列番号１１もしくは配列番号２１に記載の塩基配列のオリゴヌクレオチドプライマーを用いることができる。 In the present invention, IPF, OPF, IPB, and OPB can be used as one set of oligonucleotide primers for use in the LAMP method, and in addition to these, loop primers may be used in combination. As a result, the starting point of nucleic acid synthesis is increased, and the reaction time can be shortened and the detection sensitivity can be increased.
As the loop primer, an oligonucleotide primer having a base sequence described in SEQ ID NO: 10 or 20 as LPF and SEQ ID NO: 11 or 21 as LPB can be used.
　さらに，本発明においては，単独のプライマーセットを用いて核酸増幅操作を行うこともできるし，複数を組み合わせて核酸増幅操作を行うこともできる。複数のプライマーセットを組み合わせて核酸増幅操作を行うことにより，アフリカ豚コレラウイルスの多様性をよりカバーしうるとともに，検出感度や特異度，迅速性を向上させる効果を有する。
　これらの組み合わせの例として，前述のプライマーセット１ならびにプライマーセット２，これらを組み合わせて，アフリカ豚コレラウイルスの検出を行うなどである。 Furthermore, in the present invention, a nucleic acid amplification operation can be performed using a single primer set, or a plurality of nucleic acid amplification operations can be performed in combination. By performing nucleic acid amplification operation by combining multiple primer sets, the diversity of African swine fever virus can be covered, and the detection sensitivity, specificity, and speed can be improved.
Examples of these combinations include the detection of African swine fever virus by combining primer set 1 and primer set 2 described above.
　続いてLAMP法の一連の流れについて説明を行う。
　LAMP法によるアフリカ豚コレラウイルス検出のためには，検体試料が必要である。この検体試料としては，通常用いられるあらゆる検体試料を用いることが可能であり，例えば，ウイルス，ウイルスを含む培養細胞ならびに培養液，豚，猪，イノブタなどの動物由来の検体などが挙げられる。この場合，動物由来の検体を検体試料として用いることがより好ましく，例えば血液，血清，体液，組織抽出液，あるいは組織拭い液ならびに組織拭き取り検体を検体試料として用いることができる。これにより，迅速なアフリカ豚コレラウイルス検出が可能となるという効果を有する。 Subsequently, a series of flows of the LAMP method will be described.
Samples are required for detection of African swine fever virus by the LAMP method. As the specimen sample, any specimen specimen that is usually used can be used. Examples thereof include viruses, cultured cells containing viruses, and specimens derived from animals such as culture fluids, pigs, rabbits, and lobsters. In this case, it is more preferable to use an animal-derived specimen as the specimen sample. For example, blood, serum, body fluid, tissue extract, or tissue wiping solution and tissue wiping specimen can be used as the specimen sample. This has the effect of enabling rapid detection of African swine fever virus.
　アフリカ豚コレラウイルス検出のための検体試料については，通常用いられる前処理を行い後述するLAMP法に用いる際の試料とすることができる。例えば，タンパク質分解酵素等による組織細胞由来タンパク質を分解後，フェノール及びクロロホルムを用いて核酸抽出や精製を行ったり，市販されている抽出キット（例えばQiagen社のRNeasy Mini Kit）を用いて得られた核酸を抽出し検体として用いるなどである。 Specimen samples for detecting African swine fever virus can be used as samples for use in the LAMP method described below after pretreatment that is normally used. For example, after degrading tissue cell-derived protein with proteolytic enzymes, etc., nucleic acid extraction and purification using phenol and chloroform, or using a commercially available extraction kit (for example, RNeasy Mini Mini Kit from Qiagen) For example, nucleic acid is extracted and used as a specimen.
　検体試料の準備ができたら，前述のプライマーセットを用いて，核酸増幅のための操作を行う。この核酸増幅の際の操作として，例えば，反応Buffer中，少なくとも４種類のプライマー（IPF，IPB，OPF，OPB），鋳型依存性核酸合成酵素，デオキシヌクレオチド3リン酸などの基質を，60から65℃で15分から1時間程度インキュベートすればよい。また，上述の通り，ループプライマーをさらに用いることもできる。 When the specimen sample is ready, perform the procedure for nucleic acid amplification using the primer set described above. As an operation for this nucleic acid amplification, for example, at least 4 types of primers (IPF, IPB, OPF, OPB), template-dependent nucleic acid synthase, deoxynucleotide triphosphate, etc. in the reaction buffer are used in 60 to 65. Incubate at 15 ° C for 15 minutes to 1 hour. Further, as described above, a loop primer can be further used.
　核酸合成で使用する酵素は，鎖置換活性を有する鋳型依存性核酸合成酵素であれば特に限定する必要はない。このような酵素としては，Bst DNAポリメラーゼ（ラージフラグメント），Bca（exo-）DNAポリメラーゼ，Csa DNAポリメラーゼ，Gsp DNAポリメラーゼ（ラージフラグメント），GspSSD DNAポリメラーゼ（ラージフラグメント），Tin DNAポリメラーゼ（ラージフラグメント），大腸菌DNAポリメラーゼＩのクレノウフラグメント等が挙げられ，好ましくはBst DNAポリメラーゼ（ラージフラグメント）が挙げられる。 The enzyme used in nucleic acid synthesis is not particularly limited as long as it is a template-dependent nucleic acid synthase having strand displacement activity. Such enzymes include Bst DNA polymerase (large fragment), Bca (exo-) DNA polymerase, Csa DNA polymerase, Gsp DNA polymerase (large fragment), GspSSD DNA polymerase (large fragment), Tin DNA polymerase (large fragment) , Klenow fragment of E. coli DNA polymerase I and the like, preferably Bst DNA polymerase (large fragment).
　核酸増幅反応操作が終了したら，核酸増幅産物の検出を行う。核酸増幅産物の検出には，通常用いられるあらゆる検出方法を用いることができる。例えば，増幅された塩基配列を特異的に認識する標識オリゴヌクレオチドを用いた検出や蛍光性インターカレーター法，アガロースゲル電気泳動法による検出などである。アガロースゲル電気泳動法においてLAMP増幅産物は，塩基長の異なる多数のバンドがラダー（はしご）状に検出される。
　加えて，LAMP法では目視による検出ができる場合があり，迅速なアフリカ豚コレラウイルス検出が必要な場合に好ましい。すなわち，LAMP法では，核酸の合成により基質が大量に消費され，副産物であるピロリン酸が，共存するマグネシウムと反応してピロリン酸マグネシウムとなり，反応液が肉眼で確認できる程度に白濁することからアフリカ豚コレラウイルスの検出が可能である。この場合，反応中の濁度上昇経過や反応終了後の濁度を光学的に観察できる分光光度計等の測定機器を用いて，アフリカ豚コレラウイルスの検出を確認することも可能である。 When the nucleic acid amplification reaction operation is completed, the nucleic acid amplification product is detected. Any commonly used detection method can be used to detect the nucleic acid amplification product. For example, detection using a labeled oligonucleotide that specifically recognizes the amplified base sequence, detection by a fluorescent intercalator method, agarose gel electrophoresis, and the like. In the agarose gel electrophoresis method, a large number of bands with different base lengths are detected in a ladder form in the LAMP amplification product.
In addition, the LAMP method may allow visual detection, which is preferable when rapid detection of African swine fever virus is required. In other words, in the LAMP method, a large amount of substrate is consumed by nucleic acid synthesis, and pyrophosphate, a by-product, reacts with coexisting magnesium to become magnesium pyrophosphate, and the reaction solution becomes cloudy to the extent that it can be visually confirmed. Detection of swine cholera virus is possible. In this case, it is also possible to confirm the detection of African swine fever virus using a measuring instrument such as a spectrophotometer that can optically observe the turbidity increase during the reaction and the turbidity after the reaction is completed.
　本発明のプライマーを用いて核酸増幅の検出を行う際に必要な各種の試薬類は，あらかじめパッケージングしてキット化することができる。具体的には，本発明のプライマーあるいはループプライマーとして必要な各種のオリゴヌクレオチド，核酸合成の基質となる４種類のdNTP，鎖置換活性を有する鋳型依存性核酸合成酵素，酵素反応に好適な条件を与える緩衝液や塩類，酵素や鋳型を安定化する保護剤，さらに必要に応じて反応生成物の検出に必要な試薬類がキットとして提供される。 Various reagents necessary for detection of nucleic acid amplification using the primer of the present invention can be packaged in advance to form a kit. Specifically, various oligonucleotides necessary as a primer or loop primer of the present invention, four types of dNTPs serving as a substrate for nucleic acid synthesis, a template-dependent nucleic acid synthase having a strand displacement activity, and conditions suitable for enzymatic reaction Provided buffers and salts, protecting agents for stabilizing enzymes and templates, and reagents necessary for detection of reaction products as necessary are provided as kits.
＜＜実験例１．LAMP法による検出感度の検討＞＞
　設計を行った各種プライマーセットについて，LAMP法における検出感度を調べることを目的として実験を行った。また，比較対象として，PCR法による検出を合わせて行った。
　なお，本実験例において検出感度は，１反応チューブあたりの検出可能な最小コピー数として示してあり，遺伝子検出における感度を示す指標の一つとして常用されるものである。 << Experimental Example 1 Examination of detection sensitivity by LAMP method >>
For the designed primer sets, experiments were conducted to investigate the detection sensitivity in the LAMP method. For comparison, PCR detection was also performed.
In this experimental example, the detection sensitivity is shown as the minimum detectable copy number per reaction tube, and is commonly used as one of the indices indicating the sensitivity in gene detection.
＜実験方法＞
［検出対象試料の調製］
１．アフリカ豚コレラウイルスの配列（GenBank accession No.AY578689）の，1503番から2072番と同一の人工塩基配列（配列番号２２）をユーロフィン社から購入した。
２．この人工塩基配列を，TE緩衝液で溶解し，これを10倍ずつ，段階希釈して調整した各溶解液を，検出対象試料の鋳型DNAとして用いた。なお，各検出対象試料については，予めコピー数の算出を行った。 <Experiment method>
[Preparation of sample to be detected]
1. An artificial base sequence (SEQ ID NO: 22) identical to the sequences 1503 to 2072 of the African swine fever virus sequence (GenBank accession No. AY578689) was purchased from Eurofin.
2. This artificial base sequence was dissolved in TE buffer, and each lysate prepared by serial dilution 10 times was used as template DNA for the detection target sample. Note that the copy number was calculated in advance for each detection target sample.
［LAMP法］
１．LAMP法では，下記に示すプライマーセットを用いた。なお，プライマーの合成は北海道システムサイエンス社に依頼し，簡易カラム精製したものを使用した。
(1)　プライマーセット１
ASF-IPF-1:5'-CGTTTACACGCTTGTGGATCGTACGCATTCTTTGTGCCG-3' （配列番号8)
ASF-OPF-1:5'-ACTCGCAGTAGTAAACCAA-3' （配列番号2）
ASF-IPB-1:5'-TCCGTTGCGAGGAAACGTTTGATGCTCCGATTCAGGGC-3' （配列番号9）
ASF-OPB-1:5'-TGTCATTCGTCCTGGCA-3' （配列番号７）
ASF-LPF-1:5'-CTTTTGGGAGACCCATTGT-3' （配列番号１０）
ASF-LPB-1:5'-CCCCCATCTGGGATGA-3' （配列番号１１）
(2)　プライマーセット２
ASF-IPF-2:5'-TTGAGTCAAATCGAAGAAACACATACACTTTATTGTATTCAAACCCTA-3' （配列番号18）
ASF-OPF-2:5'-GGAAAAAGTCTCCGTACTG-3' （配列番号１２）
ASF-IPB-2:5'-GCAAGTCTTGGGCCAAGATACTTTTTGTCTTATTGCTAACGATGG-3' （配列番号19）
ASF-OPB-2:5'-ATATGGCATCAGGAGGAG-3' （配列番号１７）
ASF-LPF-2:5'-GCATTTTAATGCACATTTTAAGCCTT-3' （配列番号２０）
ASF-LPB-2:5'-AATCTTGTCGGCCTTCC-3' （配列番号２１） [LAMP method]
1. In the LAMP method, the following primer set was used. Primer synthesis was requested from Hokkaido System Science Co., Ltd., and simple column purification was used.
(1) Primer set 1
ASF-IPF-1: 5'-CGTTTACACGCTTGTGGATCGTACGCATTCTTTGTGCCG-3 '(SEQ ID NO: 8)
ASF-OPF-1: 5'-ACTCGCAGTAGTAAACCAA-3 '(SEQ ID NO: 2)
ASF-IPB-1: 5'-TCCGTTGCGAGGAAACGTTTGATGCTCCGATTCAGGGC-3 '(SEQ ID NO: 9)
ASF-OPB-1: 5'-TGTCATTCGTCCTGGCA-3 '(SEQ ID NO: 7)
ASF-LPF-1: 5'-CTTTTGGGAGACCCATTGT-3 '(SEQ ID NO: 10)
ASF-LPB-1: 5'-CCCCCATCTGGGATGA-3 '(SEQ ID NO: 11)
(2) Primer set 2
ASF-IPF-2: 5'-TTGAGTCAAATCGAAGAAACACATACACTTTATTGTATTCAAACCCTA-3 '(SEQ ID NO: 18)
ASF-OPF-2: 5'-GGAAAAAGTCTCCGTACTG-3 '(SEQ ID NO: 12)
ASF-IPB-2: 5'-GCAAGTCTTGGGCCAAGATACTTTTTGTCTTATTGCTAACGATGG-3 '(SEQ ID NO: 19)
ASF-OPB-2: 5'-ATATGGCATCAGGAGGAG-3 '(SEQ ID NO: 17)
ASF-LPF-2: 5'-GCATTTTAATGCACATTTTAAGCCTT-3 '(SEQ ID NO: 20)
ASF-LPB-2: 5'-AATCTTGTCGGCCTTCC-3 '(SEQ ID NO: 21)
２．LAMP法による増幅のため，最終反応溶液25μＬ中の各試薬濃度が下記になるよう反応試薬を調製した。
　
　・LAMP反応溶液組成
　　15.0μL Isothermal Master Mix for GenieIII（Optigene社製）
　　5.0μL 精製水
　　1.6μM IPF（配列番号８，又は配列番号１８）
　　1.6μM IPB（配列番号９，又は配列番号１９）
　　0.2μM OPF（配列番号２，又は配列番号１２）
　　0.2μM OPB（配列番号７，又は配列番号１７）
　　0.8μM LPF（配列番号１０，又は配列番号２０）
　　0.8μM LPB（配列番号１１，又は配列番号２１）
 
３．上記反応試薬20μＬに，鋳型DNAを含む各濃度の試料溶液5.0μＬを加え，最終反応溶液25.0μＬとして，各LAMP反応を２回ずつ行った。
４．専用チューブ内で65℃60分間，リアルタイム蛍光測定装置GenieIII（Optigene社製）を用いて，リアルタイムに反応を検出した。合わせて，リアルタイム蛍光測定装置GenieIIIに内蔵されているモニター上のグラフで，反応結果をリアルタイムに視認した。
５．所定時間内Amplification rateの蛍光強度比（Fluorescence Ratio）が0.02を超えた検体を陽性と判定した。 2. For amplification by the LAMP method, reaction reagents were prepared so that each reagent concentration in 25 μL of the final reaction solution was as follows.

・ LAMP reaction solution composition 15.0μL Isothermal Master Mix for GenieIII (manufactured by Optigene)
5.0 μL purified water 1.6 μM IPF (SEQ ID NO: 8 or SEQ ID NO: 18)
1.6 μM IPB (SEQ ID NO: 9 or 19)
0.2 μM OPF (SEQ ID NO: 2 or SEQ ID NO: 12)
0.2 μM OPB (SEQ ID NO: 7 or SEQ ID NO: 17)
0.8 μM LPF (SEQ ID NO: 10 or SEQ ID NO: 20)
0.8 μM LPB (SEQ ID NO: 11 or SEQ ID NO: 21)

3. To 20 μL of the reaction reagent, 5.0 μL of each concentration of sample solution containing template DNA was added to make 25.0 μL of the final reaction solution, and each LAMP reaction was performed twice.
4). The reaction was detected in real time using a real-time fluorescence analyzer GenieIII (manufactured by Optigene) at 65 ° C for 60 minutes in a dedicated tube. In addition, the results of the reaction were visually confirmed in real time using a graph on the monitor built in Genie III, a real-time fluorescence measurement device.
5). A specimen having a fluorescence ratio of amplification rate within a predetermined time exceeding 0.02 was determined to be positive.
［PCR法］
１．非特許文献４に開示されている方法を参考に行った。すなわち，PCR法で使用するプライマーとして，非特許文献４に開示されているPPA-1（配列番号２３）とPPA-2（配列番号２４）の組み合わせを用いた。 [PCR method]
1. The method disclosed in Non-Patent Document 4 was used as a reference. That is, a combination of PPA-1 (SEQ ID NO: 23) and PPA-2 (SEQ ID NO: 24) disclosed in Non-Patent Document 4 was used as a primer used in the PCR method.
２．PCR法による増幅のため，最終反応溶液25μＬ中の各試薬濃度が下記になるよう反応試薬を調製した。
 
　・PCR反応溶液組成
　　PCR buffer×10（Qiagen社製）2.5μL
　　2mM dNTPs（Qiagen社製）2μL
　　dDW（滅菌超純水） 20.98μL
 
　　100pmol/μL OPF（配列番号23) 0.16μL
　　100pmol/μL OPB（配列番号24) 0.16μL
　　5U/μL Taq Polymerase（Qiagen社製）0.1μL
 
３．上記反応試薬20μＬに，鋳型DNAを含む各濃度の試料溶液5.0μＬを加え，最終反応溶液25.0μＬとして，各PCR反応を２回ずつ行った。
(1)　PCR反応の温度サイクル条件は，95℃2分静置後，熱変性95℃15秒，アニーリング62℃30秒，ポリメラーゼ伸長反応72℃30秒，これらの一連の反応を１サイクルとして合計30サイクル行い，最後に72℃にて7分間静置後，反応を終了した。所要時間は約２時間であった。
(2)　反応終了後の反応溶液３μＬを２％アガロースゲルで電気泳動を行い，エチジウムブロマイド染色した。 2. For amplification by PCR, reaction reagents were prepared so that the concentration of each reagent in 25 μL of the final reaction solution was as follows.

PCR reaction solution composition PCR buffer × 10 (Qiagen) 2.5 μL
2 mM dNTPs (Qiagen) 2 μL
dDW (sterilized ultrapure water) 20.98μL

100pmol / μL OPF (SEQ ID NO: 23) 0.16μL
100pmol / μL OPB (SEQ ID NO: 24) 0.16μL
5U / μL Taq Polymerase (Qiagen) 0.1μL

3. To 20 μL of the reaction reagent, 5.0 μL of each sample solution containing the template DNA was added to make 25.0 μL of the final reaction solution, and each PCR reaction was performed twice.
(1) The temperature cycle conditions for the PCR reaction were 95 ° C for 2 minutes, heat denaturation at 95 ° C for 15 seconds, annealing at 62 ° C for 30 seconds, and polymerase extension reaction at 72 ° C for 30 seconds. After 30 cycles, the reaction was terminated after standing at 72 ° C for 7 minutes. The time required was about 2 hours.
(2) 3 μL of the reaction solution after completion of the reaction was electrophoresed on a 2% agarose gel and stained with ethidium bromide.
＜実験結果＞
１．図１から図３に，LAMP法を用いた際の実験結果を示す。
(1)　それぞれの図中，横軸は核酸増幅反応を行った時間（分）を，縦軸は蛍光強度比を示す。核酸が増幅されればされるほど，反応溶液中の蛍光強度比が上昇することとなり，結果として，対象となるウイルスの検出が可能となる。
(2)　また，反応チューブ中の人工塩基配列のコピー数が多いほど，核酸増幅反応が容易に進むとともに，蛍光強度比の上昇が速く検出できる。逆にコピー数が少ないほど，核酸増幅反応は進みにくくなるため蛍光強度比の上昇が遅くなり，検出に時間がかかることになる。 <Experimental result>
1. FIG. 1 to FIG. 3 show the experimental results when using the LAMP method.
(1) In each figure, the horizontal axis represents the time (minutes) during which the nucleic acid amplification reaction was performed, and the vertical axis represents the fluorescence intensity ratio. The more the nucleic acid is amplified, the higher the fluorescence intensity ratio in the reaction solution. As a result, the target virus can be detected.
(2) In addition, the larger the number of copies of the artificial base sequence in the reaction tube, the easier the nucleic acid amplification reaction proceeds, and the faster the fluorescence intensity ratio can be detected. Conversely, the smaller the copy number, the more difficult the nucleic acid amplification reaction proceeds, so the increase in the fluorescence intensity ratio becomes slower and the detection takes longer.
２．図１はプライマーセット１を用いた際の結果である。
(1)　1378.0コピー（well1ならびにwell2），137.8コピー（well3およびwell4）の反応チューブにおいて蛍光強度が，0.02を超えていた。
(2)　また，13.8コピーの反応チューブにおいては，0.02を超えるもの（well6）と超えないもの（well5）がそれぞれ見られた。
(3)　なお，1.4コピーのサンプルでは，0.02を超えず，蛍光強度の上昇は全く見られなかった（well7ならびにwell8）。
３．図２は，プライマーセット２を用いた際の結果である。図１と同様の結果であった。
４．図３は，プライマーセット１とプライマーセット２を組み合わせて用いた際の結果である。
(1)　1378.0コピー（well1ならびにwell2），137.8コピー（well3およびwell4），13.8コピー（well5ならびにwell6）の反応チューブにおいて蛍光強度が，0.02を超えていた。
(2)　また，1.4コピーのサンプルでは，0.02を超えず，蛍光強度の上昇は全く見られなかった（well7ならびにwell8）。
５．比較として行ったPCR法は，LAMP反応チューブあたり13.8コピーのものまで検出可能であった（不図示）。
６．上記より，各検討における検出感度をまとめた結果を，表１に示す。 2. FIG. 1 shows the results when primer set 1 was used.
(1) The fluorescence intensity exceeded 0.02 in the reaction tubes of 1378.0 copies (well 1 and well 2) and 137.8 copies (well 3 and well 4).
(2) In the 13.8 copies of the reaction tube, those exceeding 0.02 (well 6) and those not exceeding (well 5) were observed.
(3) The 1.4 copy sample did not exceed 0.02, and no increase in fluorescence intensity was observed (well 7 and well 8).
3. FIG. 2 shows the results when Primer Set 2 was used. The result was the same as in FIG.
4). FIG. 3 shows the results when primer set 1 and primer set 2 are used in combination.
(1) The fluorescence intensity exceeded 0.02 in the reaction tubes of 1378.0 copies (well 1 and well 2), 137.8 copies (well 3 and well 4), and 13.8 copies (well 5 and well 6).
(2) In addition, the 1.4 copy sample did not exceed 0.02, and no increase in fluorescence intensity was observed (well 7 and well 8).
5). The PCR method used as a comparison was able to detect up to 13.8 copies per LAMP reaction tube (not shown).
6). Based on the above, the results of the detection sensitivity in each study are summarized in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
７．これらの結果から，下記のことが示された。
(1)　本発明のプライマーセットを単独でLAMP法に用いることにより，PCR法と同程度の検出感度を達成しうることが示された。
(2)　本発明のプライマーセットを組み合わせてLAMP法にて用いることにより，PCR法と同程度の検出感度を達成することが示された。
(3)　本発明のプライマーセットを用いた際の所要時間が，11分から39分であり，そのほとんどは，30分以内に検出可能であった。一方，PCRを用いた際の所要時間は，約2時間の増幅反応に加え，約30分の電気泳動分析を必要とする。これらのことから，本発明のLAMP法は，PCR法よりも，迅速かつ簡易に検出が可能であることが示された。 7). From these results, the following was shown.
(1) By using the primer set of the present invention alone for the LAMP method, it was shown that detection sensitivity comparable to that of the PCR method can be achieved.
(2) It was shown that by using the primer set of the present invention in combination with the LAMP method, detection sensitivity comparable to that of the PCR method can be achieved.
(3) The time required for using the primer set of the present invention was 11 to 39 minutes, most of which could be detected within 30 minutes. On the other hand, the time required to use PCR requires about 30 minutes of electrophoretic analysis in addition to the amplification reaction of about 2 hours. From these results, it was shown that the LAMP method of the present invention can be detected more quickly and easily than the PCR method.
＜＜実験例２，プライマーセットを用いた性能比較＞＞
　各プライマーセットについて，アフリカ豚コレラウイルスの検出が可能かどうか，また，その検出時間などを確認することを目的に検討を行った。 << Experimental Example 2, Performance Comparison Using Primer Set >>
Each primer set was examined for the purpose of confirming the detection of African swine fever virus and its detection time.
＜実験方法＞
　実験例１に準じて実験を行った。 <Experiment method>
The experiment was conducted according to Experimental Example 1.
＜実験結果＞
１．結果を表２に示す。
(1)　表中，LAMP1がプライマーセット１，LAMP2がプライマーセット２，LAMP Multiprexがプライマーセット１と２いずれもを用いて，LAMP法により検出を行った結果を示す。
(2)　また，リアルタイムPCRのカットオフ値を35サイクル（Ct），LAMP法のカットオフ値を30分に設定し，この時間以内に陽性判定が行えなかった場合は，表中，「No Ct」で記してある。
(3)　なお，いずれの検討においても，2回検討を行い，２回ともカットオフ値内の増幅があった場合にのみ，陽性と判定している。 <Experimental result>
1. The results are shown in Table 2.
(1) In the table, LAMP1 is the primer set 1, LAMP2 is the primer set 2, and LAMP Multiprex is the primer set 1 and 2, and the results of detection by the LAMP method are shown.
(2) If the cut-off value for real-time PCR is set to 35 cycles (Ct) and the cut-off value for the LAMP method is set to 30 minutes and a positive determination cannot be made within this time, “No Ct” ".
(3) In each study, the study was conducted twice, and only when there was amplification within the cut-off value in both trials, it was judged as positive.
２．検討を行ったサンプル各種について，いずれもリアルタイムPCRよりも，本発明のプライマーセットを用いたLAMP法の方がより早い検出が可能であった。
３．また，プライマーセットを組み合わせて検出を行った方が，単独で検出を行うよりも早い検出がおおむね可能であった。 2. For each of the various samples examined, the LAMP method using the primer set of the present invention could be detected earlier than real-time PCR.
3. In addition, detection using a combination of primer sets was generally capable of faster detection than detection alone.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
＜考察＞
１．本願における実験結果と，先行技術３に開示された実験結果の簡単な比較を，表３に示す。
２．リアルタイムPCRでの陽性平均時間について，本願が25.35サイクル，先行技術が21.42サイクルであり，通常のリアルタイムPCRを考慮すると，本願の方が４サイクルほど高いことから，サンプルにおけるウイルス量は，本願における検討が総じて少ないと考えられた。
３．しかるに，プライマーセットを用いた結果において，陽性平均所要時間は，本願の方が19分から22分ほど先行技術より短かった。
４．これの比較から，本願技術の方が，先行技術と比較すると，迅速に検出が可能であると考えられた。 <Discussion>
1. Table 3 shows a simple comparison between the experimental results in the present application and the experimental results disclosed in Prior Art 3.
2. The positive average time in real-time PCR is 25.35 cycles for the present application and 21.42 cycles for the prior art. In consideration of normal real-time PCR, this application is about 4 cycles higher. It was thought that there were generally few.
3. However, in the results using the primer set, the average positive time required for the present application was 19 to 22 minutes shorter than the prior art.
4). From this comparison, it was considered that the technology of the present application could be detected more quickly than the prior art.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
５．さらに，本発明のプライマーセットと先行技術文献３におけるプライマーセットにおいて，対象配列とのミスマッチを調べた結果を表４から表６に示す。なお，各表における配列のアンダーラインが記されている塩基が，対象のミスマッチ部位である。
６．プライマーセット１においては，ミスマッチ数が合計10あったものの，反応重要領域におけるミスマッチはなかった。
７．プライマーセット２においては，ミスマッチ数が合計11あり，反応重要領域におけるミスマッチが3つあったが，配列としては，LPBの１配列のみに見られただけであった。
８．先行技術においては，ミスマッチ数が合計10あり，反応重要領域におけるミスマッチが4つ見られた。また，この反応重要領域におけるミスマッチは，F2，LF，LBの３つの配列に及んでいた。
９．これらより，本発明のプライマーセットは，先行技術と比較して，反応重要領域におけるミスマッチが無い，もしくは少ないものであり，この点が，前述の迅速な検出時間に関与しているものと考えられた。 5). Furthermore, Table 4 to Table 6 show the results of examining mismatches with the target sequences in the primer set of the present invention and the primer set in Prior Art Document 3. In addition, the base in which the underline of the sequence in each table is written is the target mismatch site.
6). In primer set 1, although there were a total of 10 mismatches, there was no mismatch in the reaction critical region.
7). In primer set 2, the total number of mismatches was 11, and there were 3 mismatches in the reaction critical region, but the sequence was found only in one sequence of LPB.
8). In the prior art, there were a total of 10 mismatches, and 4 mismatches were found in the reaction critical areas. In addition, the mismatch in this reaction critical region extended to three sequences, F2, LF, and LB.
9. From these, the primer set of the present invention has no or few mismatches in the reaction important region as compared with the prior art, and this point is considered to be related to the above-mentioned rapid detection time. It was.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006

Claims (8)

  1. アフリカ豚コレラウイルス検出を目的とした下記(i)から(iv)のオリゴヌクレオチドプライマーからなることを特徴とするオリゴヌクレオチドプライマーセット。
    (i)　インナープライマーF
    　(a)　5’－（配列番号３の塩基配列に相補的な塩基配列）－（塩基数０～５０の任意の塩基配列）－（配列番号４の塩基配列）－3’
    又は，
    　(b)　5’－（配列番号１３の塩基配列に相補的な塩基配列）－（塩基数０～５０の任意の塩基配列）－（配列番号１４の塩基配列）－3’
    (ii)　アウタープライマーF
    　(c)　配列番号２，又は，(d)　配列番号１２
    (iii)　インナープライマーB
    　(e)　5’－（配列番号５の塩基配列に相補的な塩基配列）－（塩基数０～５０の任意の塩基配列）－（配列番号６の塩基配列）－3’
    又は，
    　(f)　5’－（配列番号１５の塩基配列に相補的な塩基配列）－（塩基数０～５０の任意の塩基配列）－（配列番号１６の塩基配列）－3’
    (iv)　アウタープライマーB
    　(g)　配列番号７，又は，(h)　配列番号１７
          An oligonucleotide primer set comprising oligonucleotide primers (i) to (iv) below for the purpose of detecting African swine fever virus.
    (i) Inner primer F
    (a) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 3)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 4) -3'
    Or
    (b) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 13)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 14) -3'
    (ii) Outer primer F
    (c) SEQ ID NO: 2 or (d) SEQ ID NO: 12
    (iii) Inner primer B
    (e) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 5)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 6) -3'
    Or
    (f) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 15)-(arbitrary base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 16) -3'
    (iv) Outer primer B
    (g) SEQ ID NO: 7 or (h) SEQ ID NO: 17
  2. 前記(i)，(ii)，(iii)，(iv)のオリゴヌクレオチドプライマーが，それぞれ配列番号８，配列番号２，配列番号９，配列番号７の塩基配列からなることを特徴とする請求項１に記載のオリゴヌクレオチドプライマーセット。
          The oligonucleotide primers (i), (ii), (iii), and (iv) are composed of the nucleotide sequences of SEQ ID NO: 8, SEQ ID NO: 2, SEQ ID NO: 9, and SEQ ID NO: 7, respectively. 2. The oligonucleotide primer set according to 1.
  3. 前記(i)，(ii)，(iii)，(iv)のオリゴヌクレオチドプライマーが，それぞれ配列番号１８，配列番号１２，配列番号１９，配列番号１７の塩基配列からなることを特徴とする請求項１に記載のオリゴヌクレオチドプライマーセット。
          The oligonucleotide primers (i), (ii), (iii), and (iv) are composed of the nucleotide sequences of SEQ ID NO: 18, SEQ ID NO: 12, SEQ ID NO: 19, and SEQ ID NO: 17, respectively. 2. The oligonucleotide primer set according to 1.
  4. 前記配列番号２から９および１２から１９，もしくはこれらと相補的な塩基配列からなるオリゴヌクレオチドプライマーであって，
    これら塩基配列と同一の少なくとも連続した15塩基を含む塩基配列のオリゴヌクレオチドプライマーからなることを特徴とする請求項１に記載のオリゴヌクレオチドプライマーセット。
          An oligonucleotide primer comprising the above SEQ ID NOs: 2 to 9 and 12 to 19, or a complementary nucleotide sequence thereto,
    2. The oligonucleotide primer set according to claim 1, comprising an oligonucleotide primer having a base sequence containing at least 15 consecutive bases identical to these base sequences.
  5. 請求項１から４に記載のオリゴヌクレオチドプライマーセットを，単独もしくは組み合わせて用いることにより，アフリカ豚コレラウイルスの標的核酸領域の増幅反応を行うことを特徴とするアフリカ豚コレラウイルスの検出方法。
          A method for detecting African swine fever virus, comprising amplifying the target nucleic acid region of swine cholera virus by using the oligonucleotide primer set according to claim 1 alone or in combination.
  6. アフリカ豚コレラウイルスの標的核酸領域の増幅反応が，LAMP法であることを特徴とする請求項５に記載のアフリカ豚コレラウイルスの検出方法。
          The method for detecting African swine fever virus according to claim 5, wherein the amplification reaction of the target nucleic acid region of African swine fever virus is the LAMP method.
  7. 請求項２に記載のオリゴヌクレオチドプライマーセットおよび配列番号１０，配列番号１１のオリゴヌクレオチドプライマーの組み合わせをプライマーセット１，
    請求項３に記載のオリゴヌクレオチドプライマーセットおよび配列番号２０，配列番号２１のオリゴヌクレオチドプライマーをプライマーセット２として，
    これらプライマーセット１と２を組み合わせてアフリカ豚コレラウイルスの検出を行うことを特徴とする請求項６に記載のアフリカ豚コレラウイルスの検出方法。
          A combination of the oligonucleotide primer set according to claim 2 and the oligonucleotide primer of SEQ ID NO: 10 and SEQ ID NO: 11 is used as primer set 1,
    The oligonucleotide primer set according to claim 3 and the oligonucleotide primers of SEQ ID NO: 20 and SEQ ID NO: 21 as primer set 2,
    The method for detecting African swine fever virus according to claim 6, wherein the detection of African swine fever virus is performed by combining these primer sets 1 and 2.
  8. アフリカ豚コレラウイルス感染症の検出方法において，請求項１ないし４に記載のオリゴヌクレオチドプライマーセットを含むことを特徴とする検出キット。

          In the detection method of African swine fever virus infection, the detection kit characterized by including the oligonucleotide primer set of Claims 1 thru | or 4.

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