JP5137096B2 - Identification method of specific gonococci - Google Patents

Description

  The present invention relates to a method for identifying a strain lacking a part of a genomic sequence similar to the aflatoxin biosynthesis gene cluster from among Aspergillus oryzae species, and a primer set for identifying the strain .

  Aspergillus oryzae has been used in the Japanese brewing industry since ancient times, and safety to humans has been recognized as GRAS (Generally Recognized As Safe: US FDA). On the other hand, Aspergillus flavus, a filamentous fungus of Aspergillus oryzae, has a strain that produces aflatoxin with strong carcinogenicity. It has already been verified by various chemical methods that Aspergillus does not produce aflatoxins. However, the existence of Aspergillus oryzae having a region similar to the aflatoxin biosynthesis gene cluster (Non-patent Document 1) covering about 70 Kb including 25 genes including enzymes presumed to be necessary for aflatoxin production possessed by Aspergillus flavus has been reported. (Non-patent document 2), the aflatoxin non-productivity of Neisseria gonorrhoeae has not yet been fully proved from the genome structure of the gene.

  However, the presence of strains that may lack a part of the region similar to the aflatoxin biosynthesis gene cluster has been reported in Neisseria gonorrhoeae (Non-patent Documents 2 and 3). It is definite that these strains have no ability to biosynthesize aflatoxins from the gene (genomic) structure. Therefore, using these strains in the food industry can give consumers a sense of security and is more desirable than using strains that have almost the same region as the aflatoxin biosynthesis gene cluster. However, there is currently no established method for easily identifying a koji strain lacking a region similar to this aflatoxin biosynthesis gene cluster.

Appl. Environ. Microbiol. 70, 1253-1262 (2004) Curr. Genet. 37, 104-111 (2000) Appl. Environ. Microbiol. 72, 484-490 (2006)

  An object of the present invention is to provide a method for easily identifying a strain lacking a region similar to an aflatoxin biosynthetic gene cluster in Neisseria gonorrhoeae, and a primer set therefor.

  As a result of intensive studies to solve the above problems, the present inventors have found that a group of Aspergillus oryzae lacking a part of the region similar to the aflatoxin biosynthetic gene cluster is included in the partial sequence of the gene cluster similar region. It was found that it has a telomere sequence, and by using this gene structure as an index, I thought that it was possible to identify Neisseria gonorrhoeae lacking a part of the region similar to the aflatoxin biosynthesis gene cluster, The present invention has been completed.

That is, according to the present invention, there is a fragmentation site A between 3018nt and 3019nt in the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing in the genomic DNA of Aspergillus or in the nucleotide sequence shown in SEQ ID NO: 2. Provided is a method for identifying koji molds, which comprises identifying koji molds that lack a part of the aflatoxin biosynthetic gene group-like sequence using as an index the presence of a cleavage site B between 2453 nt and 2454 nt. In addition, the present invention relates to a primer having a size of 15 bases or more that hybridizes to a partial region in the region of 1nt to 3018nt in the base sequence shown in SEQ ID NO: 1 in the sequence listing, and in the base sequence shown in SEQ ID NO: 1. A nucleic acid amplification method using a primer having a size of 15 bases or more that hybridizes to a partial region in a telomere region region after 3019 nt, and performing a nucleic acid amplification method that amplifies a region having a size of 3000 bp or less. A method for identifying Aspergillus oryzae comprising identifying Aspergillus oryzae that lacks a part of aflatoxin biosynthetic gene group-like sequences is performed based on whether or not a fragment of the size of a. Furthermore, the present invention relates to a primer having a size of 15 bases or more that hybridizes to a partial region within the region of 1 nt to 2453 nt in the base sequence shown in SEQ ID NO: 2, and a telomere after 2454 nt in the base sequence shown in SEQ ID NO: 2. A nucleic acid amplification method using a primer having a size of 15 bases or more that hybridizes to a partial region within a region region, and performing a nucleic acid amplification method that amplifies a region having a size of 3000 bp or less, and a fragment having a size of 3000 bp or less The present invention provides a method for identifying koji molds, which comprises identifying koji molds that lack a part of the aflatoxin biosynthetic gene group-like sequence, based on whether or not amplification occurs. Furthermore, the present invention relates to a primer having a size of 15 bases or more that hybridizes to a partial region in the region of 1 nt to 3018 nt in the base sequence shown in SEQ ID NO: 1 in the sequence listing, and a base sequence shown in SEQ ID NO: 1 A primer having a size of 15 bases or more that hybridizes to a partial region including a cleavage site A between 3018nt and 3019nt, and a portion that hybridizes to a partial region within the region of 1nt to 3018nt, and the cleavage site A An aflatoxin comprising a primer having a base number ratio of 3: 7 to 7: 3 or a partial region in the telomere region and a primer having a size of 15 or more bases Provided is a primer set for identifying Neisseria gonorrhoeae that lacks part of a biosynthetic gene group-like sequence. Furthermore, the present invention relates to a primer having a size of 15 bases or more that hybridizes to a partial region within the region of 1 nt to 2453 nt in the base sequence shown in SEQ ID NO: 2, and 2453 nt and 2454 nt in the base sequence shown in SEQ ID NO: 2. A primer having a size of 15 bases or more that hybridizes to a partial region including a cleavage site B between the region, and a portion that hybridizes to a partial region within the region of 1nt to 2453nt, and downstream of the cleavage site B Aflatoxin biosynthetic gene comprising a primer having a base number ratio of 3: 7 to 7: 3 or a partial region in the telomere region and a primer having a size of 15 bases or more Provided is a primer set for identifying Neisseria gonorrhoeae that lacks part of a group-like sequence.

  According to the present invention, it is possible to easily identify a gonococci that is definitive that there is no ability to biosynthesize aflatoxin, which lacks a part of the aflatoxin biosynthesis gene group-like sequence. Therefore, a koji mold that is identified by applying the method of the present invention and lacks a part of the aflatoxin biosynthetic gene group-like sequence and has no ability to biosynthesize aflatoxins is easily used in the food industry. And, in turn, can give consumers a sense of security.

  Details of the aflatoxin biosynthetic gene group similar sequence (hereinafter sometimes referred to as “AF cluster”) in the genomic DNA of Aspergillus oryzae RIB 40 strain, a conserved strain of the National Research Institute for Liquors The base sequence is disclosed (DDBJ accession number: AB071288, AB076803, AB076804, AB182368). The RIB40 strain was found to have almost the same structure as the Aspergillus flavus for the entire region of the aflatoxin biosynthetic gene cluster existing in Aspergillus flavus. The inventors of the present application select 7 genes (aflT, nor-1, aflR, norA, avnA, verB, vbs) distributed in the cluster of RIB 40 strain, and based on the respective base sequences, the koji mold genome A primer set for selectively amplifying and detecting the seven genes in the PCR by the PCR method was prepared, and 210 strains of Aspergillus oryzae stored at the National Institute of Liquors, Using the primer set, the presence of the 7 genes in the genome was investigated by PCR. As a result, for 122 strains, all 7 genes were amplified and it was confirmed that all of these genes were present on the genome. These 122 shares were designated as Group 1. As for 75 strains, amplification of 4 genes of aflT, nor-1, aflR, and norA was not observed, and these 75 strains were classified as group 2. Furthermore, in 8 strains, amplification of only vbs was observed, and a large-scale lack of clusters was expected, so these 8 strains were classified as Group 3.

  Analysis of the genomic structure (base sequence) around the AF cluster for RIB 143 strains in Group 3 revealed that the AF cluster was cleaved within the ordA gene in the AF cluster, followed by chromosomal telomeres. became.

  We designed a primer set that can amplify the region containing the fragmentation site of AF cluster (region containing telomere sequence) found in RIB 143 strain, and when PCR was performed using this primer set, all groups except RIB 1353 In the strain belonging to 3, an amplification product of the same size was obtained, and no amplification product was obtained in the group 1 and group 2 strains. From the above results, it was concluded that the seven genomes belonging to Group 3 have the same structure from the AF cluster to the telomere.

  Furthermore, when the RIB 1353 strain belonging to Group 3 was analyzed for the genomic structure (base sequence) around the AF cluster, the AF cluster was cleaved within the ordA gene in the AF cluster 566nt upstream from the cleavage site in RIB 143. It was revealed that chromosomal telomere continued.

  Therefore, we designed a primer set that can amplify the region including the fragmentation site of AF cluster (region containing telomere sequence) found in RIB 1353 strain. When PCR was performed using this primer set, all groups 3 An amplification product having a size of about 1.5 bp or 1.0 bp expected for the strain to which the strain belongs was obtained, and no amplification product was obtained for the group 1 and group 2 strains. From the above, it became clear that the strains belonging to Group 3 are classified into RIB 143 type or RIB 1353 type from the chromosome structure. In addition, the koji mold belonging to group 3 is deficient in more than half of the AF cluster, and it became clear that the ability to produce aflatoxin was lost due to its genomic structure. Although the koji mold has already been established in safety, it is extremely effective to use koji mold belonging to group 3 in the food industry in order to give consumers a sense of security. The present invention provides a method for identifying koji molds that can easily identify koji molds belonging to group 3, and a nucleic acid therefor.

  The structure of a partial region within the AF cluster of Aspergillus oryzae RIB 40 strain, RIB 143 strain and RIB 1353 strain is shown in FIG. In FIG. 1, light gray arrows indicate the direction of gene and transcription. The black part is the repeating sequence of the telomeric region of Aspergillus oryzae. The telomere sequence as one unit of the repeat is shown in SEQ ID NO: 3 in the sequence listing. As shown in FIG. 1, genes such as aflR essential for aflatoxin synthesis are deleted in group 3 RIB 143 strain and RIB 1353.

  The base sequence of the region from the vbs gene to the telomere in RIB 143 strain is shown in SEQ ID NO: 1 in the sequence listing. 3019 nt to 3151 nt in SEQ ID NO: 1 is a telomeric region, and a site between 3018 nt and 3019 nt is a division site between a part of an AF cluster in the strain and a telomeric region adjacent thereto (this specification and claims) In this range, it may be referred to as “partition site A”).

  The base sequence of the region from the vbs gene to the telomere in RIB 1353 strain is shown in SEQ ID NO: 2 in the sequence listing. 2454 nt to 2585 nt in SEQ ID NO: 2 are telomeric regions, and the region between 2453 nt and 2454 nt is a fragmentation site between a part of the AF cluster in the strain and the telomeric region adjacent thereto (this specification and claims) In this range, it may be referred to as “partition site B”).

  By clarifying the genomic structure around the AF cluster of the RIB 143 and RIB 1353 strains belonging to Group 3, the presence of a fragmentation site between a part of the AF cluster and the adjacent telomere region was used as an index. It has become possible to identify Neisseria gonorrhoeae that lacks part of the AF cluster belonging to 3. The presence of the cleavage site can be examined by various methods known per se using the nucleotide sequences shown in SEQ ID NOs: 1 and 2 disclosed by the present invention.

  The presence of the fragmentation site is, for example, whether or not amplification occurs by performing a nucleic acid amplification method such as PCR using a genomic DNA of Aspergillus as a template and a primer capable of amplifying a partial region containing the fragmentation site. You can look up on the basis. In this case, one of the primers may be set to hybridize across the split site, and the other may be set to hybridize to a partial region upstream or downstream (i.e., within the telomere region) of the split site, Or you may set a pair of primer so that it may each hybridize with the partial area | region upstream of a parting part, and the partial area | region in a telomere area | region. In the former case, amplification does not occur because the primer does not hybridize unless the above-mentioned fragmentation site exists, and in the latter case, amplification does not occur unless the above-mentioned fragmentation site exists (Group 1 complete with AF cluster). Then, both primers hybridize with the template DNA, but amplification does not occur because the distance between the two hybridized primers is too far). Therefore, in any case, the fact that amplification occurs indicates that the fragmentation site exists. Here, “hybridize” means to hybridize under the conditions in the normal PCR annealing step based on complementarity of base sequences.

  That is, using a primer that hybridizes to a partial region within the region of 1nt to 3018nt in the base sequence shown in SEQ ID NO: 1 and a primer that hybridizes to a partial region containing the fragmentation site A, the genomic DNA of Aspergillus oryzae is used as a template. The presence of the fragmentation site A can be detected based on whether amplification occurs or not. The primer is preferably a nucleic acid consisting of a region of 15 bases or more in the sequence of SEQ ID NO: 1 or its complementary strand. Usually, amplification occurs even when a primer having a sequence substituted with 10% or less of the base is used. There are many cases. Therefore, as a primer that hybridizes to a partial region in the region of 1nt to 3018nt, a region of 15 or more consecutive bases in the region of 1nt to 3018nt in the base sequence shown in SEQ ID NO: 1 or 10% or less of those bases As a primer having the same base sequence as the region substituted by, a primer hybridizing to the partial region including the fragmentation site A, a region complementary to the region of 15 bases or more including the fragmentation site A or 10 of them The nucleic acid amplification method can be preferably carried out using a primer having the same base sequence as the region where% or less bases are substituted. Similarly, using a primer that hybridizes to a partial region within the region of 1nt to 2453nt in the base sequence shown in SEQ ID NO: 2 and a primer that hybridizes to a partial region containing the fragmentation site B, The nucleic acid amplification method using the template can be performed, and the presence of the cleavage site B can be detected based on whether amplification occurs. As in the case of the fragmentation site A, as a primer that hybridizes to a partial region in the region of 1nt to 2453nt, a region of 15 or more consecutive bases in the region of 1nt to 2453nt in the base sequence shown in SEQ ID NO: 2 or those As a primer having the same base sequence as the region substituted with 10% or less of the base, and a primer hybridizing to the partial region containing the fragmentation site B, complementary to a region of 15 or more consecutive bases containing the fragmentation site B The above-mentioned nucleic acid amplification method can be preferably performed using a primer having the same base sequence as that of the above-described region or a region where 10% or less of the base thereof is substituted. In the case where there is no fragmentation site A or fragmentation site B shown in SEQ ID NO: 1 or SEQ ID NO: 2, in order to ensure that the primer that hybridizes to the partial region across the fragmentation site does not hybridize, The primer is composed of a portion that hybridizes to the 1 nt to 3018 nt region in the base sequence shown in SEQ ID NO: 1 or the 1 nt to 2453 nt region in the base sequence shown in SEQ ID NO: 2 and a portion that hybridizes to the telomere region. The ratio (ratio of the number of bases) is preferably 3: 7 to 7: 3, more preferably 4: 6 to 6: 4.

  In addition, a primer that hybridizes to a partial region in the region of 1nt to 3018nt in the base sequence shown in SEQ ID NO: 1 in the sequence listing, and a partial region in the telomere region region after 3019nt in the base sequence shown in SEQ ID NO: 1 A primer that hybridizes with a nucleic acid amplification method using a genomic DNA of Aspergillus as a template, or a primer that hybridizes to a partial region in the region of 1nt to 2453nt in the base sequence shown in SEQ ID NO: 2; Nucleic acid amplification using a primer that hybridizes to a partial region within the telomere region after 2454 nt in the base sequence shown, and amplifying a region of 3000 bp or less in size using a genomic DNA of Aspergillus as a template And identify gonococci that lack a part of the aflatoxin biosynthesis gene group-like sequence based on whether amplification of fragments of 3000 bp or less occurs It is also possible to perform identification of koji mold including Rukoto. In this case, the two primers used in the nucleic acid amplification method both hybridize to the gonococcal genomic DNA complete with AF clusters, but the distance between the two primers is much greater than 3000 bp, so amplification does not occur, at least Amplification does not occur under conditions that amplify a region of 3000 bp or less. Also in this method, the primer is preferably a nucleic acid consisting of a region of 15 bases or more in SEQ ID NO: 1 or its complementary strand, but usually a primer having a sequence substituted with 10% or less of bases. Even when used, amplification often occurs. Therefore, as a primer that hybridizes to a partial region within the 1 nt to 3018 nt region in the base sequence shown in SEQ ID NO: 1, a region of 15 or more consecutive bases in the 1 nt to 3018 nt region in the base sequence shown in SEQ ID NO: 1 Alternatively, a primer having the same base sequence as the region substituted with 10% or less of those bases is used, and a primer that hybridizes to a partial region in the telomeric region in the base sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 1. The above nucleic acid amplification method can be preferably carried out using a primer having the same base sequence as a region of 15 bases or more complementary to the telomere region or a region substituted with 10% or less of those bases. Similarly, as a primer that hybridizes to a partial region in the region of 1nt to 2453nt in the base sequence shown in SEQ ID NO: 2, 15 or more consecutive bases in the region of 1nt to 2453nt in the base sequence shown in SEQ ID NO: 2 As a primer that hybridizes to a partial region in the telomeric region in the base sequence shown in SEQ ID NO: 2, using a primer having the same base sequence as that of the above region or a region in which 10% or less of those bases are substituted The above nucleic acid amplification method can be preferably carried out using a primer having the same base sequence as the region of 15 bases or more complementary to the telomere region shown in 2 or a region where 10% or less of those bases are substituted.

  In any of the above nucleic acid amplification methods, the size of the primer is not particularly limited, but is usually about 15 to 50 bases, preferably about 18 to 40 bases. The size of the amplification region is not particularly limited, but if it is too large, it takes time for amplification and the amount of reagent used increases. Therefore, the size of the amplification region is preferably 3000 bp or less, and more preferably 1500 bp or less. On the other hand, since the amplified fragment can be easily detected by electrophoresis or the like, the amplification region preferably has a size of 50 bp or more. Nucleic acid amplification methods such as PCR are well known, and kits and devices for such methods are also commercially available, and can be easily implemented using them. Whether or not amplification has occurred can be easily examined by subjecting the amplification reaction product to electrophoresis and detecting or not detecting a band by a conventional method. Furthermore, if necessary, the base sequence of the amplification product (preferably a short one of several tens of bp) may be directly determined just in case to confirm the presence of the cleavage site.

  The present invention also provides a set of primers used in the nucleic acid amplification method described above.

  In addition, the presence of a fragmentation site is determined by using a nucleic acid probe that hybridizes to a partial region including the fragmentation site (region straddling the fragmentation site), and whether or not the nucleic acid probe hybridizes to the genomic DNA of Aspergillus oryzae that serves as a sample. You can also check by checking. Here, “whether or not to hybridize” means a stringent that is used to determine the presence or absence of a target nucleic acid based on whether or not a nucleic acid probe hybridizes in a conventional method such as Southern blotting. It means to hybridize under various conditions. Such conditions are well known to those skilled in the art. In this case, in order to prevent hybridization when there is no fragmentation site A or fragmentation site B shown in SEQ ID NO: 1 or SEQ ID NO: 2, the structure of the nucleic acid probe is the base sequence shown in SEQ ID NO: 1 The ratio of the portion that hybridizes to the region of 1nt to 3018nt in the region or the region of 1nt to 2453nt in the base sequence shown in SEQ ID NO: 2 and the portion that hybridizes to the telomere region (base number ratio) is preferably 3: 7 to 7: 3, more preferably 4: 6 to 6: 4. As is well known, the nucleic acid probe is usually labeled with a well-known label such as a hapten label, a fluorescent label, a radiolabel, an enzyme label, or a biotin label.

  The detection method itself using a nucleic acid probe is well known in this field, and for example, Southern blotting can be exemplified. In order to increase the detection sensitivity, the sample DNA may be amplified by a nucleic acid amplification method such as PCR (PCR-Southern blot) prior to the Southern blot method. Alternatively, it can also be carried out by immobilizing the nucleic acid probe, labeling the sample DNA, and examining whether the sample DNA hybridizes to the solid phase probe. In this case as well, in order to increase the detection sensitivity, the sample DNA may be amplified by a gene amplification method such as PCR prior to hybridization with the solid phase probe. The amplified DNA can also be labeled by using nucleotides labeled with a radiolabel or the like.

  In the present specification, the “partial region” refers to a partial region in the base sequence shown in SEQ ID NO: 1 or 2, and is preferably a continuous region of 15 bases or more. Note that, according to the rules, only one strand is described in the sequence listing even in the case of double-stranded DNA. Therefore, only one strand is described in SEQ ID NO: 1 and SEQ ID NO: 2. This region is double-stranded, and therefore the single-stranded complementary strands shown in SEQ ID NO: 1 and SEQ ID NO: 2 can also be used for the detection. That is, “SEQ ID NO: 1” and “SEQ ID NO: 2” are intended to include specifically described single strands and their complementary strands and duplexes unless otherwise apparent from the context. Used.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Genomic features of 3 strains of Neisseria gonorrhoeae lacking a part of the sequence similar to aflatoxin biosynthetic genes The sequence similar to the aflatoxin biosynthetic genes for group 3 RIB 143 and RIB 1353 The surrounding genomic base sequences were examined in detail. As a result, compared with the genome sequence of RIB 40 strain belonging to Group 1, it is similar to the aflatoxin biosynthesis gene group including 6 genes of aflT, nor-1, aflR, norA, avnA and verB existing in RIB 40 strain. It was revealed that about 56 kb was missing from the sequence, followed by a chromosomal telomere sequence. The decoded base sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 as described above. The telomere sequence is shown in SEQ ID NO: 3. This is the first time in the world that the genome sequence of this part of Aspergillus oryzae belonging to Group 3 lacking a part of the sequence similar to the aflatoxin biosynthetic gene group has been decoded. From the results of genome structure analysis of RIB 143 and RIB 1353 strains, RIB 143 and RIB 1353 strains were divided into chromosomes upstream of the vbs sequence that is common with the RIB 40 strain of Group 1, and the heterotelomer sequence was It was estimated that it was added. A schematic diagram of a cluster structure similar to the aflatoxin biosynthesis gene group of RIB 40 strain, RIB 143 strain and RIB 1353 strain is shown in FIG. 1 as described above. Here, aflR is an important regulatory factor that positively controls the biosynthesis gene of aflatoxin in Aspergillus flavus, and it has been confirmed that aflatoxin is not produced by disruption of this gene. In group 3, the deletion of the aflR gene was confirmed, thus demonstrating that the Aspergillus oryzae strain belonging to group 3 has no ability to produce aflatoxins.

2. Identification of group 3 strains by PCR Genomes that are cleaved in the AF cluster in the ordA gene of RIB 143 and RIB 1353 strains belonging to group 3, followed by chromosomal telomeres. It had a structure (base sequence). Therefore, primers for specifically amplifying the region containing the fragmented partial structures of RIB 143 strain and RIB 1353 strain by PCR were designed (sequence a: SEQ ID NO: 4, sequence b: SEQ ID NO: 5). The sequence of SEQ ID NO: 4 is the same sequence as the antisense strand of 3023 nt to 3052 nt of SEQ ID NO: 1, and the same sequence as the antisense strand of 2457 nt to 2486 nt of SEQ ID NO: 2, and SEQ ID NO: 5 Is the same sequence as 1515nt to 1542nt of the sense strand of SEQ ID NO: 1 and SEQ ID NO: 2. Therefore, when PCR is performed using the sequence of SEQ ID NO: 1 as a template using this primer set, a 1538 bp region sandwiched between these primers is amplified, and when PCR is performed using the sequence of SEQ ID NO: 2 as a template, A region of 972 bp sandwiched between these primers is amplified. Using this primer set, PCR was carried out using as a template the genomes of koji molds classified into all groups 3. As an internal control of PCR, primers capable of amplifying 312 bp tubulin gene fragments obtained from all strains (sequence c: 5′-ccaagaacatgatggctgct-3 ′, sequence d: 5′-cttgaagagctcctggatgg-3 ′) It was used. Specifically, PCR was performed as follows. For PCR, each genomic DNA, primer (a), primer (b), primer (c), primer (d), 13 μL Insert Check-Ready-Mix (Code No .: PIK-251, TOYOBO Co. Ltd. PCR, using GeneAmp PCR sysytems 9700 (Applied Biosystems) for 4 minutes, followed by denaturation (94 ° C, 20 seconds), annealing (94 ° C, 20 seconds) 25 cycles of 68 ° C., 5 seconds) and extension reaction (72 ° C., 30 seconds). A 1% agarose gel electrophoresis was performed on 3 μL of the PCR reaction-terminated solution using 1 × Tris-Acetate-EDTA (TAE) buffer, and then treated with ethidium bromide for 20 minutes, and the gel was irradiated with ultraviolet rays to detect PCR products.

  As a result, no PCR reaction product was detected in the strains belonging to Group 1 and Group 2, and PCR amplification products having the same size as expected from the sequences were detected in RIB 143 and RIB 1353 strains. Moreover, PCR amplification products having the same size as the RIB 143 strain were detected in all of the 8 strains belonging to Group 3 except the RIB 1353 strain. Since these 7 strains in Group 3 have the same size of the amplification product, it is clear that they have a chromosomal genomic structure in which a sequence deletion similar to the RIB 143 aflatoxin biosynthetic gene group has occurred. It became.

  In addition, since a PCR product using primers (a) and (b) can produce a reaction product only in group 3, the PCR reaction using this primer results in a sequence similar to the aflatoxin biosynthetic gene group. It was proved that the strain of group 3 lacking a part can be specifically and easily identified and classified.

  Furthermore, a primer for specifically amplifying a region containing a fragmented partial structure only from a strain having a chromosome structure of RIB 143 among strains belonging to group 3 was designed (sequence e: SEQ ID NO: 6, Sequence f: SEQ ID NO: 7). The sequence of SEQ ID NO: 6 is the same sequence as the antisense strand of SEQ ID NO: 3005nt to 3031, and the sequence of SEQ ID NO: 7 is the same as 2417nt to 2438nt of the sense strand of SEQ ID NO: 1 and SEQ ID NO: 1. Is an array of Therefore, when PCR is carried out using this primer set with the sequence of SEQ ID NO: 1 as a template, a 615 bp region sandwiched between these primers is amplified. Using this primer set, PCR was carried out using as a template the genomes of koji molds classified into all groups 3. Specifically, PCR was performed as follows. For PCR, each genomic DNA, primer (e), primer (f), primer (c), primer (d), 13 μL Insert Check-Ready-Mix (Code No .: PIK-251, TOYOBO Co. Ltd. PCR, using GeneAmp PCR systems 9700 (Applied Biosystems) for 4 minutes, followed by denaturation (94 ° C, 20 seconds), annealing using GeneAmp PCR systems 9700 (Applied Biosystems) (65 ° C., 5 seconds) and elongation reaction (72 ° C., 30 seconds) for 25 cycles. A 1% agarose gel electrophoresis was performed on 3 μL of the PCR reaction-terminated solution using 1 × Tris-Acetate-EDTA (TAE) buffer, and then treated with ethidium bromide for 20 minutes, and the gel was irradiated with ultraviolet rays to detect PCR products.

  As a result, PCR reaction products were not detected in strains belonging to Group 1 and Group 2, and PCR amplification of the same size as RIB 143 strain was performed only in 6 strains belonging to Group 3 and having the same chromosome structure as RIB 143 strain. A product was detected.

  Since PCR products using primers (e) and (f) can produce reaction products only in strains belonging to Group 3 and having the same chromosomal structure as RIB 143, the PCR reaction using this primer produced aflatoxin production. It was proved that a strain having a common chromosomal structure with the RIB 143 strain can be specifically and easily identified and classified among the strains of group 3 lacking a part of the sequence similar to the synthetic gene group.

  According to the method of the present invention, it is possible to easily identify a gonococcus definitive that there is no ability to biosynthesize aflatoxin from a gene (genome) structure. The use of koji molds that are identified using the method of the present invention and have a definite ability to biosynthesize aflatoxins in the food industry or the like can give a sense of security to consumers.

Structure of part of the Aspergillus oryzae RIB 40 strain having a region similar to the aflatoxin biosynthesis gene cluster and part of the region similar to the aflatoxin biosynthesis gene cluster, RIB 143 and RIB 1353 It is a figure which shows the structure of a part.

Claims (20)

  In the genomic DNA of Neisseria gonorrhoeae, there is a cleavage site A between 3018nt and 3019nt in the base sequence shown in SEQ ID NO: 1 in the sequence listing, or between 2453nt and 2454nt in the base sequence shown in SEQ ID NO: 2. A method for identifying koji molds, comprising identifying koji molds that lack a part of the aflatoxin biosynthetic gene group-like sequence, using as an index the presence of a split site B. A primer that hybridizes to a partial region in the region of 1nt to 3018nt in the base sequence shown in SEQ ID NO: 1 and that has a size of 15 bases or more, and hybridizes to a partial region that includes the cleavage site A, and that has a size of 15 bases or more of a primer, the hybridizing portion in the partial regions within the region of the 1Nt～3018nt, base ratio of the number of the downstream side and the hybridizing portion than the split site a is 3: 7 to 7: 3 The method according to claim 1, wherein a nucleic acid amplification method is performed using a primer that is a gonococcal genomic DNA as a template, and the amplification is performed based on whether amplification occurs. The primer that hybridizes to a partial region in the region of 1nt to 3018nt has the same base sequence as a region of 15 or more bases in the region of 1nt to 3018nt in the base sequence shown in SEQ ID NO: 1, The method according to claim 2 , wherein the primer that hybridizes to the partial region containing the split site A has the same base sequence as a region complementary to a continuous region of 15 or more bases containing the split site A. The method according to claim 2 or 3, wherein the size of each primer is 18 bases or more. The method according to claim 4 , wherein the method is carried out using a pair of primers having the base sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively. Hybridizing to a partial region within the region of 1nt~2453nt in the base sequence shown in SEQ ID NO: 2, size hybridizes to a partial area including the above primer 15 bases, the split site B, and the size The ratio of the number of bases of a primer having 15 bases or more and a portion that hybridizes to a partial region within the region of 1nt to 2453nt and a portion that hybridizes to the downstream side of the cleavage site B is from 3: 7 to The method according to claim 1, wherein a nucleic acid amplification method using a 7: 3 primer and a genomic DNA of Aspergillus oryzae as a template is performed, and the amplification is performed based on whether or not amplification occurs. The primer that hybridizes to a partial region in the region of 1nt to 2453nt has the same base sequence as a region of 15 or more bases continuous in the region of 1nt to 2453nt in the base sequence shown in SEQ ID NO: 2, The method according to claim 6 , wherein the primer that hybridizes to a partial region including a split site has the same base sequence as a region complementary to a continuous region of 15 bases or more including the split site B. The method according to any one of claims 5 to 7 , wherein the size of each primer is 18 bases or more. A primer having a size of 15 bases or more that hybridizes to a partial region in the region of 1nt to 3018nt in the base sequence shown in SEQ ID NO: 1 in the sequence listing, and a telomere region region after 3019nt in the base sequence shown in SEQ ID NO: 1. A nucleic acid amplification method using a primer having a size of 15 bases or more that hybridizes to a partial region of the DNA and amplifying a region having a size of 3000 bp or less, and amplifying fragments having a size of 3000 bp or less A method for identifying Aspergillus oryzae, comprising identifying Aspergillus oryzae that lacks a part of an aflatoxin biosynthetic gene group-like sequence, which is performed based on whether or not the occurrence occurs. A primer that hybridizes to a partial region in a region of 1nt to 3018nt in the base sequence shown in SEQ ID NO: 1 is a region of 15 or more consecutive bases in a region of 1nt to 3018nt in the base sequence shown in SEQ ID NO: 1. A primer having the same base sequence and hybridizing to a partial region in the telomeric region in the base sequence shown in SEQ ID NO: 1 is the same as a region of 15 bases or more complementary to the telomeric region shown in SEQ ID NO: 1. The method of Claim 9 which has a base sequence. Hybridizing to a partial region within the region of 1nt~2453nt in the base sequence shown in SEQ ID NO: 2, size 15 and a base or more primers, 245 4 nt subsequent telomeric region within the region of nucleotide sequence shown in SEQ ID NO: 2 A nucleic acid amplification method using a primer having a size of 15 bases or more that hybridizes to a partial region of the nucleic acid and amplifying a region having a size of 3000 bp or less. A method for identifying Neisseria gonorrhoeae comprising identifying Neisseria gonorrhoeae that lacks a part of aflatoxin biosynthetic gene group-like sequences, based on whether or not it occurs. A primer that hybridizes to a partial region in the region of 1nt to 2453nt in the nucleotide sequence shown in SEQ ID NO: 2 is a region of 15 or more consecutive bases in the region of 1nt to 2453nt in the nucleotide sequence shown in SEQ ID NO: 2. A primer having the same base sequence and hybridizing to a partial region in the telomere region in the base sequence shown in SEQ ID NO: 2 is the same as a region of 15 bases or more complementary to the telomere region shown in SEQ ID NO: 2 The method according to claim 11 , which has a base sequence. The method according to any one of claims 9 to 12, wherein the size of each primer is 18 bases or more. The method of Claim 9 or 10 performed using a pair of primer which has a base sequence each shown to sequence number 4 and sequence number 5. A primer having a size of 15 bases or more that hybridizes to a partial region within the region of 1nt to 3018nt in the base sequence shown in SEQ ID NO: 1 of the sequence listing, and between 3018nt and 3019nt in the base sequence shown in SEQ ID NO: 1. A primer having a size of 15 bases or more that hybridizes to a partial region including the fragmentation site A, and a portion that hybridizes to a partial region within the region of 1 nt to 3018 nt, and to the downstream side of the fragmentation site A Aflatoxin biosynthetic gene group-like sequence comprising a primer having a base number ratio of 3: 7 to 7: 3 or a primer having a size of 15 bases or more that hybridizes to a partial region in the telomere region Primer set for identifying Neisseria gonorrhoeae that lacks a part of The primer that hybridizes to a partial region in the region of 1nt to 3018nt has the same base sequence as a region of 15 or more bases in the region of 1nt to 3018nt in the base sequence shown in SEQ ID NO: 1, The primer that hybridizes to the partial region including the fragmentation site A has the same base sequence as the region complementary to the continuous region of 15 bases or more including the fragmentation site A, and hybridizes to the partial region in the telomere region. The primer set according to claim 15 , wherein the soybean primer has the same base sequence as a region of 15 bases or more complementary to the telomere region shown in SEQ ID NO: 1. The primer set according to claim 15 or 16, wherein the size of each primer is 18 bases or more. A primer having a size of 15 bases or more that hybridizes to a partial region within the region of 1nt to 2453nt in the base sequence shown in SEQ ID NO: 2 and a cleavage site B between 2453nt and 2454nt in the base sequence shown in SEQ ID NO: 2 A primer having a size of 15 bases or more, which hybridizes to a partial region containing 1 part to 2453 nt, and a part which hybridizes to a downstream side of the cleavage site B Part of aflatoxin biosynthetic gene group-like sequence consisting of a primer having a base ratio of 3: 7 to 7: 3 or a primer having a size of 15 bases or more that hybridizes to a partial region in the telomere region Primer set for identifying Neisseria gonorrhoeae that lacks. The primer that hybridizes to a partial region in the region of 1nt to 2453nt has the same base sequence as a region of 15 or more bases continuous in the region of 1nt to 2453nt in the base sequence shown in SEQ ID NO: 2, The primer that hybridizes to the partial region including the fragmentation site B has the same base sequence as the region complementary to the continuous region of 15 bases or more including the fragmentation site B, and hybridizes to the partial region in the telomere region. The primer set according to claim 18 , wherein the soybean primer has the same base sequence as a region of 15 bases or more complementary to the telomere region shown in SEQ ID NO: 2. The primer set according to claim 18 or 19, wherein the size of each primer is 18 bases or more.

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