JP7390736B2 - Simultaneous detection method and primer kit for rot fungi - Google Patents

Description

The present invention relates to a method and a primer kit for simultaneous detection of ERIC fungi, and more specifically, to a method for simultaneous detection of ERIC fungi, and more specifically to an American ERIC fungus with genotype ERIC type I, an American ERIC fungus with genotype ERIC type II, and a method for detecting ERIC fungi other than ERIC types I and II. The present invention relates to a method for simultaneous detection of a genotypic American rot fungus, a phenotypically atypical European rot fungus, and a phenotypically typical European rot fungus, and a primer kit.

Two types of bacteria are known to be responsible for Paenibacillus larvae, which is a monitored infectious disease of honeybees: Paenibacillus larvae and Melissococcus plutonius. These organisms have different toxicity and resistance to disinfectants depending on their genotype and phenotype, so it is important to know which type of fungus causes rot disease that occurs in the field, and to identify bee colonies that have not yet developed the disease. It is extremely important to know which type of rot fungus the plant is currently contaminated with in order to take disease control measures. In addition, in order to use the only currently available anti-rot disease preventive agent (Tylosin) effectively for a long time without spreading resistant bacteria, we need to understand the contamination status of the rot fungi in bee colonies and decide which groups should be treated. It is important to identify the symptoms and use preventive drugs appropriately.

Currently, five types of American rot fungi are known, with genotypes ranging from ERIC type I to ERIC type V, and European rot fungi have typical and atypical phenotypes. Two types are known: Among these, the American scab fungus with the genotype ERIC type I, the American scab fungus with the genotype ERIC type II, the phenotypically atypical European scab fungus, and the phenotypically typical European scab fungus are currently the most common in the world. It is rampant in Japan, and is causing rot maggot disease in Japan as well. In particular, the number of domestic cases caused by the American rot fungus of genotype ERIC type II has been rapidly increasing in recent years.

As a detection method for European rot fungi, a PCR method that distinguishes between typical strains and atypical strains has been reported (Arai et al., 2014, J. Vet. Med. Sci., 76 :491-498 (Non-Patent Document 1)). Additionally, a PCR method for detecting the ERIC type I strain has been reported for the American rot fungus (Beims et al., 2020, Open Vet. J., 10:53-58 (Non-patent Document 2)). A PCR method that can specifically detect the American rot fungus of genotype ERIC type II, which is highly toxic to larvae and is becoming increasingly important in Japan, has not yet been reported.

In addition, although it has been common practice to detect each of the two rot fungi individually, Japanese Patent Application Laid-Open No. 2019-062745 (Patent Document 1) describes a method for simultaneously detecting pathogenic bacteria including the two rot fungi. is listed. However, a method that can simultaneously detect two rot fungi after distinguishing their genotypes and phenotypes has not yet been reported.

JP2019-062745A

Arai et al. , 2014, J. Vet. Med. Sci. , 76:491-498 Beims et al. , 2020, Open Vet. J. , 10:53-58

The present invention has been made in view of the above-mentioned problems, and provides a method for simultaneously and easily detecting two rot fungi after distinguishing their genotypes and phenotypes. The American rot fungus with genotype ERIC type II, the American rot fungus with genotypes other than ERIC types I and II, the phenotypically atypical European fungus, and the phenotypically typical European fungus. The object of the present invention is to provide a method for simultaneous detection of rot fungi that can be detected simultaneously and easily, and a primer kit that can be suitably used for this method.

The present inventors have conducted extensive research to achieve the above objective, and have designed a multiplex PCR system that can simultaneously detect two rot fungi after distinguishing their genotypes and phenotypes. That is, first, specific target sequences to be detected by multiplex PCR were selected for each genotype or phenotype of the two rot fungi, American rot fungus and European rot fungus. In particular, as a sequence specific to the American rot fungus of genotype ERIC type II, Funfhaus et al. , 2009, Environ. Microbiol. Rep. , 1:240-250, the present inventors determined that the genotype of ERIC type II was determined by comparing the genome information of multiple American rot fungus strains with known genotypes. It was discovered that there is only one sequence that is truly specific to the American rot fungus. In addition, for the American rot fungus of genotype ERIC type I or type II, which is currently particularly rampant, target sequences specific to each genotype were selected, and in addition, target sequences for all genotypes including these were selected. By selecting the 16S rRNA gene sequence as a common target sequence of the American rot fungus, and further combining these sequences, the American rot fungus of any genotype other than ERIC types I and II (hereinafter referred to as In addition, in some cases, it was also possible to detect American rot fungi with genotypes other than ERIC types I and II.

Next, the present inventors designed primers for the target sequences of each of the rot fungi selected above. Target sequences and phenotypes of all genotypes (currently genotypes ERIC types I to V) of the American rot fungus (herein sometimes referred to as "all genotypes of the American rot fungus") and phenotypes typical of Europe. Previously reported primers were used for each target sequence of the rot fungus, but multiple primers were newly designed for each of the other target sequences. Furthermore, from among multiple combinations of these primers, we selected positive controls (genotype ERIC type I American scab fungus, genotype ERIC type II American scab fungus, phenotypic typical European scab fungus, and phenotype When performing multiplex PCR using a sample containing all the genomic DNA of an atypical European rot fungus as a template, all of the desired specific PCR products derived from each target sequence can be confirmed, and 5 We found a combination in which the PCR products of each species all have different sizes.

As a result, multiplex PCR is performed using such a specific combination of primers, that is, a combination of primers that can amplify a specific target region on the target sequence, and the PCR products are separated and detected by, for example, electrophoresis. Therefore, the American rot fungus with genotype ERIC type I, the American rot fungus with genotype ERIC type II, the American rot fungus with genotypes other than ERIC types I and II, and the phenotypically atypical European rot fungus. The present inventors have discovered that it is possible to simultaneously and easily detect all of the phenotypic typical European rot fungi, and have completed the present invention.

Aspects of the present invention obtained from this knowledge are as follows.
[1]
Paenibacillus larvae ERIC I with genotype ERIC type I, Paenibacillus larvae ERIC II with genotype ERIC type II, and Paenibacillus larvae ERIC II with genotypes other than ERIC type I and II in the sample. A method for simultaneously detecting a phenotypic atypical Melissococcus plutonius, and a phenotypic typical Melissococcus plutonius,
Using the DNA derived from the sample as a template, the following five target regions:
The first target region from 53rd to 1024th in the nucleotide sequence set forth in SEQ ID NO: 1,
a second target region from positions 794 to 2101 in the nucleotide sequence set forth in SEQ ID NO: 2;
the third target region from positions 2 to 339 in the nucleotide sequence set forth in SEQ ID NO: 3;
A fourth target region from positions 109 to 367 in the nucleotide sequence set forth in SEQ ID NO: 4, and a fifth target region from positions 36 to 222 in the nucleotide sequence set forth in SEQ ID NO: 5,
A PCR step in which multiplex PCR is performed using five types of primer sets, each of which is a combination of primers that can amplify the
a detection step of detecting the PCR product obtained in the PCR step;
A method for simultaneously detecting rot fungi, including:
[2]
The five types of primer sets are
A primer that is a combination of forward primer I that hybridizes to the 53rd to 75th sites in the nucleotide sequence of SEQ ID NO: 1 and reverse primer I that hybridizes to the 1002nd to 1024th sites in the nucleotide sequence of SEQ ID NO: 1. set I,
A primer that is a combination of forward primer II that hybridizes to the 1369th to 1396th sites in the nucleotide sequence of SEQ ID NO: 2 and reverse primer II that hybridizes to the 1895th to 1922nd sites in the nucleotide sequence of SEQ ID NO: 2. Set II,
A primer that is a combination of a forward primer III that hybridizes to positions 3 to 30 in the nucleotide sequence of SEQ ID NO: 3 and a reverse primer III that hybridizes to positions 312 to 335 in the nucleotide sequence of SEQ ID NO: 3. Set III,
A primer that is a combination of a forward primer IV that hybridizes to positions 111 to 138 in the nucleotide sequence of SEQ ID NO: 4 and a reverse primer IV that hybridizes to positions 340 to 367 in the nucleotide sequence of SEQ ID NO: 4. Set IV, and a forward primer V that hybridizes to the 36th to 55th sites in the nucleotide sequence of SEQ ID NO: 5, and a reverse primer V that hybridizes to the 201st to 222nd sites in the nucleotide sequence of SEQ ID NO: 5. Primer set V, which is a combination,
The method for simultaneous detection of rot fungi according to [1].
[3]
Simultaneous detection of rot fungi according to [2], wherein the primer set II is a combination of a forward primer II consisting of the nucleotide sequence of SEQ ID NO: 8 and a reverse primer II consisting of the nucleotide sequence of SEQ ID NO: 9. Method.
[4]
The rotten maggot according to [2] or [3], wherein the primer set III is a combination of a forward primer III consisting of the nucleotide sequence of SEQ ID NO: 10 and a reverse primer III consisting of the nucleotide sequence of SEQ ID NO: 11. Method for simultaneous detection of disease bacteria.
[5]
Any one of [2] to [4], wherein the primer set IV is a combination of a forward primer IV consisting of the nucleotide sequence of SEQ ID NO: 12 and a reverse primer IV consisting of the nucleotide sequence of SEQ ID NO: 13. The method for simultaneous detection of rot fungi according to item 1.
[6]
The five types of primer sets are
Primer set I, which is a combination of forward primer I consisting of the nucleotide sequence of SEQ ID NO: 6 and reverse primer I consisting of the nucleotide sequence of SEQ ID NO: 7;
Primer set II, which is a combination of forward primer II consisting of the nucleotide sequence of SEQ ID NO: 8 and reverse primer II consisting of the nucleotide sequence of SEQ ID NO: 9;
Primer set III, which is a combination of forward primer III consisting of the nucleotide sequence of SEQ ID NO: 10 and reverse primer III consisting of the nucleotide sequence of SEQ ID NO: 11;
Primer set IV is a combination of forward primer IV consisting of the nucleotide sequence of SEQ ID NO: 12 and reverse primer IV consisting of the nucleotide sequence of SEQ ID NO: 13; and forward primer V consisting of the nucleotide sequence of SEQ ID NO: 14. A primer set V, which is a combination of a reverse primer V consisting of the nucleotide sequence of SEQ ID NO: 15,
The method for simultaneous detection of rot fungi according to any one of [1] to [5].
[7]
The method for simultaneous detection of rot fungi according to any one of [1] to [6], wherein the detection step includes a step of separating the PCR product by electrophoresis.
[8]
Primer set II, which is a combination of forward primer II consisting of the nucleotide sequence of SEQ ID NO: 8 and reverse primer II consisting of the nucleotide sequence of SEQ ID NO: 9;
Primer set III is a combination of forward primer III consisting of the nucleotide sequence of SEQ ID NO: 10 and reverse primer III consisting of the nucleotide sequence of SEQ ID NO: 11; and forward primer IV consisting of the nucleotide sequence of SEQ ID NO: 12. Reverse primer IV consisting of the nucleotide sequence of SEQ ID NO: 13, and primer set IV, which is a combination of
A primer kit comprising at least one primer set selected from the group consisting of:
[9]
Primer set I, which is a combination of forward primer I consisting of the nucleotide sequence of SEQ ID NO: 6 and reverse primer I consisting of the nucleotide sequence of SEQ ID NO: 7;
Primer set II, which is a combination of forward primer II consisting of the nucleotide sequence of SEQ ID NO: 8 and reverse primer II consisting of the nucleotide sequence of SEQ ID NO: 9;
Primer set III, which is a combination of forward primer III consisting of the nucleotide sequence of SEQ ID NO: 10 and reverse primer III consisting of the nucleotide sequence of SEQ ID NO: 11;
Primer set IV is a combination of forward primer IV consisting of the nucleotide sequence of SEQ ID NO: 12 and reverse primer IV consisting of the nucleotide sequence of SEQ ID NO: 13; and forward primer V consisting of the nucleotide sequence of SEQ ID NO: 14. A primer set V, which is a combination of a reverse primer V consisting of the nucleotide sequence of SEQ ID NO: 15,
The primer kit according to [8], comprising:
[10]
The primer kit according to [8] or [9], which is a primer kit for use in the method for simultaneous detection of rot fungi according to any one of [1] to [7].

According to the present invention, pathogenic bacteria that infect honey bees include the American rot fungus of genotype ERIC type I, the American rot fungus of genotype ERIC type II, and the American rot fungus of genotypes other than ERIC types I and II. A method for simultaneous detection of a European rot fungus that can simultaneously and easily detect a diseased fungus, a phenotypically atypical European rot fungus, and a phenotypical European rot fungus, and a method that can be suitably used for this method. It becomes possible to provide primer kits.

FIG. 1 is a schematic diagram showing the design site in the nucleotide sequence of SEQ ID NO: 1 of a primer for detecting the target sequence (16S rRNA gene) of all genotypes of the American rot fungus. FIG. 2 is a schematic diagram showing the design site in the nucleotide sequence of SEQ ID NO: 5 of a primer for detecting a target sequence (Na ⁺ /H ⁺ antiporter gene) of a phenotypic typical European rot fungus. FIG. 2 is a schematic diagram showing the design site in the nucleotide sequence of SEQ ID NO: 2 of a primer for detecting the target sequence (Toxin 1 gene) of the American rot fungus of genotype ERIC type I. FIG. 2 is a schematic diagram showing the design site in the nucleotide sequence of SEQ ID NO: 3 of a primer for detecting the target sequence of the American rot fungus of genotype ERIC type II (sequence registered with GenBank accession number FI763267). FIG. 2 is a schematic diagram showing a design site in the nucleotide sequence of SEQ ID NO: 4 of a primer for detecting a target sequence (Fur family transcriptional regulator gene) of a phenotypically atypical European rot fungus. 1 is an electrophoretic image obtained by 2% agarose gel electrophoresis after multiplex PCR using each primer set combination in Test Example 1. 1 is an electrophoretic image obtained by 2% agarose gel electrophoresis after multiplex PCR using each primer set combination in Test Example 1. This is an electrophoretic image obtained by 2% agarose gel electrophoresis after multiplex PCR using the primer set combination 1-Aa of Test Example 1. This is an electrophoresis image obtained by 2% agarose gel electrophoresis after multiplex PCR using genomic DNA extracted from each strain (strain No. 1 to 24) of Test Example 2 as template DNA. This is an electrophoresis image obtained by 2% agarose gel electrophoresis after multiplex PCR using genomic DNA extracted from each strain (strain No. 25 to 48) of Test Example 2 as template DNA. This is an electrophoresis image obtained by 2% agarose gel electrophoresis after multiplex PCR using genomic DNA extracted from each strain (strain No. 49 to 72) of Test Example 2 as template DNA. This is an electrophoresis image obtained by 2% agarose gel electrophoresis after multiplex PCR using genomic DNA extracted from each strain of Test Example 2 (strain Nos. 73 to 96) as template DNA. This is an electrophoretic image obtained by 2% agarose gel electrophoresis after multiplex PCR using genomic DNA extracted from each strain of Test Example 2 (strain Nos. 97 to 105) as template DNA. This is an electrophoretic image obtained by 2% agarose gel electrophoresis after multiplex PCR using each serially diluted genomic DNA of Test Example 3 as a template DNA. This is an electrophoretic image obtained by 2% agarose gel electrophoresis after multiplex PCR using the honey sample of Test Example 4. This is a pie chart showing the results of an investigation of the contamination status of domestic honey with rot fungi using multiplex PCR in Test Example 5 (a: contamination status with two rot fungi, b: genotype ERIC type I or type II American). Contamination status with a European rot fungus, c: Contamination status with a phenotypically typical or atypical European rot fungus).

Hereinafter, the present invention will be explained in detail based on its preferred embodiments.

<Root fungus>
According to the method for simultaneous detection of Paenibacillus larvae ERIC I of the present invention, Paenibacillus larvae ERIC I of genotype ERIC type I, Paenibacillus larvae ERIC II of genotype ERIC II, ERIC I It is possible to simultaneously detect genotypes of American rot fungi other than type II and type II, atypical European rot fungi (atypical Melissococcus plutonius), and phenotypically typical European rot fungi (Typical Melissococcus plutonius). It is. The rot fungus to be detected may be a part of the fungus body or a dead fungus, as long as its DNA can be present.

(Paenibacillus larvae)
The American rot fungus is a Gram-positive spore-forming bacillus, and five genotypes (genotypes ERIC types I to V) are known at present. The ERIC type was described by Genersch et al. , 2006, Int. J. Syst. Evol. Microbiol. 56:501-511 and Beims et al. , 2020, Int. J. Med. Microbiol. 310:151394. All genotype strains are highly toxic to bee larvae, but the genotype ERIC type I strain is relatively less toxic to bee broods and takes a long time to develop, so the hive cell is covered when it pupates. Mortality rate during the later larva stage is high. On the other hand, the genotype ERIC type II strain is highly toxic to bee larvae and causes the larvae to die early before the hive is capped. In addition, for bee colonies, with genotype ERIC type I strains, infected bee larvae often die during the operculum stage, which delays the discovery and removal of dead larvae by worker bees, resulting in damage to the entire bee colony. There is a tendency for the bacteria to spread quickly and the colony to collapse quickly. On the other hand, with the genotype ERIC type II strain, infected bee larvae die early and are detected early by worker bees and removed from the hive, making it difficult for the bacterium to spread throughout the colony, and the disease is severe enough to cause the colony to collapse. It tends to take some time for it to happen. Furthermore, although both genotype strains have strong resistance to disinfectants (e.g., slightly acidic hypochlorous acid water and chlorous acid water), the genotype ERIC type II strain is known to be particularly resistant (Ohashi et al. et al., 2020, J. Vet. Med. Sci. 82:261-271).

(European rot fungus, Melissococcus plutonius)
The European rot fungus is commonly isolated from cases of European rot and has long been known as a typical strain (typical strain), and a strain with characteristics significantly different from the typical strain (atypical strain). type strain) is known. Phenotypic typical strains were described by Haynes et al. , 2013, Environ. Microbiol. Rep. , 5:525-529, and the phenotypic atypical strain is classified as CC12 according to the same MLST classification. All phenotypic strains can grow under anaerobic or microaerophilic conditions, but phenotypic atypical strains grow relatively quickly and can also grow under aerobic conditions in a medium with a Na/K ratio <1. be. Furthermore, phenotypic atypical strains have the ability to produce acids from L-arabinose, D-cellobiose, and salicin, as well as the ability to hydrolyze esculin, and many have β-glucosidase activity, whereas phenotypic typical strains has neither.

<Method for simultaneous detection of rot fungi>
The method for simultaneously detecting ERIC fungi of the present invention includes, in a sample, American ERIC fungi having genotype ERIC type I; A method for simultaneously detecting a European rot fungus, a phenotypically atypical European rot fungus, and a phenotypically typical European rot fungus,
Using the DNA derived from the sample as a template, the following five target regions:
The first target region from 53rd to 1024th in the nucleotide sequence set forth in SEQ ID NO: 1,
a second target region from positions 794 to 2101 in the nucleotide sequence set forth in SEQ ID NO: 2;
the third target region from positions 2 to 339 in the nucleotide sequence set forth in SEQ ID NO: 3;
A fourth target region from positions 109 to 367 in the nucleotide sequence set forth in SEQ ID NO: 4, and a fifth target region from positions 36 to 222 in the nucleotide sequence set forth in SEQ ID NO: 5,
A PCR step in which multiplex PCR is performed using five types of primer sets, each of which is a combination of primers that can amplify the
a detection step of detecting the PCR product obtained in the PCR step;
This is a method that includes

[sample]
There is no particular restriction on the "sample" to be subjected to the simultaneous detection method of the rot fungus of the present invention, as long as DNA derived from the rot fungus can be present; for example, honey bees (adults, pupae, larvae), Dead bees, bee maggots (decomposing bee larvae and pupae); beehives, honey, royal jelly, propolis, pollen, soil, bee droppings, various things that have fallen to the bottom of beehives. Examples include environmental materials collected from inside or around the nest, such as sediment (debris). The sample according to the present invention may be any of these, but for example, honey is preferable when the purpose is to investigate the contamination status of bee colonies with rot fungi. The rot fungus is known to contaminate honey, which can become a source of infection for larvae and spread throughout the farm, or several years before the onset of rot. This is because it has been reported that honey begins to be contaminated with rot fungi. According to the present invention, even when using a sample such as honey that contains a relatively small amount of DNA of the rot fungus, the DNA derived from the rot fungus can be efficiently amplified by the multiplex PCR described below. It is possible to detect the target rot fungus quickly, easily, and with high precision without using culture.

The sample may be one that has been subjected to treatments such as pulverization or freezing, or it may be diluted or suspended with a diluent as appropriate. Examples of the diluent include water; physiological saline; buffers such as phosphate buffer, Tris buffer, Good buffer, and borate buffer.

[Target area]
According to the present invention, the following five target regions discovered by the present inventors:
The first target region from 53rd to 1024th in the nucleotide sequence set forth in SEQ ID NO: 1,
a second target region from positions 794 to 2101 in the nucleotide sequence set forth in SEQ ID NO: 2;
the third target region from positions 2 to 339 in the nucleotide sequence set forth in SEQ ID NO: 3;
A fourth target region from positions 109 to 367 in the nucleotide sequence set forth in SEQ ID NO: 4, and a fifth target region from positions 36 to 222 in the nucleotide sequence set forth in SEQ ID NO: 5,
By designing primers that can amplify each of the above-mentioned rot fungi, it is possible to simultaneously detect all of the above-mentioned rot fungi using multiplex PCR.

The first target region is common to all genotypes of the American rot fungus (currently, all genotypes of the American rot fungus of genotypes ERIC I to V), and is common to all genotypes of the American rot fungus, and 16S rRNA gene specific to type V American rot fungus (GenBank accession number: X60619 (Govan et al., 1999, Appl. Environ. Microbiol., 65:2243-2245); This sequence is referred to as the "first target sequence"). A typical nucleotide sequence of a portion of the first target sequence, including the first target region, is shown in SEQ ID NO:1. In the nucleotide sequence shown in SEQ ID NO: 1, "n" represents a, c, g, or t.

The second target region is the Toxin 1 gene (plx1) specific to the American rot fungus of genotype ERIC type I (GenBank accession number: KC456421 (Beims et al., 2020, Open Vet. J., 10: 53-58); hereinafter, the sequence of this gene will be referred to as the "second target sequence" in some cases). A typical nucleotide sequence of the second target sequence is shown in SEQ ID NO:2. As the second target region, positions 1369 to 1922 in the nucleotide sequence set forth in SEQ ID NO: 2 are more preferable.

The third target region is a sequence registered with GenBank accession number FI763267 (hereinafter referred to as " The region on the third target sequence (referred to as "third target sequence"). A typical nucleotide sequence of the third target sequence is shown in SEQ ID NO:3. As the third target region, positions 3 to 335 in the nucleotide sequence set forth in SEQ ID NO: 3 are more preferable.

The fourth target region is the Fur family transcriptional regulator gene (GenBank accession number: AP012282 (locus tag: MPD5_0863)), which is a gene specific to the phenotypic atypical European rot fungus (Arai et al., 2014, J .Vet.Med.Sci., 76:491-498); hereinafter, the sequence of this gene will be referred to as the "fourth target sequence" in some cases). A typical nucleotide sequence of the fourth target sequence is shown in SEQ ID NO:4. The fourth target region is more preferably positions 111 to 367 in the nucleotide sequence set forth in SEQ ID NO: 4.

The fifth target region is the Na ⁺ /H ⁺ antiporter gene (napA) (GenBank accession number: AP012200 (locus tag: MPTP_0420)), which is a gene specific to the phenotypical European rot fungus (Arai et al. , 2014, J. Vet. Med. Sci., 76:491-498); hereinafter, the sequence of this gene will be referred to as the "fifth target sequence" in some cases). A typical nucleotide sequence of the fifth target sequence is shown in SEQ ID NO:5.

It should be understood that individual differences may occur at the strain level due to polymorphisms and the like in the nucleotide sequences of each of the first to fifth target sequences.

In each of the first to fifth target regions, the range amplified by the following primer sets is a range of different base numbers, that is, a range of nucleotide fragments of different lengths for each of the five target regions, that is, the following: It is sufficient that the PCR product sizes (number of bases (bp)) obtained by multiplex PCR are in different ranges, and may be a part or the entire range of each target region independently. In each of the first to fifth target regions, the number of bases to be amplified is different from each other, preferably 100 to 1500 bases, more preferably 150 to 1000 bases.

Among these, in each of the first to fifth target regions, the range selected for amplification with the following primer set is particularly effective in reducing non-specific reactions and making it easier to differentiate each PCR product by electrophoresis etc. From the viewpoint of clarity and the amplification efficiency of each target region by PCR being equivalent to each other, the following ranges are respectively:
In the first target region, the range from 53rd to 1024th in the nucleotide sequence set forth in SEQ ID NO: 1 (972 bases in the same sequence),
In the second target region, the 1369th to 1922nd range in the nucleotide sequence set forth in SEQ ID NO: 2 (554 bases in the same sequence),
In the third target region, the range from 3rd to 335th in the nucleotide sequence set forth in SEQ ID NO: 3 (333 bases in the same sequence),
In the fourth target region, the range from 111 to 367 in the nucleotide sequence set forth in SEQ ID NO: 4 (257 bases in the same sequence), and in the fifth target region, the range from 36 to 222 in the nucleotide sequence set forth in SEQ ID NO: 5. range (187 bases in the same sequence)
It is preferable that

[Primer design site]
In the present invention, in order to be able to amplify each of the first to fifth target regions, that is, two different sites (hereinafter referred to as "primer primer" in some cases) on each of the first to fifth target regions are Primers are designed so that they hybridize to the respective designated sites (referred to as "designed sites"). Two primer design sites on one target region may partially overlap with each other.

The number of bases in the primer design site is preferably 15 or more bases, more preferably 17 to 30 bases, and even more preferably 20 to 28 bases.

There are no particular restrictions on such primer design sites as long as they satisfy the conditions for the amplification range (number of bases) described above in each of the first to fifth target regions, and preferably, the following primers are used for other sites. These primers can be appropriately selected so that they do not hybridize with other primers, and their GC contents are close to each other and within a preferable range.

Among these, the primer design site is particularly selected from the viewpoints of reducing non-specific reactions, making it clearer to differentiate each PCR product by electrophoresis, etc., and making the amplification efficiency of each target region equal to each other. , respectively, the following parts:
In the first target region, the 53rd to 75th sites in the nucleotide sequence of SEQ ID NO: 1 and the 1002nd to 1024th sites in the nucleotide sequence of SEQ ID NO: 1, preferably a combination thereof,
In the second target region, the 1369th to 1396th sites in the nucleotide sequence of SEQ ID NO: 2 and the 1895th to 1922nd sites in the nucleotide sequence of SEQ ID NO: 2, preferably a combination thereof,
In the third target region, the 3rd to 30th sites in the nucleotide sequence of SEQ ID NO: 3 and the 312nd to 335th sites in the nucleotide sequence of SEQ ID NO: 3, preferably a combination thereof,
In the fourth target region, the 111th to 138th sites in the nucleotide sequence of SEQ ID NO: 4 and the 340th to 367th sites in the nucleotide sequence of SEQ ID NO: 4, preferably a combination thereof,
In the fifth target region, the 36th to 55th sites in the nucleotide sequence of SEQ ID NO: 5, the 201st to 222nd sites in the nucleotide sequence of SEQ ID NO: 5, preferably a combination thereof,
can be mentioned.

[Primer set]
The primer set according to the present invention is a combination of primers that hybridize to two different primer design sites among the first to fifth target regions.

In the present invention, "hybridizing" of a primer to the primer design site means that the primer hybridizes to the sense strand of the primer design site (for example, a site on the nucleotide sequence shown in SEQ ID NOS: 1 to 5). In addition to hybridizing to its complementary strand.

In addition, in the present invention, the expression that a primer "hybridizes" with the primer design site may be any nucleotide sequence that allows at least a portion of the primer and the primer design site to form a double strand; They do not have to be completely complementary. In the present invention, such conditions for hybridization include that the sequence complementarity of the nucleotide sequence between the primer and the primer design site is 80% or more, 90% or more, 95% or more (for example, 96% or more, 97% or more). 98% or more, 99% or more). Note that the sequence complementarity can be calculated as appropriate by those skilled in the art using a known method (for example, BLAST (NCBI)). In addition, in the present invention, the hybridization conditions include that the nucleotide sequence of the primer is continuous at one or more positions of a nucleotide sequence that is completely complementary to the entire length of the nucleotide sequence of the primer design site. 1 to 5 bases (preferably 1 to 3 bases, eg, 3 bases, 2 bases, 1 base) may be inserted, deleted, or substituted.

The nucleotide sequence constituting such a primer should be matched to the nucleotide sequence of the primer design site so as to satisfy the above-mentioned hybridization conditions, and more preferably so as not to hybridize to other sites or other primers. Furthermore, the GC contents can be appropriately designed so that they are close to each other and within a preferable range.

The length of the primers according to the present invention is independently preferably 15 bases or more, more preferably 17 to 30 bases, and even more preferably 20 to 28 bases. Furthermore, the GC content of the primers according to the present invention is preferably 40 to 60%, more preferably 45 to 55%.

The primer according to the present invention can be synthesized using, for example, a commercially available oligonucleotide synthesizer. Furthermore, the primer according to the present invention does not need to be composed only of natural nucleotides (deoxyribonucleotides and/or ribonucleotides), and includes, for example, PNA (polyamide nucleic acid), LNA (registered trademark, locked nucleic acid), ENA (Registered trademark, 2'-O,4'-C-Ethylene-bridged nucleotides) may be partially or entirely composed of non-natural nucleotides.

More specifically, as such a primer set,
A primer that is a combination of forward primer I that hybridizes to the 53rd to 75th sites in the nucleotide sequence of SEQ ID NO: 1 and reverse primer I that hybridizes to the 1002nd to 1024th sites in the nucleotide sequence of SEQ ID NO: 1. set I,
A primer that is a combination of forward primer II that hybridizes to the 1369th to 1396th sites in the nucleotide sequence of SEQ ID NO: 2 and reverse primer II that hybridizes to the 1895th to 1922nd sites in the nucleotide sequence of SEQ ID NO: 2. Set II,
A primer that is a combination of a forward primer III that hybridizes to positions 3 to 30 in the nucleotide sequence of SEQ ID NO: 3 and a reverse primer III that hybridizes to positions 312 to 335 in the nucleotide sequence of SEQ ID NO: 3. Set III,
A primer that is a combination of a forward primer IV that hybridizes to positions 111 to 138 in the nucleotide sequence of SEQ ID NO: 4 and a reverse primer IV that hybridizes to positions 340 to 367 in the nucleotide sequence of SEQ ID NO: 4. Set IV, and a forward primer V that hybridizes to the 36th to 55th sites in the nucleotide sequence of SEQ ID NO: 5, and a reverse primer V that hybridizes to the 201st to 222nd sites in the nucleotide sequence of SEQ ID NO: 5. Primer set V, which is a combination,
can be mentioned.

As mentioned above, individual differences may occur between bacterial strains due to deletions or insertions of several bases, so according to each primer set, the following PCR products:
According to primer set I, preferably corresponds to the 53rd to 1024th range (972 bases) in the nucleotide sequence set forth in SEQ ID NO: 1, or the 53rd to 1024th range in the nucleotide sequence set forth in SEQ ID NO: 1. A PCR product amplified in a range (preferably 972 to 974 bases);
According to Primer Set II, preferably corresponds to the 1369th to 1922nd range (554 bases) in the nucleotide sequence set forth in SEQ ID NO: 2, or the 1369th to 1922nd range in the nucleotide sequence set forth in SEQ ID NO: 2. A PCR product amplified in a range (preferably 551 to 557 bases);
According to primer set III, preferably corresponds to the range from 3rd to 335th (333 bases) in the nucleotide sequence set forth in SEQ ID NO: 3, or the range from 3rd to 335th in the nucleotide sequence set forth in SEQ ID NO: 3. PCR products amplified in a range (preferably 330 to 336 bases);
According to Primer Set IV, preferably corresponds to the 111st to 367th range (257 bases) in the nucleotide sequence set forth in SEQ ID NO: 4, or the 111st to 367th range in the nucleotide sequence set forth in SEQ ID NO: 4. PCR products amplified in a range (preferably 254 to 260 bases);
According to primer set V, preferably the range corresponding to the 36th to 222nd range (187 bases) in the nucleotide sequence set forth in SEQ ID NO: 5 or the range corresponding to the 36th to 222nd range in the nucleotide sequence set forth in SEQ ID NO: 4. (preferably 184 to 190 bases) is obtained.

In the above, the range that "corresponds" to each range of the nucleotide sequence is defined as BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information). ) etc. (for example, parameter: default value (i.e., initial setting value)), when the nucleotide sequences are aligned, the range of each control (for example, in the case of primer set I above, 53 to 53 in the nucleotide sequence set forth in SEQ ID NO: 1) 1024th range).

Primer set I is preferably a combination of forward primer I consisting of the nucleotide sequence of SEQ ID NO: 6 and reverse primer I consisting of the nucleotide sequence of SEQ ID NO: 7,
Primer set II is preferably a combination of forward primer II consisting of the nucleotide sequence of SEQ ID NO: 8 and reverse primer II consisting of the nucleotide sequence of SEQ ID NO: 9,
Primer set III is preferably a combination of forward primer III consisting of the nucleotide sequence of SEQ ID NO: 10 and reverse primer III consisting of the nucleotide sequence of SEQ ID NO: 11,
Primer set IV is preferably a combination of forward primer IV consisting of the nucleotide sequence of SEQ ID NO: 12 and reverse primer IV consisting of the nucleotide sequence of SEQ ID NO: 13,
Primer set V is preferably a combination of forward primer V consisting of the nucleotide sequence of SEQ ID NO: 14 and reverse primer V consisting of the nucleotide sequence of SEQ ID NO: 15.

Furthermore, the combination of the five types of primer sets is particularly important from the viewpoint of reducing non-specific reactions, making it clearer to distinguish each PCR product by electrophoresis, etc., and achieving the same amplification efficiency by PCR for each target region. The preferred primer set III (SEQ ID NO: 8 and 9, SEQ ID NO: 10 and 11, SEQ ID NO: 12 and 13) is preferred. 10 and 11), more preferably all of the above preferred primer sets II to IV, and even more preferably all of the above preferred primer sets IV.

[DNA extraction process]
The method for simultaneous detection of rot fungi of the present invention may further include a step of extracting DNA from the sample before the PCR step. The DNA extraction method is not particularly limited, and conventionally known methods can be used as appropriate, such as the alkaline-SDS method, phenol extraction method, and commercially available nucleic acid extraction/purification kits (e.g., InstaGene Matrix (Bio-Rad)). , DNeasy PowerSoil Kit (QIAGEN), DNeasy Plant Mini Kit (QIAGEN), Johnespin (Fasmac), Johnes Purespin (Fasmac)), and methods similar thereto. Among these, a preferred method when using honey as the sample is to extract the DNA of the American rot fungus, which exists in honey in the form of spores covered with a hard shell that is resistant to external stimuli, and to A method using Johnespin and Johnepurespin can be mentioned from the viewpoint of being able to more efficiently and simultaneously extract the DNA of the European rot fungus, which exists in the form of a trophozoite that is relatively vulnerable to stimulation, from honey.

[PCR process (amplification process)]
The method for simultaneous detection of rot fungi of the present invention includes a PCR step of performing multiplex PCR using five types of primer sets, each of which is a combination of primers capable of amplifying each of the first to fifth target regions. include.

In the PCR step according to the present invention, for example, the five types of primer sets and complementary strand synthase (e.g., DNA polymerase) are added to the DNA extracted from the sample to perform a multiplex PCR reaction. If there is a desired target region, that is, a desired target sequence specific to the desired rot fungus, the primers hybridize to this, and the region sandwiched between the primer sets is used as a template to target the target region. The above nucleotide fragments are synthesized, and an amplification product (PCR product) in which such nucleotide fragments are amplified can be obtained. According to multiplex PCR using five types of primer sets according to the present invention, if target sequences specific to five types of rot fungi are present in the sample, five types of PCR products are Obtainable.

Reaction conditions such as the type of complementary strand synthase, the composition of the reaction solution, the reaction temperature, time, and number of cycles in the multiplex PCR can be appropriately selected without particular limitations. For example, in the present invention, when using the above-mentioned preferred primer sets IV (SEQ ID NO: 6 and 7, SEQ ID NO: 8 and 9, SEQ ID NO: 10 and 11, SEQ ID NO: 12 and 13, SEQ ID NO: 14 and 15), , general PCR conditions can be adopted, for example, the temperature for complementary strand synthesis, more preferably DNA elongation by DNA polymerase, can be in the range of 50 to 75°C, more preferably 52 to 72°C. can.

[Detection process]
The PCR product obtained in the PCR step can be detected appropriately by a known method or a method analogous thereto. This makes it possible to detect the desired target sequence, that is, to detect the presence of a sequence specific to the desired rot fungus, and indirectly to detect the target rot fungus in the sample. becomes.

Examples of the detection method include a method of separating and detecting the PCR product by electrophoresis such as agarose gel; a DNA array method of measuring using a substrate immobilized with an oligonucleotide probe that hybridizes to the PCR product, etc. can be mentioned.

In addition, the obtained PCR products can be detected by a method of intercalating a fluorescent dye (e.g., ethidium bromide, Syber Green (registered trademark), SYTO63, GelRed, GelGreen, etc.) and detecting the fluorescence; Fluorescent substances (FITC, FAM, DEAC, R6G, TexRed, Cy5, BODIPY FL, etc.), radioisotopes ( ^3H , ^14C , ^32P , ^35S , ^123I , etc.), luminescent substances (luminol, luciferin, lucigenin, etc.) Confirmation can be performed by labeling with a labeling substance such as (e.g.) and detecting a signal (coloration (coloring), reflected light, luminescence, quenching, fluorescence, radiation due to radioactive isotopes, etc.) according to the labeling substance. . Furthermore, if necessary, the intensity of the signal may be measured to quantify the PCR product to estimate the amount of the target sequence of interest or the gene expressed therein.

The detection method is not limited to the above method, and the above methods and conventionally known methods may be combined as appropriate. Among them, when using the five types of primer sets according to the present invention, it is possible to separate the PCR products particularly easily, so the detection method includes a step of separating the PCR products by the electrophoresis. It is preferable. In this case, for example, the obtained PCR product is electrophoresed on an agarose gel (e.g., 2%), stained with the fluorescent dye (e.g., ethidium bromide, GelRed, GelGreen, etc.) and separated. By confirming the presence or absence of a band of the PCR product using fluorescence, it is possible to detect the presence or absence of the desired PCR product, that is, the presence or absence of the desired target sequence, that is, the presence or absence of the desired rot fungus in the sample. According to multiplex PCR using the five types of primer sets according to the present invention, even if all five types of PCR products are obtained, they can be clearly separated and detected separately.

<Primer kit>
The present invention
Primer set II, which is a combination of forward primer II consisting of the nucleotide sequence of SEQ ID NO: 8 and reverse primer II consisting of the nucleotide sequence of SEQ ID NO: 9;
Primer set III is a combination of forward primer III consisting of the nucleotide sequence of SEQ ID NO: 10 and reverse primer III consisting of the nucleotide sequence of SEQ ID NO: 11; and forward primer IV consisting of the nucleotide sequence of SEQ ID NO: 12. Reverse primer IV consisting of the nucleotide sequence of SEQ ID NO: 13, and primer set IV, which is a combination of
Also provided is a primer kit comprising at least one primer set selected from the group consisting of:

As described above, the primer sets included in the primer kit of the present invention each include:
Primer set II detects the second target sequence by amplifying the 1369th to 1922nd range (554 bases) or the corresponding range (preferably 551 to 557 bases) in the nucleotide sequence set forth in SEQ ID NO: 2. , that is, a primer set that can specifically detect the American rot fungus of genotype ERIC type I,
Primer set III detects the third target sequence by amplifying the 3rd to 335th range (333 bases) or the corresponding range (preferably 330 to 336 bases) in the nucleotide sequence set forth in SEQ ID NO: 3. , that is, a primer set that can specifically detect the American rot fungus of genotype ERIC type II,
Primer set IV detects the fourth target sequence by amplifying the 111st to 367th range (257 bases) or the corresponding range (preferably 254 to 260 bases) in the nucleotide sequence set forth in SEQ ID NO: 4. , that is, a primer set that can specifically detect a phenotypically atypical European rot fungus.

All of these primer sets are newly discovered by the present inventors. The primer kit of the present invention preferably includes at least primer set III, and more preferably includes all of primer sets II, III, and IV.

Furthermore, the primer kit of the present invention includes:
Primer set I is a combination of forward primer I consisting of the nucleotide sequence of SEQ ID NO: 6 and reverse primer I consisting of the nucleotide sequence of SEQ ID NO: 7, and/or forward primer V consisting of the nucleotide sequence of SEQ ID NO: 14. and a reverse primer V consisting of the nucleotide sequence of SEQ ID NO: 15, a primer set V, which is a combination of
It is also preferable to further include.

Primer set I detects the first target sequence by amplifying the 53rd to 1024th range (972 bases) or the corresponding range (preferably 972 to 974 bases) in the nucleotide sequence set forth in SEQ ID NO: 1. In other words, this is a primer set that can detect all genotypes of the American rot fungus. By combining the above-mentioned ERIC type I or II genotype genotypes with primers (primer set II and primer set III) that can specifically detect American rot fungi, genes other than ERIC type I and II can be detected. type of American rot fungus can be detected. In other words, if a PCR product is not confirmed with any of primer sets II to III, and a PCR product is confirmed only with primer set I, it is possible to confirm that a PCR product with a genotype other than ERIC type I or type II is detected. It can be determined that the maggot pathogen has been detected.

Primer set V detects the fifth target sequence by amplifying the 36th to 222nd range (187 bases) or the corresponding range (preferably 184 to 190 bases) in the nucleotide sequence set forth in SEQ ID NO: 5. In other words, it is a primer set that can specifically detect a phenotypic typical European rot fungus.

The primer kit of the present invention can be suitably used in the above-described method for simultaneously detecting rot fungi of the present invention, in addition to the methods for detecting each rot fungus described above. Each primer included in the primer kit of the present invention may contain other components such as a buffer, a stabilizer, a preservative, and a preservative. In addition, the primer kit of the present invention includes a diluted solution of the sample; reagents for extracting and purifying DNA; reagents such as enzymes, buffers, and pH adjusters necessary for multiplex PCR; reagents necessary for the detection process. ; standard nucleic acid; instructions for use, etc. may further be provided.

Hereinafter, the present invention will be explained in more detail based on test examples, but the present invention is not limited to the following test examples.

1. Selection of target sequence for multiplex PCR 1.1. Target sequence of American rot fungus (ERIC type I)
As a target sequence specific to the American rot fungus of genotype ERIC type I, Beims et al. , 2020, Open Vet. J. The sequence of the Toxin 1 gene (plx1) described in 10:53-58 was used. A typical nucleotide sequence of the target sequence (Toxin 1 gene) of the American rot fungus of genotype ERIC type I is shown in SEQ ID NO: 2.

(ERIC type II)
A target sequence specific to the American rot fungus of genotype ERIC type II was selected as follows. That is, first, Funfhaus et al. , 2009, Environ. Microbiol. Rep. , 1:240-250, nine sequences reported to be present only in ERIC type II strains were obtained from the GenBank database. Next, we compared the full-length genomes of American rot fungus strains of other genotypes (ERIC types I, III, IV, and V) registered in the GenBank database, and found that only ERIC type II strains possessed the genomes. It was found that there was only one sequence (registered with GenBank accession number FI763267), which was selected as the target sequence. In addition, strain No. 1 in Tables 11 and 12 below. The presence or absence of the same target sequence in the American rot fungus strains described in 72 to 86 with known genotypes was investigated by PCR using the following primer set III, and it was found that the target sequence was indeed true for ERIC type II strains. It was confirmed that the sequence was specific to . A typical nucleotide sequence of the target sequence of the American rot fungus of genotype ERIC type II is shown in SEQ ID NO: 3.

(all genotypes)
The sequence of the 16S rRNA gene, which is commonly possessed by all genotypes of American saprophytic fungi, including ERIC types I and II (ERIC types I to V), was combined with the sequence of the 16S rRNA gene, which is common to all genotypes of American saprophytic fungi, including ERIC types I and II. This was used as the target sequence. A typical nucleotide sequence of the target sequence (16S rRNA gene) of all genotypes of the American rot fungus is shown in SEQ ID NO: 1. By combining this target sequence with the target sequences of the above-mentioned ERIC type I and type II American rot fungi, both of the target sequences of the above-mentioned genotype ERIC type I and II can be detected. By detecting only the target sequence (16S rRNA gene) of all genotypes of American rot fungi, it is possible to detect American rot fungi of genotypes other than ERIC type I and II. .

1.2. Target sequence of European rot fungus (atypical)
Target sequences specific to the phenotypic atypical European rot fungus include Arai et al. , 2014, J. Vet. Med. Sci. The sequence of the Fur family transcriptional regulatory gene described in J. Phys., 76:491-498 was used. A typical nucleotide sequence of the target sequence (Fur family transcriptional regulator gene) of the phenotypically atypical European rot fungus is shown in SEQ ID NO: 4.

(typical)
As a target sequence specific to a phenotypic typical European rot fungus, Arai et al. , 2014, J. Vet. Med. Sci. The sequence of the Na ⁺ /H ⁺ antiporter gene (napA) described in J. Phys., 76:491-498 was used. A typical nucleotide sequence of the target sequence (Na ⁺ /H ⁺ antiporter gene) of a phenotypic typical European rot fungus is shown in SEQ ID NO: 5.

2. Design of Primer Set 2.1 Target Sequence Common to All Genotypes of American Scabies As a primer that specifically detects the target sequence (16S rRNA gene) of all genotypes of American Scabies, Govan et al. , Appl. Environ. Microbiol. , 65:2243-2245, and de Graaf et al. , 2013, J. Apic. Res. , 52:1-28 were used. Primers for detecting the target sequence (16S rRNA gene) of all genotypes of the American rot fungus, the sequence number indicating the nucleotide sequence, and the expected nucleotide fragment that can be amplified with the primer set (primer set I) The size (PCR product size) is shown in Table 1 below, and a schematic diagram showing the design site in the nucleotide sequence of SEQ ID NO: 1 of each primer is shown in FIG.

2.2 Target sequence of a phenotypically typical European rot fungus As a primer for specifically detecting a target sequence (Na ⁺ /H ⁺ antiporter gene) of a phenotypically typical European rot fungus, Arai et al. , 2014, J. Vet. Med. Sci. , 76:491-498 were used. Primers for detecting the target sequence (Na ⁺ /H ⁺ antiporter gene) of the European rot fungus with a typical phenotype, the sequence number indicating the nucleotide sequence thereof, and the nucleotide fragment that can be amplified with the same primer set (primer set V) The expected size (PCR product size) of each primer is shown in Table 2 below, and a schematic diagram showing the design site in the nucleotide sequence of SEQ ID NO: 5 of each primer is shown in FIG.

2.3 Target sequences of genotype ERIC type I American rot fungus, genotype ERIC type II American rot fungus, and phenotypically atypical European rot fungus Target sequences other than those in 2.1 to 2.2 above For this, multiple forward primers (F) and reverse primers (R) were designed in order to construct multiplex PCR with higher accuracy. Gene sequence analysis software Sequencher 5.4.6 (Gene Codes Corp.) was used to design each primer, and the primers were designed to have a length of 20 to 28 bp and a GC content of 40 to 60%. In addition, in order to increase the specificity of the primers, we avoided sequences in which the base at the 3' end is T, so that when multiplex PCR was performed, nucleotide fragments of different sizes for each target sequence were amplified. Primers were designed so that the resulting PCR products were of different sizes. In addition, by comparing the gene sequences of all bacteria on the GenBank database through BLAST search (http://blast.ncbi.nlm.nih.gov), we can confirm that the designed primers have high specificity for the target bacteria. It was confirmed.

The primers (12 types) for detecting the target sequence (Toxin 1 gene) of the American rot fungus of genotype ERIC type I and the sequence numbers indicating their nucleotide sequences are shown in Table 3 below. A schematic diagram showing the design site in the nucleotide sequence of is shown in FIG. In addition, the table below lists primers (six types) for detecting the target sequence of the American rot fungus of genotype ERIC type II (sequence registered with GenBank accession number FI763267) and the sequence numbers indicating their nucleotide sequences. 4, and a schematic diagram showing the designed site in the nucleotide sequence of SEQ ID NO: 3 of each primer is shown in FIG. Furthermore, the primers (four types) for detecting the target sequence (Fur family transcriptional regulator gene) of the phenotypic atypical European rot fungus and the sequence numbers indicating their nucleotide sequences are shown in Table 5 below. A schematic diagram showing the design site in the nucleotide sequence of SEQ ID NO: 4 is shown in FIG.

2.4 Combination of Primer Sets In order to select the optimal combination of primer sets for multiplex PCR, the multiple primers listed in Tables 3 to 5 designed above were tested for the American rot fungus of genotype ERIC type II. 9 sets (1-9) for the target sequence, 10 sets (A-J) for the target sequence of the American rot fungus with genotype ERIC type I, and 4 sets (a) for the target sequence of the phenotypically atypical European rot fungus. Primer sets for ~d) were designed. Table 6 below shows the combination of forward primer (F) and reverse primer (R) in each primer set, and the expected size of the nucleotide fragment that can be amplified with each primer set (PCR product size).

3. multiplex PCR
The QIAGEN Multiplex PCR Kit (QIAGEN) was used as the enzyme for the PCR reaction, and each reaction consisted of a total 20 μL system containing 10 μL of 2×QIAGEN Multiplex PCR Master Mix, 2 μL of Q solution, and 2 μL of template DNA. carried out in did. The final concentration of each primer in the reaction solution was 0.1 μM for the primers (primers listed in Table 1) that detect the target sequence (16S rRNA gene) of all genotypes of the American rot fungus, and 0.1 μM for the other primers (primers listed in Table 1). The primers described in 2 to 5) were all 0.2 μM.

The PCR reaction conditions were as follows: 98°C for 15 minutes; 94°C for 30 seconds, 60°C for 1 minute and 30 seconds, 72°C for 1 minute (x35 cycles); and 72°C for 10 minutes. All PCR reactions were performed on a T100 ^™ Thermal Cycler (Bio-Rad Laboratories), and 6 μL of the PCR reaction solution was subjected to 2% agarose gel electrophoresis. After 2% agarose gel electrophoresis was performed at 100V for 30 minutes, the DNA was stained with ethidium bromide and the nucleotide fragments derived from each target sequence were amplified by observing the PCR product bands under UV irradiation. It was confirmed.

<Test Example 1> Examination of the optimal primer set combination A representative strain of the American rot fungus with the genotype ERIC type I (DTK386 strain) and a representative strain of the American rot fungus with the genotype ERIC type II, as shown in Table 7 below. InstaGene Matrix (Bio-Rad ) to extract genomic DNA according to its instruction manual. After measuring the concentration of genomic DNA extracted from these four strains using NanoDrop ND-1000 (Thermo Scientific), 2 μL of a mixture of genomic DNA of each strain adjusted to a final concentration of 0.25 ng/μL was used as a positive control. It was used as a template DNA for.

One primer set was selected for each target sequence from the primer sets listed in Table 6, and primer set I (Table 1) and In combination with primer set V (Table 2) that detects the target sequence (Na ⁺ /H ⁺ antiporter gene) of the European rot fungus with a typical phenotype, a total of 360 combinations were prepared using the above positive control as a template and in accordance with 3 above. Multiplex PCR was performed under the conditions described.

In this test example, if all the primer sets correctly amplified the desired nucleotide fragment on the target sequence of the positive control by multiplex PCR, the electrophoresis image shows, from the top, all genotypes of the American rot fungus (PCR (Product size: 973 bp), Genotype ERIC type I of American saprophyte (PCR product size: 475-836 bp), Genotype ERIC type II of American saprophyte (PCR product size: 326-338 bp), Phenotype atypical Five specific PCR products derived from a phenotypically typical European rot fungus (PCR product size: 187 bp) are amplified.

Part of the electrophoretic image obtained by 2% agarose gel electrophoresis after multiplex PCR is shown in FIGS. 6 and 7. As a result of the electrophoresis, if a non-specific reaction (amplification of non-specific DNA from a source other than the target sequence) is confirmed, if the five types of desired specific PCR products cannot be confirmed, or if the five types of target specific PCR products cannot be confirmed, Even if the desired specific PCR product was confirmed, if the amplification efficiency of some of the PCR products was poor and the brightness of the electrophoretic image was low, it was determined that the primer set combination was not suitable for multiplex PCR. On the other hand, when five specific PCR products of interest are confirmed with the expected size and all PCR products are confirmed with similar brightness in the electrophoretic image, primers particularly suitable for multiplex PCR are determined. It was determined that it was a combination of sets.

As a result, non-specific reactions were not observed in any of the combinations, but most of the combinations did not amplify one or two types of PCR products. However, the combination 1-Aa, that is, the primer set for detecting the target sequence of the American rot fungus of genotype ERIC type II, is P1-II-F1 (forward primer III) consisting of the nucleotide sequence of SEQ ID NO: 10. ) and P1-II-R1 (reverse primer III) consisting of the nucleotide sequence of SEQ ID NO: 11. The primer set to be detected is a combination of P1-I-F1 (forward primer II) consisting of the nucleotide sequence of SEQ ID NO: 8 and P1-I-R1 (reverse primer II) consisting of the nucleotide sequence of SEQ ID NO: 9. A certain primer set A (primer set II), a primer set for detecting a target sequence of a phenotypically atypical European rot fungus, is Mp-A-F1 (forward primer IV) consisting of the nucleotide sequence of SEQ ID NO: 12, The combination of Mp-A-R1 (reverse primer IV) consisting of the nucleotide sequence of SEQ ID NO: 13 and primer set a (primer set IV) is a combination of primer sets particularly suitable for multiplex PCR. It was confirmed that there is. As shown in Figures 6 and 7, unless the combination 1-A-a is used, the stability of multiplex PCR is low. When any of the primer sets was replaced with another primer set, the five target specific PCR products tended not to be stably amplified.

Using the combination of the primer set 1-A-a above, multiplex PCR was performed under the conditions described in 3 above using the above positive control or sterilized water (negative control) as template DNA, and the results were obtained by 2% agarose gel electrophoresis. The electrophoretic image is shown in FIG.

<Test Example 2> Multiplex PCR specificity confirmation test In order to confirm the specificity of the primer set combination of 1-A-a, 105 strains including the rot fungi listed in Tables 8 to 12 below were used. Multiplex PCR was performed using genomic DNA extracted from the following as template DNA.

All of the rot fungi listed in Tables 11 and 12 can be divided into more detailed genotypes (sequence types (ST)) using a genotyping method called Multilocus Sequence Typing, but in this study, we used a genotyping method called Multilocus Sequence Typing. In order to cover all genotypes isolated to date, 15 strains of American rot fungi (9 strains of ERIC type I and 6 strains of ERIC type II) and 19 strains of European rot fungi (typically 10 strains and 9 atypical strains) were used. ATCC 35311 strain (strain No. 89), which is a type strain of European rot fungus, was purchased from American Type Culture Collection. Furthermore, as bacterial species that are not rot maggots, 58 strains isolated from domestic honey (Okamoto et al., 2021, Front. Microbiol. 12:667096) and 13 strains isolated from honey bee larvae (Arai et al. ., 2014, J. Vet. Med. Sci., 76:491-498).

Genomic DNA of each strain was extracted using InstaGene Matrix (Bio-Rad) according to its instruction manual. After measuring the concentration of each extracted genomic DNA using NanoDrop ND-1000 (Thermo Scientific), the concentration was adjusted to 0.25 ng/μL and used as template DNA. In addition, a mixture prepared from the four strains listed in Table 7 above in the same manner as in Test Example 1 was used as template DNA for positive control (P).

Multiplex with the combination of the primer set 1-A-a above under the conditions described in 3 above using template DNA prepared from each strain, positive control (P), or sterile water (negative control, N) as template DNA. PCR was performed and electrophoretic images obtained by 2% agarose gel electrophoresis are shown in FIGS. 9A to 9E.

In addition, the electrophoresis revealed that the genotype ERIC type I (expected PCR product size: 972-974 bp and 551-557 bp) and the genotype ERIC type II (expected PCR product) size: 972-974 bp and 330-336 bp), phenotypically atypical European rot fungus (expected PCR product size: 254-260 bp), phenotypically typical European rot fungus (expected PCR product size: 184 The presence or absence of a band of a specific PCR product derived from 190 bp) was confirmed, and the strains in which the band was confirmed were judged to be positive (+) for the said rot fungus, and the strains that were not confirmed were determined to be positive (+) for the said rot fungus. It was judged to be negative (-). The results are shown in Tables 8 to 12 below.

As shown in Figures 9A to 9E and Tables 8 to 12, in this multiplex PCR specificity confirmation test, all of the tested bacterial strains were accurately identified as ERIC type I American sarcoptic bacteria. , the genotype was ERIC type II, the American rot fungus, the phenotypically atypical European rot fungus, or the phenotypically typical European rot fungus (strain No. 72 in Figures 9C to 9E and Tables 11 to 12). ~105). Furthermore, no non-specific DNA amplification was observed from the genomic DNA of bacteria that were not pathogenic fungi (strains No. 1 to 71 in Figures 9A to 9C and Tables 8 to 11).

<Test Example 3> Multiplex PCR Sensitivity Confirmation Test As template DNA, DNA extracted from the four strains listed in Table 7 above by the following method was used. That is, the American rot fungus was cultured on MYPGP agar medium at 35°C under 5% _CO2 conditions for 2 days, and the European rot fungus was cultured on KSBHI agar medium at 35°C under anaerobic conditions for 3 days. After that, the genomic DNA was obtained from Okamoto et al. , 2021, Front. Microbiol. , 12:667096, and Arai et al. , 2012, PLoS One, 7: e33708.

The MYPGP agar medium used for culturing the American rot fungus was prepared by adding 10 g of Mueller-Hinton broth (Oxoid CM0405), 15 g of yeast extract, 3 g of K ₂ HPO ₄ , 1 g of sodium pyruvate, and 20 g of powdered agar to 1 L of distilled water. After sterilization and dissolution in an autoclave at 121° C. for 15 minutes, 20 mL of a filter-sterilized 10% glucose solution was added, and the mixture was dispensed into a plastic petri dish. In addition, the KSBHI agar medium used for culturing European rot fungus was prepared by adding 37 g of Brain heart infusion broth, 10 g of soluble starch, 20.4 g of KH ₂ PO ₄ , and 15 g of powdered agar to 1 L of distilled water, and incubating at 115°C for 15 minutes. After sterilization and dissolution in an autoclave, the solution was prepared by dispensing it into a plastic petri dish.

After measuring the concentration of the extracted genomic DNA using NanoDrop ND-1000 (Thermo Scientific), it was serially diluted, and 2 μL of each was used as template DNA. Multiplex PCR was performed using the combination of primer sets 1-Aa above under the conditions described in 3 above, and the electrophoretic image obtained by 2% agarose gel electrophoresis is shown in FIG.

As shown in Figure 10, in this multiplex PCR sensitivity confirmation test, the target sequence (16S rRNA gene) of all genotypes of the American saprophytic fungus was extracted from at least 100 fg of the genomic DNA of each saprophytic fungus and at least 1 pg of the target sequence (16S rRNA gene). All other target sequences could be detected from the genomic DNA of each rot fungus.

<Test Example 4> Multiplex PCR using honey sample
(1) Preparation of samples (1-1) Preparation of spore liquid of American scab fungus As a representative strain of American scab fungus of genotype ERIC type I and a representative strain of American scab fungus of genotype ERIC type II, The DTK386 strain (alias: I strain) and DTK384 strain (alias: N strain) (both derived from honey bee rot) listed in Table 7 were used. These strains were cultured for a long period of time on MYPGP agar medium at 35°C and 5% _CO2 , and 70-90% of the bacterial cells turned into spores by spore staining using a Wiltz spore staining kit (Muto Chemical). After confirming that the spores were on the agar medium, the spores were collected. The recovered spores were washed with sterile water and then suspended in sterile water again to obtain a spore solution. Each spore solution was stored at 4°C until used in experiments. The spore concentration was calculated by counting the number of spores on a hemocytometer using an inverted microscope (CKX41, OLYMPUS).

(1-2) Preparation of bacterial suspension of European rot fungus The DAT606 strain listed in Table 7 was used as a representative strain of a phenotypically typical European rot fungus and a representative strain of a phenotypically atypical European rot fungus. and DAT561 strain (all derived from bee rot maggots). After culturing these strains on KSBHI agar medium at 35°C under anaerobic conditions for 3 days, the colonies that grew were collected with a sterile cotton swab, suspended in KSBHI liquid medium supplemented with 10% glycerin, and each bacterial solution was incubated at -80°C. Stored at °C. The colony forming unit concentration (Colony Forming Unit [CFU]/mL) of the bacterial solution was determined by diluting the bacterial solution serially 10 times on Basal agar medium (yeast extract 10 g, glucose 10 g, soluble starch 10 g, L-cysteine 0.25 g, powder). 20g of agar, 100mL/L of 1M KH ₂ PO ₄ [pH 6.6] was autoclaved at 115°C for 15 minutes; Forsgren et al., 2013, J.Apic.Res., 52:1-14). After dripping and culturing anaerobically at 35°C for 3 days, the number of colonies that grew was counted. European rot fungi are arranged in chains, and each chain forms one colony, so each Gram-stained bacterial solution was observed under a microscope and the average number of streptococcal cells in 100 chains was counted. The accurate bacterial concentration (bacterial cells/mL) was calculated using the formula "CFU/mL x average number of streptococcal cells".

(1-3) Selection of honey that is not contaminated with European rot fungi We use honey produced in New Zealand, where the occurrence of European rot fungi has not been reported, that is, there is no contamination with European rot fungi, and we use honey produced in New Zealand that is not contaminated with European rot fungi. Contamination with rot fungi was confirmed. That is, first, honey diluted to 50% (v/v) with sterile water is centrifuged at 12,000 x g for 15 minutes, and the resulting precipitate is centrifuged at 80°C, 85°C, 90°C, 95°C, or 100°C. The mixture was heat-treated for 10 minutes. The precipitate after heat treatment was inoculated onto a MYPGP agar medium supplemented with 20 μg/mL of nalidixic acid and 10 μg/mL of pipemidic acid, and cultured for 6 days under 5% CO ₂ conditions to determine the presence or absence of colonies of the American rot fungus on the agar medium. confirmed. Honey produced in New Zealand, in which no growth of American scab fungi was observed, was selected as honey for artificially inoculating with scab fungi (honey not contaminated with scab fungi).

(1-4) Inoculation of honey with S. saprophyllum The honey that is not contaminated with the S. saprophyllum selected in (1-3) above is injected with the spore solution of the S. albicans prepared in (1-1), and Add the European rot fungus bacterial solution prepared in (1-2) so that the concentration of each bacterial strain in honey is 1,000, 100, 10, or 1 [bacterial cell/mL], Honey contaminated with a known concentration of S. rot fungi (S. rot fungus-inoculated honey) was prepared.

(2) DNA extraction from the sample 5 mL of the honey inoculated with the rot fungus prepared in (1-4) above was diluted to 50% (v/v) with sterile water and centrifuged at 12,000 x g for 15 minutes. The resulting precipitate was suspended in 500 μL of sterile water to prepare a DNA extraction material. DNA was extracted from the DNA extraction material using Johnespin (Fasmac) or Johnes Pure Spin (Fasmac) according to their respective instruction manuals, and 2 μL of the extracted DNA was used as template DNA to infuse the following multi It was subjected to plex PCR.

(3) Multiplex PCR
Multiplex PCR was performed using the combination of primer set 1-Aa above under the conditions described in 3 above. In addition, multiplex PCR was performed in the same manner using a mixture (positive control) or sterilized water (negative control) prepared from the four strains listed in Table 7 in the same manner as in Test Example 1 or sterilized water (negative control) as template DNA. FIG. 11 shows an electrophoretic image obtained by 2% agarose gel electrophoresis after multiplex PCR.

As shown in Figure 11, DNA can be extracted from honey inoculated with rot fungi at a concentration of 100 to 1,000 bacterial cells/mL, and multiplex Five types of target specific PCR products could be detected by PCR.

<Test Example 5> Investigating the contamination status of domestic honey with rot fungi As an application example of multiplex PCR using the combination of the primer set 1-A-a above, we investigated the contamination of bee colonies or apiaries with honey with rot fungi. In order to confirm the usefulness of the method for surveying the situation, we investigated the percentage of domestically produced honey that is contaminated with rot fungi. As samples, we collected 116 types of honey shown in Tables 13 to 15 below, with as many different origins (place of origin, producer, and honey source) as possible. All honey was obtained between 2017 and 2020 by purchase or transfer. J12, J24, and J42 are honeys produced by Japanese honeybees, and all others are honeys produced by Western honeybees. All honey was stored at room temperature. DNA was extracted using Johne Pure Spin (Fasmac) in the same manner as in Test Example 4 (2), and 2 μL of the extracted DNA was used as template DNA for multiplex PCR. Multiplex PCR was performed using the combination of primer sets 1-Aa above under the conditions described in 3 above.

From the electrophoretic image obtained by 2% agarose gel electrophoresis after the multiplex PCR, all genotypes of the American rot fungus (expected PCR product size: 972-974 bp), and the genotype ERIC type I of the American rot fungus were found. P. aeruginosa (expected PCR product size: 972-974 bp and 551-557 bp), genotype ERIC type II American P. aeruginosa (expected PCR product size: 972-974 bp and 330-336 bp), phenotypic atypical Confirm the presence or absence of bands of PCR products derived from the European rot fungus (expected PCR product size: 254 to 260 bp) and the phenotypic typical European rot fungus (expected PCR product size: 184 to 190 bp). The honey in which the band was confirmed was determined to be positive for the rot fungus (+), and the honey in which the band was not confirmed was determined to be negative for the rot fungus (-). The results are shown in Tables 13 to 15 below. In addition, the proportion of honey that was positive for each of the 116 types of domestically produced honey (the positive rate of the scrofulous fungi in domestically produced honey) was calculated. The results are shown in FIG.

As shown in Tables 13 to 15 and Figure 12, in a survey of 116 types of domestically produced honey containing rot fungi, some type of rot fungus was detected in 94.0% of the honey; 6.0% of the honey was below the detection limit (undetected) ((a) in FIG. 12). Focusing on American rot fungi, 80.2% of honey was detected with genotype ERIC type I or II, and 46.6% of honey had genotype ERIC type I or II. Pathogenic bacteria were detected at the same time (FIG. 12(b)). European rot fungi were detected in 88.8% of the honey, both typical and atypical types were detected in 56.9% of the honey, and only the typical type was detected in 31.9% of the honey (see Figure 12). c)). Furthermore, 40 honey samples (34.5%) contained all types of rot fungi (genotype ERIC type I and II American rot fungi, phenotypic typical and atypical European rot fungi). were detected at the same time (Tables 13 to 15). Thus, the multiplex PCR of the present invention was shown to be extremely useful as a method for detecting all types of rot fungi from samples such as honey at once.

According to the present invention, pathogenic bacteria that infect honey bees include the American rot fungus of genotype ERIC type I, the American rot fungus of genotype ERIC type II, and the American rot fungus of genotypes other than ERIC types I and II. A method for simultaneously detecting a pathogenic fungus, a phenotypic atypical European fungus, and a phenotypical European fungus that can be simultaneously and easily detected, and primers that can be suitably used for this method kits can be provided.

Sequence number: 6
<223> Plarvae1/Forward Primer I
Sequence number: 7
<223> Plarvae2/Reverse Primer I
Sequence number: 8
<223> Pl-I-F1/Forward Primer II
Sequence number: 9
<223> Pl-I-R1/Reverse Primer II
Sequence number: 10
<223> Pl-II-F1/Forward Primer III
Sequence number: 11
<223> Pl-II-R1/Reverse Primer III
Sequence number: 12
<223> Mp-A-F1/Forward Primer IV
Sequence number: 13
<223> Mp-A-R1/reverse primer IV
Sequence number: 14
<223> Mp-TF/Forward Primer V
Sequence number: 15
<223> Mp-TR/Reverse Primer V
Sequence number: 16
<223> Pl-IF2
Sequence number: 17
<223> Pl-IF3
Sequence number: 18
<223> Pl-IF4
Sequence number: 19
<223> Pl-IF5
Sequence number: 20
<223> Pl-IF6
Sequence number: 21
<223> Pl-IF7
Sequence number: 22
<223> Pl-I-R2
Sequence number: 23
<223> Pl-I-R3
Sequence number: 24
<223> Pl-I-R4
Sequence number: 25
<223> Pl-I-R5
Sequence number: 26
<223> Pl-II-F2
Sequence number: 27
<223> Pl-II-F3
Sequence number: 28
<223> Pl-II-R2
Sequence number: 29
<223> Pl-II-R3
Sequence number: 30
<223> Mp-A-F2
Sequence number: 31
<223> Mp-A-R2

Claims (9)

Paenibacillus larvae ERIC I with genotype ERIC type I, Paenibacillus larvae ERIC II with genotype ERIC type II, and Paenibacillus larvae ERIC II with genotypes other than ERIC type I and II in the sample. A method for simultaneously detecting a phenotypic atypical Melissococcus plutonius, and a phenotypic typical Melissococcus plutonius,
Using the DNA derived from the sample as a template, the following five target regions:
The first target region from 53rd to 1024th in the nucleotide sequence set forth in SEQ ID NO: 1,
a second target region from positions 1369 to 1922 in the nucleotide sequence set forth in SEQ ID NO: 2;
the third target region from positions 3 to 335 in the nucleotide sequence set forth in SEQ ID NO: 3;
A fourth target region from positions 111 to 367 in the nucleotide sequence set forth in SEQ ID NO: 4, and a fifth target region from positions 36 to 222 in the nucleotide sequence set forth in SEQ ID NO: 5,
A PCR step in which multiplex PCR is performed using five types of primer sets, each of which is a combination of primers that can amplify the
a detection step of detecting the PCR product obtained in the PCR step;
including, and
The five types of primer sets are
A primer that is a combination of forward primer I that hybridizes to the 53rd to 75th sites in the nucleotide sequence of SEQ ID NO: 1 and reverse primer I that hybridizes to the 1002nd to 1024th sites in the nucleotide sequence of SEQ ID NO: 1. set I,
A primer that is a combination of forward primer II that hybridizes to the 1369th to 1396th sites in the nucleotide sequence of SEQ ID NO: 2 and reverse primer II that hybridizes to the 1895th to 1922nd sites in the nucleotide sequence of SEQ ID NO: 2. Set II,
A primer that is a combination of a forward primer III that hybridizes to positions 3 to 30 in the nucleotide sequence of SEQ ID NO: 3 and a reverse primer III that hybridizes to positions 312 to 335 in the nucleotide sequence of SEQ ID NO: 3. Set III,
A primer that is a combination of a forward primer IV that hybridizes to positions 111 to 138 in the nucleotide sequence of SEQ ID NO: 4 and a reverse primer IV that hybridizes to positions 340 to 367 in the nucleotide sequence of SEQ ID NO: 4. set IV, and
A primer that is a combination of forward primer V that hybridizes to the 36th to 55th sites in the nucleotide sequence of SEQ ID NO: 5 and reverse primer V that hybridizes to the 201st to 222nd sites in the nucleotide sequence of SEQ ID NO: 5. set V,
A method for simultaneously detecting rot fungi, characterized in that : The primer set II is a combination of a forward primer II consisting of the nucleotide sequence of SEQ ID NO: 8 and a reverse primer II consisting of the nucleotide sequence of SEQ ID NO: 9 . Method for simultaneous detection of maggot pathogens. 3. The primer set III is a combination of a forward primer III consisting of the nucleotide sequence of SEQ ID NO: 10 and a reverse primer III consisting of the nucleotide sequence of SEQ ID NO: 11. A method for simultaneous detection of rot fungi. Among claims 1 to 3, the primer set IV is a combination of a forward primer IV consisting of the nucleotide sequence of SEQ ID NO: 12 and a reverse primer IV consisting of the nucleotide sequence of SEQ ID NO: 13. The method for simultaneous detection of rot fungi according to any one of the above. The five types of primer sets are
Primer set I, which is a combination of forward primer I consisting of the nucleotide sequence of SEQ ID NO: 6 and reverse primer I consisting of the nucleotide sequence of SEQ ID NO: 7;
Primer set II, which is a combination of forward primer II consisting of the nucleotide sequence of SEQ ID NO: 8 and reverse primer II consisting of the nucleotide sequence of SEQ ID NO: 9;
Primer set III, which is a combination of forward primer III consisting of the nucleotide sequence of SEQ ID NO: 10 and reverse primer III consisting of the nucleotide sequence of SEQ ID NO: 11;
Primer set IV is a combination of forward primer IV consisting of the nucleotide sequence of SEQ ID NO: 12 and reverse primer IV consisting of the nucleotide sequence of SEQ ID NO: 13; and forward primer V consisting of the nucleotide sequence of SEQ ID NO: 14. A primer set V, which is a combination of a reverse primer V consisting of the nucleotide sequence of SEQ ID NO: 15,
The method for simultaneous detection of rot fungi according to any one of claims 1 to 4 , characterized in that: The method for simultaneous detection of rot fungi according to any one of claims 1 to 5 , wherein the detection step includes a step of separating the PCR product by electrophoresis. Primer set II, which is a combination of forward primer II consisting of the nucleotide sequence of SEQ ID NO: 8 and reverse primer II consisting of the nucleotide sequence of SEQ ID NO: 9;
Primer set III is a combination of forward primer III consisting of the nucleotide sequence of SEQ ID NO: 10 and reverse primer III consisting of the nucleotide sequence of SEQ ID NO: 11; and forward primer IV consisting of the nucleotide sequence of SEQ ID NO: 12. Reverse primer IV consisting of the nucleotide sequence of SEQ ID NO: 13, and primer set IV, which is a combination of
A primer kit comprising at least one primer set selected from the group consisting of: Primer set I, which is a combination of forward primer I consisting of the nucleotide sequence of SEQ ID NO: 6 and reverse primer I consisting of the nucleotide sequence of SEQ ID NO: 7;
Primer set II, which is a combination of forward primer II consisting of the nucleotide sequence of SEQ ID NO: 8 and reverse primer II consisting of the nucleotide sequence of SEQ ID NO: 9;
Primer set III, which is a combination of forward primer III consisting of the nucleotide sequence of SEQ ID NO: 10 and reverse primer III consisting of the nucleotide sequence of SEQ ID NO: 11;
Primer set IV is a combination of forward primer IV consisting of the nucleotide sequence of SEQ ID NO: 12 and reverse primer IV consisting of the nucleotide sequence of SEQ ID NO: 13; and forward primer V consisting of the nucleotide sequence of SEQ ID NO: 14. A primer set V, which is a combination of a reverse primer V consisting of the nucleotide sequence of SEQ ID NO: 15,
The primer kit according to claim 7 , comprising: 9. The primer kit according to claim 7 or 8 , which is a primer kit for use in the method for simultaneous detection of rot fungi according to any one of claims 1 to 6 .