Primer set, probe, method and kit for detecting causative bacterium of black spot on pearl oyster shell

Abstract

To provide a PCR primer set, a probe and the like useful for detecting Tenacibaculumsp. Pbs-1 strain.SOLUTION: A forward primer comprising a nucleotide sequence S1 of at least 15 contiguous nucleotides in the nucleotide sequence at positions 345 to 395 of SEQ ID NO: 1, or a nucleotide sequence S2 in which one or two nucleotides are inserted, deleted, or substituted in the nucleotide sequence S1; and a reverse primer comprising a nucleotide sequence S3 of at least 15 contiguous nucleotides in the nucleotide sequence at positions 555 to 589 of SEQ ID NO: 1, or a nucleotide sequence S3 in which one or two nucleotides are inserted, deleted, Or a PCR primer set comprising a reverse primer comprising a substituted nucleotide sequence S4. Disclosed is a probe for detecting Tenacibaculumsp. Pbs-1, comprising a nucleotide sequence S5 consisting of at least 15 contiguous nucleotides in the nucleotide sequence ranging from positions 380 to 445 of SEQ ID NO: 1, or a nucleotide sequence S5 resulting from insertion, deletion, or substitution of one or two nucleotides in the nucleotide sequence S6.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a primer set, a probe, a method, a kit, etc. for detecting the bacterium causing pearl oyster shell black stain disease. [Background technology]

[0002] Mie, Nagasaki, and Ehime prefectures together account for approximately 90% of Japan's pearl culture production. The Akoya pearls produced in these regions are highly valued worldwide. With annual production and exports exceeding 20 tons and 30 billion yen, respectively, they are the backbone of the local economy and industry, and an important export industry. Mie Prefecture, in particular, is the birthplace of pearl culture and a key region for the development of large pearl production techniques and the production of small and large pearls. However, in recent years, the black shell disease of pearl-forming Akoya oysters has become a serious problem, threatening the continuation of this traditional technique.

[0003] This disease was first reported in 1967 and remains unresolved as of 2024. Severe cases of black shell disease can lead to death, and in some years, fewer than half of the original number of pearls can be harvested. Even if the disease doesn't lead to death, the pearls they produce develop black stains, resulting in them being downgraded to low-quality pearls (dokuzu pearls), resulting in a dramatic difference in trading price. This makes it extremely difficult for pearl farmers to consistently produce pearls. Between 1993 and 2003, the number of pearl farms in Mie, Ehime, and Nagasaki prefectures declined by 36%, 28%, and 24%, respectively. The decline in Mie Prefecture was particularly dramatic, with the number of farms declining to 283 in 2013 (10% of its peak). Thus, black shell stain is a major problem for pearl farmers, and countermeasures against it are urgently needed. However, even the cause of the disease remains unknown. A research consortium led by Toyama University investigated the incidence of black shell stain in pearl oysters in Japan's major pearl oyster aquaculture areas (Mie, Ehime, and Nagasaki) and found that the disease may be caused by a high density of a specific bacterium (Non-Patent Document 1). Further research led to the isolation of Tenacibaculum sp. Pbs-1 strain from an individual with black shell stain and the successful reproduction of the disease by infecting healthy oysters with this strain. This identified one of the causes of the disease, which had been unknown for approximately 50 years (Non-Patent Document 2). Furthermore, a visual detection LAMP (Loop-mediated isothermal amplification) method was developed that can detect the causative bacterium in situ (Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] New developments in parasitic diseases of pearl-forming mother oysters, Pinctada martensii, Akihiro Sakano, Takuya Fujimura, Michiko Ito, Seigo Takashima, Tadashi Isshiki, Journal of the Japanese Society of Applied Biology, 30, 1-6, 2017 [Non-patent document 2] Newly isolated bacterium Tenacibaculum sp. Strain Pbs-1 from diseased pearl oysters is associated with black-spot shell disease, Sakatoku A., Fujimura T., Ito M., Takashima S., Isshiki T., Aquacluture, 493, 61-67, 2018 [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2024-002628 Summary of the Invention [Problem to be solved by the invention]

[0006] However, the LAMP method described in Patent Document 1 is not a quantitative method and is therefore not suitable for use in detailed histopathological analysis or for understanding the dynamics of causative bacteria in the environment.

[0007] Therefore, a primary objective of the present invention is to provide a PCR primer set, probe, method, kit, etc. that are useful for detecting (particularly quantitatively detecting) the Tenacibaculum sp. Pbs-1 strain, which is the bacterium that causes pearl oyster shell black stain disease. [Means for solving the problem]

[0008] As a result of extensive research aimed at solving the above problems, the present inventors have found that a specific PCR primer set and / or probe is useful for detecting (particularly quantitatively detecting) the Tenacibaculum sp. Pbs-1 strain. The present invention was completed through further research based on this finding.

[0009] The present invention includes the following aspects. [Section 1] A PCR primer set for detecting Tenacibaculum sp. Pbs-1 strain, A forward primer comprising a base sequence S1 of at least 15 consecutive bases in the base sequence from positions 345 to 395 of SEQ ID NO: 1, or a base sequence S2 in which one or two bases are inserted, deleted, or substituted in the base sequence S1; and A reverse primer comprising a base sequence S3 of at least 15 consecutive bases in the base sequence from positions 555 to 589 of SEQ ID NO: 1, or a base sequence S4 in which one or two bases are inserted, deleted, or substituted in the base sequence S3. PCR primer set comprising: [Section 2] Item 1, wherein the forward primer contains a base corresponding to position 380 of SEQ ID NO: 1, and the reverse primer contains at least one of bases corresponding to positions 569, 571, and 574 of SEQ ID NO: 1. The PCR primer set according to Item 1. [Section 3] Item 3. The PCR primer set according to Item 1 or 2, wherein the forward primer comprises the nucleotide sequence represented by SEQ ID NO: 2, and the reverse primer comprises the nucleotide sequence represented by SEQ ID NO: 3. [Section 4] Item 4. The PCR primer set according to any one of Items 1 to 3, wherein the length of the nucleotide sequence of the forward primer and the reverse primer is 20 to 40 nucleotides each. [Section 5] A probe for detecting Tenacibaculum sp. Pbs-1 strain, comprising a base sequence S5 of at least 15 consecutive bases in the base sequence from positions 380 to 445 of SEQ ID NO: 1, or a base sequence S6 in which one or two bases have been inserted, deleted, or substituted in the base sequence S5. [Section 6] Item 6. The probe according to Item 5, wherein the probe comprises the base sequence represented by SEQ ID NO:4. [Section 7] Item 7. The probe according to Item 5 or 6, wherein the length of the base sequence of the probe is 20 to 40 bases. [Section 8] A PCR primer set according to any one of Items 1 to 4, for use in combination with the probe according to any one of Items 5 to 7. [Section 9] A probe according to any one of Items 5 to 7, for use in combination with the PCR primer set according to any one of Items 1 to 4. [Section 10] A method for detecting Tenacibaculum sp. Pbs-1 strain in a sample, comprising: A step A of carrying out a PCR reaction using the PCR primer set according to any one of items 1 to 4 and nucleic acids in a sample as a template; and Step B: detecting an amplification product in the reaction product of Step A A method comprising: [Section 11] A kit for detecting Tenacibaculum sp. Pbs-1 strain, comprising the PCR primer set according to any one of Items 1 to 4 and / or the probe according to any one of Items 5 to 7. [Effects of the Invention]

[0010] The present invention provides a PCR primer set, probe, method, kit, and the like that are useful for detecting Tenacibaculum sp. Pbs-1, the bacterium that causes pearl oyster shell black spot disease. In particular, quantitative detection is possible, which allows for detailed histopathological analysis and understanding of the dynamics of the bacterium that causes pearl oyster shell black spot disease in the environment. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the results of electrophoresis of amplification products obtained by quantitative PCR (qPCR) in an example. [Figure 2] 1 is a graph showing the linearity of the amplification curve and Cq (quantification cycle) value by the qPCR method of an example. [Figure 3] 1 is a graph showing the detection of Tenacibaculum sp. Pbs-1 strain in environmental samples by the qPCR method of an example. DETAILED DESCRIPTION OF THE INVENTION

[0012] 1. PCR primer set In one embodiment, the PCR primer set can be suitably used to detect Tenacibaculum sp. strain Pbs-1 (hereinafter, may be simply referred to as "strain Pbs-1").

[0013] The Pbs-1 strain was discovered by a research consortium led by Toyama University as one of the bacteria that causes pearl oyster shell blackening disease, and can also be obtained by the method described in Non-Patent Document 2.

[0014] The PCR primer set can be preferably used to specifically detect the Pbs-1 strain. Here, "specific detection" refers to, for example, not (or substantially not) detecting closely related species. The closely related species preferably have 16S rRNA genes and / or 23S rRNA genes that share, for example, 95% or more, preferably 98% or more, more preferably 99% or more, but less than 100% identity with the nucleotide sequence of the 16S rRNA gene and / or 23S rRNA gene of the Pbs-1 strain. The nucleotide sequence identity can be calculated using the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) (http: / / www.ncbi.nlm.nih.gov / BLAST / ) with default (initial setting) parameters. Examples of closely related species include T. mesophilum, T. amylolyticum, T. sediminilitoris, T. maritimum, and T. ascidiaceicola.

[0015] The PCR primer set can be used to detect (specifically detect) the ISR (Internal Spacer Region) region between the 16S rRNA gene and the 23S rRNA gene of the Pbs-1 strain. The ISR region preferably has the following sequence: ctagagaaag atggtgagtt acaatagagg ttaattttgc tctttgctgt taattttaaa aaatagacta agatcttaaa aatgagataa tttttagatg tttgatatta atatctaagc tgtctaagac agtctcgtag ctcagctggt tagagcgcta cactgataat gtagaggtcg gcagttcgag tctgcccgag actacaattt taagactaag taaaggaaat tctagaagtt gggattcaca attctgaatt cataattttg aatttgcaat tgggggatta gctcagctgg ctagagcgct tgccttgcac gcaagaggtc atcggttcga ctccgatatt ctccactggg caatgtccag agataataag aattattatt tctgagagta ttgcaggcag attgtaacta acaagtttaa ttacttgtca gtggcgactc gccacaacgt tcattgacat attggtaaaa tgatatcgta aagaatcaag atagagagtt agataacatc taacaacata tttttataag aacaagaatt ataaagagct cgttgtagcg caagctacag caaaaagta (SEQ ID NO: 1) This sequence is registered in NCBI's BLAST under accession number LC724053.

[0016] The PCR primer set may amplify a portion of the base sequence of SEQ ID NO: 1 or the entire length of the base sequence of SEQ ID NO: 1, but it is preferable that the PCR primer set amplifies at least a region of the base sequence of SEQ ID NO: 1 that is specific to the Pbs-1 strain (for example, a region that contains bases different from those of the related species, such as the region containing bases 380, 569, 571, or 574 of SEQ ID NO: 1).

[0017] The PCR primer set preferably comprises one or more forward primers and one or more reverse primers.

[0018] In one embodiment, the forward primer preferably comprises a base sequence S1 of at least 15 consecutive bases in the base sequence from positions 345 to 395 of SEQ ID NO: 1, or a base sequence S2 in which one or two bases (preferably one base) have been inserted, deleted, or substituted (preferably substituted) in the base sequence S1, and more preferably comprises the base sequence S1.

[0019] The forward primer (or base sequence S1 or S2) preferably contains the base corresponding to position 380 of SEQ ID NO: 1, and more preferably contains the base sequence corresponding to positions 358 to 381 of SEQ ID NO: 1 (the base sequence represented by SEQ ID NO: 2).

[0020] The length of the forward primer (or nucleotide sequence S1 or S2) is preferably 16 bases or more, more preferably 17 bases or more, even more preferably 18 bases or more, still more preferably 19 bases or more, and particularly preferably 20 bases or more. The length of the forward primer (or nucleotide sequence S1 or S2) is preferably 50 bases or less, more preferably 45 bases or less, and even more preferably 40 bases or less. The length of the forward primer (or nucleotide sequence S1 or S2) is, for example, 16 to 50 bases, preferably 18 to 45 bases, and even more preferably 20 to 40 bases.

[0021] In one embodiment, the reverse primer preferably comprises a base sequence S3 of at least 15 consecutive bases in the base sequence from bases 555 to 589 of SEQ ID NO: 1, or a base sequence S4 in which one or two bases (preferably one) have been inserted, deleted, or substituted (preferably substituted) in the base sequence S3, and more preferably comprises the base sequence S3.

[0022] The reverse primer (or base sequence S3 or S4) preferably contains at least one (preferably two, more preferably three) of the bases corresponding to positions 569, 571, and 574 of SEQ ID NO: 1, and more preferably contains the base sequence corresponding to positions 569 to 589 of SEQ ID NO: 1 (the base sequence represented by SEQ ID NO: 3).

[0023] The length of the reverse primer (or base sequence S3 or S4) is preferably 16 bases or more, more preferably 17 bases or more, even more preferably 18 bases or more, still more preferably 19 bases or more, and particularly preferably 20 bases or more. The length of the reverse primer (or base sequence S3 or S4) is preferably 50 bases or less, more preferably 45 bases or less, and even more preferably 40 bases or less. The length of the reverse primer (or base sequence S3 or S4) is, for example, 16 to 50 bases, preferably 18 to 45 bases, and even more preferably 20 to 40 bases.

[0024] The PCR primer set is preferably used in combination with the probe described in 2 below.

[0025] Each primer included in the PCR primer set can be prepared by chemical synthesis, genetic engineering techniques, or by using a commercially available DNA synthesizer.

[0026] 2. Probe In one embodiment, the probe is suitable for use in detecting the Pbs-1 strain.

[0027] The probe preferably recognizes the ISR region of the Pbs-1 strain, and more preferably recognizes the region amplified by the PCR primer set described in 1 above (e.g., the region between the forward primer and the reverse primer).

[0028] In one embodiment, the probe preferably comprises a base sequence S5 of at least 15 consecutive bases in the base sequence from positions 380 to 445 of SEQ ID NO: 1, or a base sequence S6 in which one or two (preferably one) bases have been inserted, deleted, or substituted (preferably substituted) in base sequence S5, more preferably comprises base sequence S5, and even more preferably comprises a base sequence corresponding to positions 397 to 427 of SEQ ID NO: 1 (the base sequence represented by SEQ ID NO: 4).

[0029] The length of the probe (or base sequence S5 or S6) is preferably 16 bases or more, more preferably 17 bases or more, even more preferably 18 bases or more, still more preferably 19 bases or more, and particularly preferably 20 bases or more. The length of the probe (or base sequence S5 or S6) is preferably 50 bases or less, more preferably 45 bases or less, and even more preferably 40 bases or less. The length of the probe (or base sequence S5 or S6) is, for example, 16 to 50 bases, preferably 18 to 45 bases, and even more preferably 20 to 40 bases.

[0030] The probe is preferably labeled with a labeling substance (e.g., a fluorescent substance). In one embodiment, the 5' end of the probe is preferably modified with a fluorescent substance and the 3' end of the probe is preferably modified with a quencher substance. Examples of fluorescent substances include Cy5, FAM, HEX, JOE, ROX, TET, Texas Red, and VIC. Examples of quencher substances include 6-carboxytetramethylrhodamine (TAMRA), BHQ (Black Hole Quencher), and IBFQ (Iowa Black FQ).

[0031] The probe is preferably used in combination with the PCR primer set described in 1 above.

[0032] The probes can be prepared by methods such as chemical synthesis or genetic engineering techniques, or by using a commercially available DNA synthesizer.

[0033] 3. Detection Method In one embodiment, the method for detecting Tenacibaculum sp. Pbs-1 strain in a sample preferably comprises the following steps A and B. (Process A) A step of carrying out a PCR reaction using the PCR primer set described in 1 above and nucleic acid in a sample as a template. (Process B) a step of detecting an amplification product in the reaction product of step A

[0034] As used herein, "detecting Tenacibaculum sp. Pbs-1 strain in a sample" not only means determining whether or not the bacteria causing the pearl oyster shell black stain disease may be present in the sample, but also includes qualitatively or quantitatively evaluating the amount of the bacteria causing the pearl oyster shell black stain disease that may be present in the sample.

[0035] The sample is not particularly limited as long as it can contain the bacterium responsible for pearl oyster shell black stain. The sample may be derived from a pearl oyster that has not developed black shell stain. According to the present invention, even healthy individuals that have not developed black shell stain can detect the bacterium responsible for black shell stain if they are carriers of the bacterium, thereby preventing the onset of black shell stain in those individuals. The sample is preferably a nucleic acid extract derived from pearl oyster shells. Nucleic acid extraction can be performed using a known or conventional kit (e.g., Wizard Genomic DNA Purification Kit (Promega), FastDNA SPIN Kit for Soil (MP-Biomedicals), or PowerSoil DNA isolation kit (Mo Bio)). The nucleic acid extract may be subjected to one or more treatments, such as concentration, dilution, and purification. The amount of nucleic acid in the sample may be, for example, 0.1 pg or more, 0.5 pg or more, or 1 pg or more. The amount of nucleic acid in the sample may be, for example, 10 ng or less. Furthermore, the amount of nucleic acid in the sample may be, for example, 0.1 pg to 10 ng. 10 pieces, preferably 10 to 10 9 The amount of nucleic acid contained in a cell.

[0036] In one embodiment, step A is preferably a step of incubating a PCR composition containing a sample, the PCR primer set described in 1 above, the probe described in 2 above, a DNA polymerase, a substrate, and an additive.

[0037] The concentrations of the forward primer and reverse primer of the PCR primer set are not particularly limited, but are each preferably 0.1 μM or more, more preferably 0.15 μM or more, and even more preferably 0.2 μM or more. Furthermore, the concentrations are preferably 0.5 μM or less, 0.4 μM or less, or 0.3 μM or less. Furthermore, the concentrations are preferably 0.1 to 0.5 μM.

[0038] The probe concentration is not particularly limited, but is preferably 0.1 μM or more, more preferably 0.15 μM or more, and even more preferably 0.2 μM or more. Furthermore, the concentration is preferably 0.5 μM or less, 0.4 μM or less, or 0.3 μM or less. Furthermore, the concentration is preferably 0.1 to 0.5 μM.

[0039] Examples of DNA polymerases include Taq, Tbr, Tfl, Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT™, DEEPVENT™, and mutants thereof.

[0040] Substrates include, for example, a mixture of deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyadenosine triphosphate (dATP), and deoxythymidine triphosphate (dTTP).

[0041] Additives include, for example, buffers, salts, and other additives.

[0042] Examples of buffers include Tris, Tricine, Bis-Tricine, Hepes, Mops, Tes, Taps, Pipes, Caps, and combinations of two or more of these. Buffers are usually dissolved in water (e.g., sterilized water) and used in the form of an aqueous solution.

[0043] Examples of salts include chlorides such as LiCl, NaCl, KCl, MgCl2, and MnCl2; acetates such as lithium acetate, sodium acetate, potassium acetate, magnesium acetate, and manganese acetate; sulfates such as K2SO4, MgSO4, and MnSO4; and combinations of two or more of these.

[0044] Other additives include, for example, betaine, bovine serum albumin, surfactants, DMSO (dimethyl sulfoxide), gelatin, glycerol, pyrophosphate, spermidine, T4Gene32 protein, formamide, and combinations of two or more of these.

[0045] The PCR composition may contain reagents contained in commercially available PCR (particularly real-time PCR) kits, such as Premix Ex Taq (trademark) from Takara Bio Inc.

[0046] In one embodiment, the incubation in step A is preferably carried out by thermal cycling. Thermal cycling typically involves repeating a cycle consisting of step A1, in which DNA is denatured (dissociated from double-stranded DNA into single-stranded DNA), step A2, in which a primer is annealed to the single-stranded DNA, and step A3, in which the primer is extended. Step A1 may be carried out at 85 to 100°C (preferably 90 to 98°C) for 1 to 100 seconds, for example, in terms of cleavage into single strands and prevention of enzyme inactivation. Step A2 may be carried out at a lower temperature (e.g., 50 to 70°C, preferably 55 to 65°C) than steps A1 and A3 for 1 to 100 seconds, for example, in terms of prevention of nonspecific binding and annealing. Step A3 may be carried out for 1 to 100 seconds at a temperature lower than that of step A1 and higher than that of step A2 (e.g., 55 to 80°C, preferably 60 to 75°C), for example, from the viewpoint of polymerase functionality. The cycle of steps A1 to A3 may be repeated, for example, 30 to 70 times. The temperature and time of steps A1 to A3 may be changed every 1 to 3 cycles.

[0047] Step B is not particularly limited as long as it can detect the amplification product in the reaction product of step A. Examples of the detection method include electrophoresis, the intercalator method, and hybridization with the probe described in 2 above. Examples of fluorescent molecules used in the intercalator method include TB Green and SYBR (trademark) Green.

[0048] 4. Kit In one embodiment, the kit can be suitably used for detecting the Pbs-1 strain. The kit preferably includes the PCR primer set described in 1 above and / or the probe described in 2 above. The kit may further include components contained in the PCR composition described in 3 above, such as DNA polymerase, substrates, and additives. The kit may further include a control. The control may be a positive control and / or a negative control. The kit may further include reagents necessary for sample preparation, instruments used for detection (e.g., reaction tubes, etc.), instructions, etc. [Example]

[0049] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0050] (Production Example 1: Preparation of DNA from Pbs-1 strain and other Tanacibaculum bacteria (related species)) The Pbs-1 strain was collected using the method described in Non-Patent Document 2. Other Tanacibaculum bacteria (T. mesophilum, T. amylolyticum, T. sediminilitoris, T. maritimum, and T. ascidiaceicola) were purchased from the National Institute of Technology and Evaluation. DNA from the Pbs-1 strain and other Tanacibaculum bacteria was extracted from the cells after approximately one day of culture in seawater Cytophaga liquid medium using a Wizard Genomic DNA Purification Kit (Promega). The DNA solution thus obtained was used as the DNA template. The concentration of the extracted DNA was quantified using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific KK, Kanagawa, Japan) and stored at -30°C until use.

[0051] Example 1 The qPCR reaction solution had a total volume of 10 μL and the following composition: 1×Premix Ex Taq (Takara Bio Inc., Shiga, Japan) 0.25 μM forward primer 0.25 μM reverse primer 0.25 μM 5'-FAM-labeled probe 3 μL DNA template (1 pg).

[0052] The qPCR reaction was performed at 95°C for 30 seconds, followed by 50 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, and finally 72°C for 1 minute. After qPCR, 2% (w / v) agarose gel electrophoresis was performed.

[0053] The results are shown in Figure 1. In Figure 1, M represents a 100-bp ladder marker, 1 represents Tanacibaculum sp. Pbs-1 strain, 2 represents Tanacibaculum mesophilum, 3 represents Tanacibaculum sediminilitoris, 4 represents Tanacibaculum ascidiaceicola, 5 represents Tanacibaculum amylolyticum, 6 represents Tanacibaculum maritimum, and NC represents a negative control (an equal volume of sterile water was added instead of the DNA template).

[0054] As is clear from FIG. 1, DNA from closely related species was not amplified, and only DNA from the Pbs-1 strain was amplified.

[0055] Example 2 The Pbs-1 strain was added to natural seawater (1 L) from Tsukumo Bay. 1 ~10 9 The resulting solution was added to the culture medium so that the total number of cells was 10, and a 10-fold serial dilution series was prepared. The entire bacteria were collected by filtration using a 0.2 μm filter, and DNA was extracted from the filter using the FastDNA SPIN Kit for Soil (MP-Biomedicals). qPCR reactions were performed using the extracted DNA (3 μL) as a template under the same conditions as in Example 1 to examine its quantitative properties.

[0056] The results are shown in Figure 2. As is clear from Figure 2, the Pbs-1 strain was quantitatively detected even in seawater samples containing other common bacteria. 1 It was also possible to quantify the number of copies of DNA. 2 ) showed high quantitative accuracy of 0.998.

[0057] Example 3 The mantle of a pearl oyster from Nagasaki Prefecture that showed no signs of disease was instilled with 100 μL of Pbs-1 culture medium (3.4 × 10 9 cells / mL) and reared (infected) at 25°C for 10 days (controls were injected with the same amount of medium only, four individuals for each). DNA was extracted from a portion of the shell after rearing (infection), and qPCR was performed using the extracted DNA as a template under the same conditions as in Example 1 to examine its quantitative properties.

[0058] The results are shown in Figure 3. As is clear from Figure 3, the Pbs-1 strain was quantitatively detected in all experimentally infected individuals. Furthermore, since it was also detected in one control individual, it is believed that it is also possible to detect it in individuals that are carriers but have not developed disease (asymptomatically infected individuals). [Industrial Applicability]

[0059] The PCR primer set and probe of the present invention have high quantitative and specific detection capabilities for Pbs-1 strains, enabling dynamic monitoring of Pbs-1 strains in cultured seawater and the preservation and succession of healthy pearl farming environments. Furthermore, quantitative detection of the causative bacterium in diseased individuals allows for detailed pathological analysis of disease infection and onset mechanisms. This is expected to contribute significantly to elucidating the nature of this disease. Furthermore, this invention allows for quantitative testing of the presence or absence of bacteria when conducting research on infection control measures, which is expected to significantly accelerate progress. Furthermore, preliminary studies have shown that black discoloration similar to this disease is observed not only in Japanese and Vietnamese pearl oysters (Pinctada fucata) cultivated in Japan, but also in other species of pearl oysters from other countries, such as Indian Ocean pearl oysters (P. radiata). Therefore, this invention is sought after not only by pearl farmers in Japan but also by pearl farmers around the world, and its widespread adoption is expected to have even greater economic benefits.

Claims

1. A PCR primer set for detecting Tenacibaculum sp. Pbs-1 strain, A forward primer comprising a base sequence S1 of at least 15 consecutive bases in the base sequence from positions 345 to 395 of SEQ ID NO: 1, or a base sequence S2 in which one or two bases are inserted, deleted, or substituted in the base sequence S1; and A reverse primer comprising a base sequence S3 of at least 15 consecutive bases in the base sequence from 555 to 589 of SEQ ID NO: 1, or a base sequence S4 in which one or two bases are inserted, deleted, or substituted in the base sequence S3. PCR primer set comprising:

2. 2. The PCR primer set according to claim 1, wherein the forward primer comprises a base corresponding to the 380th position of SEQ ID NO: 1, and the reverse primer comprises at least one of the bases corresponding to the 569th, 571st, and 574th positions of SEQ ID NO:

1.

3. 2. The PCR primer set according to claim 1, wherein the forward primer comprises the base sequence represented by SEQ ID NO: 2, and the reverse primer comprises the base sequence represented by SEQ ID NO:

3.

4. 2. The PCR primer set according to claim 1, wherein the length of the base sequence of the forward primer and the reverse primer is 20 to 40 bases each.

5. A probe for detecting Tenacibaculum sp. Pbs-1 strain, comprising a base sequence S5 of at least 15 consecutive bases in the base sequence from positions 380 to 445 of SEQ ID NO: 1, or a base sequence S6 in which one or two bases have been inserted, deleted, or substituted in the base sequence S5.

6. The probe according to claim 5, wherein the probe comprises the base sequence represented by SEQ ID NO:

4.

7. 6. The probe according to claim 5, wherein the length of the base sequence of the probe is 20 to 40 bases.

8. A PCR primer set according to any one of claims 1 to 4, for use in combination with a probe according to any one of claims 5 to 7.

9. The probe according to any one of claims 5 to 7, for use in combination with the PCR primer set according to any one of claims 1 to 4.

10. A method for detecting Tenacibaculum sp. Pbs-1 strain in a sample, comprising: Step A: carrying out a PCR reaction using the PCR primer set according to any one of claims 1 to 4 and nucleic acids in a sample as a template; and Step B: detecting an amplification product in the reaction product of Step A A method comprising:

11. A kit for detecting Tenacibaculum sp. Pbs-1 strain, comprising the PCR primer set according to any one of claims 1 to 4 and / or the probe according to any one of claims 5 to 7.

Images