Primer set, method for detecting mycolic acid-producing gene, and method for inhibiting abnormal foaming

Abstract

To provide a primer set suitable for a PCR method effective for evaluating the possibility of mycolic acid mass production, a method of setting the primer set, a method of detecting a gene that produces mycolic acid, and a method of suppressing abnormal foaming.SOLUTION: Provided is a primer set characterized by including a forward primer having a specific base sequence or a base sequence different from the base sequence by several bases on a 5' end side, and a reverse primer having a specific base sequence different from the aforementioned specific base sequence or a base sequence differing from base sequence by several bases on the 5' end side. Also provided are a method of setting the primer set, a method of using the primer set to detect a gene that produces mycolic acid, and a method of suppressing abnormal foaming.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a primer set suitable for use in the polymerase chain reaction (PCR), particularly real-time PCR, a method for designing the primer set, a method for detecting mycolic acid-producing genes using the primer set, and a method for inhibiting abnormal foaming of treated water, etc., using the detection of mycolic acid-producing genes. [Background technology]

[0002] Abnormal foaming caused by bacteria and other factors has been observed in water treatment facilities for some time now. The layer of hard bubbles generated by this foaming, also known as "scum," can cause foul odors, pest infestations, poor sludge settling, and a decline in water quality. It can also cause the treated water to overflow from the treatment tank, adversely affecting the surrounding environment and water treatment operations, creating a major problem. As soon as scum occurs, follow-up measures are taken, such as physical removal of the scum or chemical water quality adjustment, but dealing with large amounts of scum requires a great deal of effort.

[0003] The main causative bacteria for scum formation are thought to be "actinomycetes." Actinomycetes refers to a group of Gram-positive bacteria characterized by the morphological characteristics of elongated growth through the formation of hyphae. This group includes many genera under the phylum Actinobacteria, as well as some bacilli and cocci, classified based on molecular phylogeny based on the base sequence of the 16S r ribonucleic acid (RNA) gene. Among these actinomycetes, mycolic acid, secreted primarily by bacteria of the Gordonia genus, is thought to be the primary causative agent for scum formation. Mycolic acid is a general term for long-chain fatty acids with approximately 60 to 90 carbon atoms. It contains a cyclopropane structure and is a substance that forms the backbone of the cell walls of tuberculosis bacteria, acid-fast bacteria, and other bacteria, or is expressed on the surface of the cell walls.

[0004] The detection of actinomycetes, one of the bacteria that causes scum, begins with the discovery of some abnormality in the treatment tanks of water treatment facilities. Conventional methods involve sampling treated water and confirming the presence of actinomycetes under a microscope, or culturing fungi or bacteria from treated water under specified conditions to form colonies and then identifying and quantifying the actinomycetes. The former method often relies on the subjective evaluation of the individual in charge and does not allow for quantitative evaluation. Genetic analysis of the bacteria would be expensive and time-consuming. Meanwhile, the latter method requires too much time and effort for the culture itself, and even if the actinomycetes can be accurately identified and quantified, the information does not provide the necessary information to prevent mass proliferation of actinomycetes or the mass production of mycolic acids.

[0005] Patent Document 1 describes a conventional technique for quantifying specific bacteria by PCR in treatment tanks for drinking water, sewage, various types of wastewater, etc. It also describes that the technique in Patent Document 1 makes it possible to quantify Gordonia amarae, a species of the Gordonia genus that is thought to be one of the bacteria responsible for the generation of scum.

[0006] However, it is believed that abnormal foaming is not limited to specific species of bacteria such as Gordonia amarae, and there is a problem that it is difficult to effectively suppress abnormal foaming in treated water, etc., using methods that detect only specific species of bacteria, such as those described in Patent Document 1. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 11-89597 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made in light of the above-mentioned problems, and aims to provide a primer set suitable for a PCR method that is effective in evaluating the possibility of mass production of mycolic acid, a method for designing the primer set, a method for detecting mycolic acid-producing genes, and a method for inhibiting abnormal foaming. [Means for solving the problem]

[0009] In order to achieve the above object, a first aspect of the present invention is a primer set characterized by comprising a forward primer having the base sequence of SEQ ID NO: 1 or a base sequence which differs from that base sequence by several bases at the 5' end, and a reverse primer having the base sequence of SEQ ID NO: 2 or a base sequence which differs from that base sequence by several bases at the 5' end.

[0010] A second aspect of the present invention is a primer set characterized by comprising a forward primer having the base sequence of SEQ ID NO: 1 and a reverse primer having a base sequence that is 92% or more homologous to the base sequence of SEQ ID NO: 3.

[0011] A third aspect of the present invention is a method for designing a primer set, comprising: (a) a synthetic gene search step of identifying an amino acid sequence of an exon portion common to multiple species among the amino acid sequences of synthetic genes of a target enzyme, converting the identified amino acid sequence into a deoxyribonucleic acid (DNA) sequence, and designating the DNA sequence as a conservative DNA sequence; and (b) a primer creation step of creating a PCR primer set consisting of a forward primer and a reverse primer, each having a primer length of approximately 17 to 25 mer, based on the conservative DNA sequence.

[0012] A fourth aspect of the present invention is a method for detecting mycolic acid-producing genes, characterized by comprising the steps of: (a) preparing a forward primer having the base sequence of SEQ ID NO: 1 or a base sequence that differs from that base sequence by several bases at the 5' end; (b) preparing a reverse primer having the base sequence of SEQ ID NO: 2 or a base sequence that differs from that base sequence by several bases at the 5' end; and (c) detecting mycolic acid-producing genes from an aqueous solution sample by PCR using a primer set having the forward primer and the reverse primer.

[0013] A fifth aspect of the present invention is a method for detecting mycolic acid-producing genes, characterized by comprising the steps of: (a) preparing a forward primer having the base sequence of SEQ ID NO: 1; (b) preparing a reverse primer having a base sequence that is 92% or more homologous to the base sequence of SEQ ID NO: 3; and (c) detecting mycolic acid-producing genes from an aqueous solution sample by PCR using a primer set having the forward primer and the reverse primer.

[0014] A sixth aspect of the present invention is a method for inhibiting abnormal foaming in the target water, comprising the steps of: (a) collecting an aqueous solution sample from target water; (b) detecting a mycolic acid-producing gene from the aqueous solution sample by PCR using a primer set including a forward primer having the base sequence of SEQ ID NO: 1 or a base sequence that differs from that base sequence by several bases at its 5' end; and a reverse primer having the base sequence of SEQ ID NO: 2 or a base sequence that differs from that base sequence by several bases at its 5' end; (c) evaluating the possibility of mass proliferation of actinomycetes in the target water based on the detection results; and (d) if the evaluation determines that there is a possibility of mass proliferation of actinomycetes, taking measures against the proliferation of actinomycetes before abnormal foaming occurs in the target water, wherein the method is characterized by inhibiting abnormal foaming in the target water caused by actinomycetes.

[0015] According to the present invention, a primer set suitable for a PCR method that is effective for evaluating the possibility of mass production of mycolic acid, a method for designing the primer set, a method for detecting mycolic acid-producing genes, and a method for inhibiting abnormal foaming can be provided. [Brief explanation of the drawings]

[0016] [Figure 1] 1 is a flowchart showing a method for setting a primer set according to an embodiment of the present invention. [Figure 2] 1 is a flowchart showing a method for suppressing abnormal foaming according to an embodiment of the present invention. [Figure 3] This is Table 1 showing the results of confirming the amplified species using NCBI Primer BLAST. [Figure 4] This is Table 2 showing the results of confirming the amplified species using NCBI Primer BLAST. DETAILED DESCRIPTION OF THE INVENTION

[0017] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the following description of the drawings, identical or similar parts are designated by identical or similar reference numerals. However, it should be noted that the drawings are schematic. Therefore, specific methods, etc. should be determined in a more diverse manner, taking into consideration the gist of the technical ideas that can be understood from the following description.

[0018] Furthermore, the embodiments of the present invention shown below are merely examples of methods for realizing the technical idea of ​​the present invention, and the technical idea of ​​the present invention is not limited to the described methods, etc. The technical idea of ​​the present invention is not limited to the contents described in the embodiments of the present invention, and various modifications can be made within the technical scope defined by the organic combination of the invention-specifying matters described in the claims.

[0019] The primer set according to an embodiment of the present invention is a primer pair suitable for amplification by PCR, particularly real-time PCR, and comprises a forward primer having the nucleotide sequence of SEQ ID NO: 1 in the Sequence Listing or a nucleotide sequence that differs from that nucleotide sequence by several bases at the 5' end, and a reverse primer having the nucleotide sequence of SEQ ID NO: 2 in the Sequence Listing or a nucleotide sequence that differs from that nucleotide sequence by several bases at the 5' end. As shown in Table 1 and the Sequence Listing, the nucleotide sequence of SEQ ID NO: 1 is an 18-mer, and the nucleotide sequence of SEQ ID NO: 2 is a 25-mer. The second base from the 5' end of the nucleotide sequence of SEQ ID NO: 2 is C (cytosine) or T (thymine), and is therefore indicated by the symbol "Y." The fifth base is A (adenine) or G (guanine), and is therefore indicated by the symbol "R." Note that while all bases (nucleic acid bases) in this embodiment are represented by uppercase letters, the bases in the Sequence Listing are represented by lowercase letters due to the specifications of the Sequence Listing.

[0020] [Table 1]

[0021] The forward primer in the primer set according to an embodiment of the present invention may have any base sequence substantially homologous to the base sequence of SEQ ID NO: 1. In the description of the embodiment, the base sequences of the various primers are "substantially homologous" to each other means that they have sufficient fragment length and homology to function as various primers. For example, depending on the purpose and conditions of use, the various primers do not necessarily have to be 100% complementary to the target DNA region, and several bases near the 5' end of the primer for the target DNA region may differ. Even if several bases differ, it is possible to effectively amplify the target DNA fragment by adjusting various conditions such as the annealing temperature.

[0022] In the forward primer according to the embodiment, the nucleotide may be one in which a base has been inserted, deleted, or substituted in the base sequence of SEQ ID NO: 1, but it is preferable that the nucleotide sequence has 94% or more homology with the base sequence of SEQ ID NO: 1. More preferably, in the forward primer according to the embodiment, the nucleotide sequence has 100% homology with the base sequence of SEQ ID NO: 1. In the forward primer according to the embodiment, when a base has been inserted, deleted, or substituted, the position of the insertion, deletion, or substitution is not particularly limited, but it is preferable that there is no insertion, deletion, or substitution of a base on the 3'-end side.

[0023] The reverse primer of the primer set according to the embodiment of the present invention may have any base sequence substantially homologous to the base sequence of SEQ ID NO: 2. For example, three base sequences, SEQ ID NOs: 3 to 5, are shown in the sequence listing as specific examples of the base sequence of SEQ ID NO: 2, but the reverse primer according to the embodiment may be any of these, or may be a base sequence other than the three specific base sequences of SEQ ID NOs: 3 to 5. Furthermore, the reverse primer according to the embodiment may have a nucleotide sequence in which a base is inserted, deleted, or substituted in the base sequence of SEQ ID NO: 3, but preferably has a base sequence that is 92% or more homologous to the base sequence of SEQ ID NO: 3. More preferably, the reverse primer according to the embodiment has a base sequence that is 96% or more homologous to the base sequence of SEQ ID NO: 3, and even more preferably has a base sequence that is 100% homologous to the base sequence of SEQ ID NO: 3. The same concept applies to the base sequences of SEQ ID NOs: 4 and 5. When a base is inserted, deleted, or substituted in the reverse primer according to the embodiment, i.e., in each of the base sequences of SEQ ID NOs: 2 to 5, the position of the insertion, deletion, or substitution is not particularly limited, but it is preferable that there is no insertion, deletion, or substitution of a base on the 3'-end side.

[0024] The nucleotide sequence of SEQ ID NO: 3, which is a specific example of a reverse primer according to an embodiment, differs from the nucleotide sequence of SEQ ID NO: 4 in the fifth nucleotide sequence from the 5' end. The nucleotide sequence of SEQ ID NO: 3 in the fifth nucleotide sequence from the 5' end is "A," whereas the nucleotide sequence of SEQ ID NO: 4 in the fifth nucleotide sequence from the 5' end is "G." The nucleotide sequence of SEQ ID NO: 3, which is a specific example of a reverse primer according to an embodiment, differs from the nucleotide sequence of SEQ ID NO: 5 in two positions, the second and fifth nucleotides from the 5' end. The nucleotide sequence of SEQ ID NO: 3 in the second and fifth nucleotides from the 5' end are "C" and "A," respectively, whereas the nucleotide sequence of SEQ ID NO: 5 in the second and fifth nucleotides from the 5' end are "T" and "G." The nucleotide sequence of SEQ ID NO: 4, which is a specific example of a reverse primer according to an embodiment, differs from the nucleotide sequence of SEQ ID NO: 5 in the second nucleotide sequence from the 5' end. The second base from the 5' end of the base sequence of SEQ ID NO: 4 is "C," whereas the second base from the 5' end of the base sequence of SEQ ID NO: 5 is "T." The differences between the base sequences of SEQ ID NOs: 3 to 5 are present at one or two positions on the 5' end.

[0025] (Primer set design method) Regarding the design of a primer set according to an embodiment of the present invention, the procedure will be explained below using an example in which the sequence corresponding to the mycolic acid production gene among the DNA sequences of the target actinomycete or actinomycete group is unknown.

[0026] First, as a preliminary investigation, we will search public databases such as NCBI for species of the genus Gordonia, a type of actinomycete, whose full genomes or genomes close to them have been made public.

[0027] Next, in step S101 of Figure 1, a search for synthetic genes for mycolic acid synthases is performed by searching for the amino acid sequence of synthetic genes encoding mycolic acid synthases in public amino acid sequence databases such as the NCBI protein database. Based on the obtained amino acid sequence, the amino acid sequence of the exon portion common to multiple Gordonia species is deduced, and this amino acid sequence is converted into a DNA sequence. DNA sequences that can be deduced as common to multiple Gordonia species are defined as "conserved DNA sequences." The presence or absence of a portion corresponding to the conserved DNA sequence is confirmed within the full genome of Gordonia obtained in the preliminary investigation stage or a genome close to it. If a sequence highly homologous to the conserved DNA sequence is confirmed, proceed to the next step.

[0028] Next, in step S103 of FIG. 1 , primers suitable for PCR, particularly real-time PCR, are created from the conserved DNA sequence. From the perspectives of improving PCR detection (ensuring yield) and eliminating nonspecific amplification, primers preferably have a base length of approximately 17 to 25 mers. For example, the base sequence of SEQ ID NO: 1 is an 18 mer, and the base sequences of SEQ ID NOs: 2 to 5 are 25 mers, both of which fall within this preferred range. To create a primer set, various application software may be used from the prototype stage of the primer set, or the primer set sequence may be estimated and manually prototyped, followed by the use of various applications such as Primer BLAST to confirm specificity. As application software, known primer design programs such as Primer 3 Plus (http: / / www.bioinformatics.nl / cgi-bin / primer3plus / primer3plus.cgi) may be used to amplify the desired conserved DNA sequence.

[0029] Next, the species that can be amplified with the created primer set are confirmed. For example, the amplified species is confirmed using NCBI Primer BLAST or the like to check the presence and proportion of mismatches on the 3' end. The presence of mismatches on the 3' end is determined, for example, by checking whether there are one or more mutations within a few bases, for example, within five bases, from the 3' end of each primer. This is because it has been found that if there are one or more mutations within a few bases, particularly within five bases, from the 3' end of each primer, amplification of the target DNA fragment by PCR is difficult. If there are two or more mismatches within a few bases, particularly within five bases, from the 3' end of each primer, amplification of the target DNA fragment by PCR is generally considered impossible.

[0030] Finally, in the practicality verification step of step S105 shown in Fig. 1, if it is determined that the created primer set is theoretically capable of amplifying the target DNA fragment, the primer set is used to perform PCR on an actual test sample, etc., to verify the practicality of the created primer set. If it is determined that the created primer set is practically usable through PCR on an actual test sample, etc., the created primer set is used as a primer set according to an embodiment of the present invention.

[0031] The method for designing a primer set according to an embodiment of the present invention has been described above as a method for calculating a DNA sequence from an amino acid sequence encoding a target enzyme, but this need not necessarily be the case if the DNA sequence encoding the target enzyme is known from the beginning. For example, if the desired base sequence is available from GenBank provided by NCBI or the NITE Biological Resource Center (NBRC), it can be used.

[0032] (Method for detecting mycolic acid-producing genes) The method for detecting mycolic acid-producing genes using the primer set according to the embodiment is as follows. First, in the DNA extraction step, the collected sample is subjected to pretreatment such as ultrasonication to extract DNA. The sample pretreatment and DNA extraction methods may be known methods or may be other methods. The collected sample may be an aqueous solution, or may be an aqueous solution containing a foamy substance such as scum, a solid, or the like dissolved therein.

[0033] Next, in the DNA detection step, amplification conditions, i.e., denaturation temperature, annealing temperature and time, number of cycles, extension temperature and time, etc., are determined appropriately based on the designed primer length, GC content, etc., and PCR is performed. The polymerase used in PCR may be a known polymerase, such as a DNA polymerase such as Taq DNA polymerase or proofreading DNA polymerase. A calibration curve can be prepared by amplifying actinomycete-derived DNA by PCR and adjusting its concentration. DNA extracted from scum previously collected and known to contain actinomycetes may also be used for the calibration curve.

[0034] Next, in the data analysis stage, the amplified actinomycete DNA fragments in the sample are quantified. Various known or novel methods can be used as the quantification method. For example, quantification can be performed by measuring the DNA concentration of the solution after PCR using a spectrophotometer, or a quantification method using electrophoresis or DNA staining with a dye can also be used. When performing PCR, particularly real-time PCR, any known method can be used to fluorescently detect the PCR amplification product, such as the intercalation method or the fluorescently labeled probe method. From the perspective of simultaneously amplifying target DNA of multiple Gordonia species, the intercalation method is preferable to the fluorescently labeled probe method, which exhibits high specificity using probes.

[0035] (Method for suppressing abnormal foaming in treated water) An example of a method for suppressing abnormal foaming using a primer set according to an embodiment is given below. As shown in Fig. 2, in water quality testing step S201, a water quality test is performed as part of daily water quality management in a treatment tank or the like of a water treatment facility. In judgment step S203 shown in Fig. 2, a preliminary judgment is made based on the test results of water quality test items such as pH as to whether actinomycetes will grow in the future. For example, an example of an indicator is when the pH fluctuates significantly within the pH range of 7 to 8, which is considered to be the range in which actinomycetes are likely to grow.

[0036] If the water quality test indicates the possibility of actinomycete proliferation, in PCR step S205 shown in Figure 2, PCR, particularly real-time PCR, is performed on the treated water sample using the primer set according to the embodiment to detect and quantify the mycolic acid production gene. The specific real-time PCR procedure may be the same as the above-mentioned method for detecting mycolic acid production genes, or it may be any procedure commonly performed by those skilled in the art. The quantification results in decision step S207 shown in Figure 2 are evaluated, for example, by determining whether the quantification value of the amplified DNA fragment exceeds a reference value, or whether the production rate of the mycolic acid production gene calculated from the quantification results obtained at multiple time points exceeds a reference value, and the possibility of mass proliferation of actinomycetes and mass production of mycolic acid is examined.

[0037] If it is determined that there is a possibility of mass proliferation of actinomycetes and mass production of mycolic acid, appropriate preventive measures, such as adjusting the pH of the water, are taken in preventive measures step S209 shown in Figure 2. After taking preventive measures, it is preferable to conduct water quality testing again at a predetermined timing. Alternatively, instead of water quality testing, the possibility of mass proliferation of actinomycetes and mass production of mycolic acid may be determined again by PCR.

[0038] In special conditions that favor the proliferation of actinomycetes, such as when the nitrification solution circulation pump is started or restarted, when the inlet door is changed, or after a heavy rainfall, it is desirable to intensively detect mycolic acid production genes using real-time PCR and conduct frequent monitoring in cases where actinomycetes are likely to newly enter the treatment tank.

[0039] In the above example, the treated water, which is an aqueous solution, is used as the sample. However, scum, a solution of scum, or an aqueous solution containing scum may also be used as the sample.

[0040] The primer set according to an embodiment of the present invention is a primer set capable of efficiently amplifying target DNA fragments by PCR, particularly real-time PCR, focusing on species of the genus Gordonia and those related to mycolic acid production. This primer set is not limited to detecting specific Gordonia species, but can also detect a wide range of species that produce mycolic acid, the direct causative agent of scum, making it possible to effectively detect scum occurrence in advance. The advantage of detecting mass scum occurrence in advance and taking preventative measures is that it eliminates the extensive effort required for subsequent countermeasures.

[0041] Real-time PCR using the primer set according to the present invention can be performed in approximately two hours per cycle, making it possible to evaluate treated water samples from multiple locations on the same day, and even to perform repeat tests on the same sample on the same day. This allows for rapid evaluation of the possibility of mass production of mycolic acids using a method that is not dependent on the individual.

[0042] In the primer set design method according to an embodiment of the present invention, as described above, the DNA sequence is deduced from the amino acid sequence of the enzyme, and effective primers can be created by confirming that the DNA sequence is a cross-species sequence. The "back-calculation method" of deducing the DNA sequence from the amino acid sequence is unprecedented in the creation of PCR primers and is a completely novel design method. This primer set design method can be applied to other systems as long as the amino acid sequence is known, even if the DNA sequence is not known.

[0043] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. [Example]

[0044] (Primer set setting) As a preliminary investigation step in setting the primer set, we searched the NCBI nucleotide database for nucleotides of Gordonia species whose full genome information is publicly available, and found that Gordonia sp. 1D chromosome (NCBI GenBank: ACCESSION No. CP023405, 5151623 base pairs) was the matched gene.

[0045] Next, in the search for the synthetic gene for the mycolic acid-producing enzyme, a search was conducted in the NCBI protein database using the keyword "Cyclopropane fatty acid synthase gordonia." This resulted in an amino acid sequence related to the gene name "cfa" (NCBI GenBank: ACCESSION No. GAC78239, DEFINITION cyclopropane fatty acid synthase [Gordonia malaquae NBRC 108250]). Searching for the protein information obtained from this amino acid sequence revealed that it contained both the "Cfa superfamily" and "CMAS (CmaA2, MmaA2, and PcaA)."

[0046] The amino acid sequence related to "cfa" in ACCESSION No. GAC78239, which corresponds to the "Cfa superfamily," was mechanically converted to a DNA sequence. The resulting DNA sequence was then searched using NCBI Nucleotide BLAST. This DNA sequence, which corresponds to positions 369613 to 370871 in the full genome of Gordonia sp. 1D chromosome (NCBI GenBank: ACCESSION No. CP023405), is shown in SEQ ID NO: 8 in the Sequence Listing. The DNA sequence was mechanically converted to an amino acid sequence, and NCBI Protein BLAST was performed on the amino acid sequence. Sequences containing the cfa superfamily of Gordonia were found. Therefore, it was estimated that the DNA sequence of the primer candidate is likely common to multiple Gordonia species, and the DNA sequence was designated a "conserved DNA sequence."

[0047] Next, in the primer creation stage, primer candidates were selected from conservative DNA sequences, and finally, a primer set was created using the nucleotide sequence of SEQ ID NO: 1 as the forward primer and the nucleotide sequence of SEQ ID NO: 3 as the reverse primer. The nucleotide sequence of SEQ ID NO: 1 is an 18-mer, and the nucleotide sequence of SEQ ID NO: 3 is a 25-mer, which was found to be within a preferable length range from the perspectives of improving PCR detection (ensuring yield) and eliminating nonspecific amplification. After creating this primer set, specificity (confirmation of amplified species) was confirmed using NCBI Primer BLAST. As shown in Figures 3 and 4, the amplified species were confirmed to be the only sequences with no mismatches at the 3' end that belonged to Gordonia species. Therefore, this primer set was determined to have strong selectivity for Gordonia species, and this primer set was designated as the primer set of Example 1. The forward primer having the nucleotide sequence of SEQ ID NO: 1 was designated as the forward primer of Example 1, and the reverse primer having the nucleotide sequence of SEQ ID NO: 3 was designated as the reverse primer of Example 1.

[0048] 3 and 4 list combinations of nucleotides (including bacterial species names) registered in the NCBI nucleotide database and their accession numbers. In the tables of FIGS. 3 and 4, sequences highly structurally homologous to each nucleotide and the forward primer (SEQ ID NO: 1) and reverse primer (SEQ ID NO: 3) of Example 1 are extracted, and the structural homology (unit: %) with each primer is shown. For example, the "Gordonia sp. 1D chromosome (NCBI GenBank: ACCESSION No. CP023405)" shown in FIG. 3 is shown to have 100% structural homology with the forward primer (SEQ ID NO: 1) and reverse primer (SEQ ID NO: 3) of Example 1. In the case of "Gordonia sp. 1D chromosome (NCBI GenBank: ACCESSION No. CP023405)," the nucleotide sequence amplified by the forward primer (SEQ ID NO: 1) and reverse primer (SEQ ID NO: 3) of Example 1 is the 131-mer nucleotide sequence shown in SEQ ID NO: 6. Furthermore, in the case of "Gordonia ajococcus strain A2 chromosome, complete genome (NCBI GenBank: ACCESSION No. CP052884)" shown in FIG. 3, it can be seen that there is a nucleotide sequence that is 100% homologous to the forward primer (SEQ ID NO: 1) of Example 1, and a nucleotide sequence that is 96% homologous to the reverse primer (SEQ ID NO: 3) of Example 1. The only difference from the reverse primer (SEQ ID NO: 3) of Example 1 is the fifth nucleotide from the 5' end. In "Gordonia ajococcus strain A2 chromosome, complete genome (NCBI GenBank: ACCESSION No. CP052884)," the nucleotide sequence amplified by the forward primer (SEQ ID NO: 1) of Example 1 and the reverse primer (SEQ ID NO: 3) of Example 1 is the 131-mer nucleotide sequence shown in SEQ ID NO: 7.Similarly, the structural homology between the forward primer (SEQ ID NO: 1) and the reverse primer (SEQ ID NO: 3) of Example 1 can be confirmed for the nucleotides following "Gordonia rubripertincta strain SD5 chromosome, complete genome (NCBI GenBank: ACCESSION No. CP059694)" shown in Figure 3 and the nucleotides shown in Figure 4.

[0049] For example, in the case of "Gordonia ajococcus strain A2 chromosome, complete genome (NCBI GenBank: ACCESSION No. CP052884)" shown in Figure 3, when a reverse primer having the nucleotide sequence of SEQ ID NO: 4 is used as a reverse primer, structural homology becomes 100%, thereby enabling more effective amplification of the desired DNA fragment. Furthermore, in the case of "Gordonia rubripertincta strain SD5 chromosome, complete genome (NCBI GenBank: ACCESSION No. CP059694)" and "Gordonia rubripertincta strain CWB2 complete genome (NCBI GenBank: ACCESSION No. CP022580)" shown in Figure 3, when a reverse primer having the nucleotide sequence of SEQ ID NO: 5 is used as a reverse primer, structural homology becomes 100%, thereby enabling more effective amplification of the desired DNA fragment.

[0050] In the practicality verification stage, real-time PCR was performed on actual scum samples under specified conditions using the primer set (SEQ ID NOs: 1 and 3) according to Example 1 (real-time PCR machine: QuantStudio3 ("QuantStudio" is a registered trademark, manufactured by Thermo Fisher Scientific)). The DNA sequence of the real-time PCR product was decoded and NCBI BLAST was performed. The highest match rate was achieved by a species of Gordonia, suggesting that the species was effectively detected. Furthermore, comprehensive analysis was performed using a next-generation sequencer on the bacterial DNA contained in the scum sample used for amplification. Bacteria of the genus Gordonia were detected as the most prioritized species, suggesting that the species of Gordonia had been successfully detected. [Example]

[0051] (Detection of mycolic acid-producing genes) For the DNA extraction step, scum samples collected from sewage treatment tanks were sonicated for 30 seconds and then vacuum filtered using a glass filter (Whatman GF / F 47 mm) from GE Healthcare. DNA was then extracted and purified from the filter using a DNA extraction kit (DNeasy Blood and Tissue kit) from Qiagen.

[0052] Real-time PCR was performed on the obtained DNA extraction sample using the reagent materials in Table 2, the temperature conditions in Table 3, and the primer set of Example 1. A Thermo Fisher Scientific real-time PCR machine (QuantStudio3) was used, and the calibration curve sample was prepared by PCR amplification of actinomycete-derived DNA extracted from another scum sample collected in advance and adjusting the concentration. DNA quantification was performed using the intercalation method, which utilizes a fluorescent substance that binds to double-stranded nucleic acids. As a result, it was confirmed that the amplified DNA fragments effectively amplified DNA derived from the genus Gordonia in the scum sample. [Table 2] [Table 3]

[0053] (Other embodiments) As described above, the present invention has been described by the above embodiments and examples, but the descriptions and drawings that form part of this disclosure should not be understood to limit the present invention. Various alternative embodiments, examples, and operating techniques will become apparent to those skilled in the art from this disclosure.

[0054] For example, a primer set can be designed to detect a wider range of species in genera other than Gordonia that have genes involved in mycolic acid production. It is also possible to perform multiplex PCR in which multiple primer sets are used simultaneously.

[0055] For example, in the present invention, with regard to the individual sequences of the nucleic acids to be detected and various primers, matters described based on their mutual complementary relationship naturally apply to each described sequence and the sequence complementary to each sequence, unless otherwise specified. When the present invention is applied to the sequence complementary to each sequence, the sequence recognized by the complementary sequence should be read as a sequence complementary to the corresponding sequence described in this specification, within the scope of common general technical knowledge of a person skilled in the art.

[0056] As such, the present invention naturally includes various embodiments not described herein. Therefore, the technical scope of the present invention is defined only by the invention-specifying matters according to the claims, which can be interpreted appropriately from the above explanation.

Claims

1. a forward primer having the base sequence of SEQ ID NO: 1 or a base sequence different by one base from the base sequence of SEQ ID NO: 1; a reverse primer having the base sequence of SEQ ID NO: 2; A primer set comprising:

2. a forward primer having a nucleotide sequence that is 94% or more identical to the nucleotide sequence of SEQ ID NO: 1; a reverse primer having a base sequence that is 92% or more identical to the base sequence of SEQ ID NO: 3; A primer set comprising:

3. The primer set according to claim 2, wherein the base sequence of the reverse primer is identical to the base sequence of SEQ ID NO:

3.

4. providing a forward primer having the base sequence of SEQ ID NO: 1 or a base sequence that differs by one base from the base sequence of SEQ ID NO: 1; providing a reverse primer having the base sequence of SEQ ID NO: 2; detecting a mycolic acid-producing gene from an aqueous solution sample by PCR using a primer set having the forward primer and the reverse primer; A method for detecting a mycolic acid-producing gene, comprising:

5. providing a forward primer having a base sequence that is 94% or more identical to the base sequence of SEQ ID NO: 1; providing a reverse primer having a base sequence that is 92% or more identical to the base sequence of SEQ ID NO: 3; detecting a mycolic acid-producing gene from an aqueous solution sample by PCR using a primer set having the forward primer and the reverse primer; A method for detecting a mycolic acid-producing gene, comprising:

6. obtaining an aqueous sample from the water of interest; detecting a mycolic acid-producing gene from the aqueous solution sample by PCR using a primer set comprising a forward primer having the base sequence of SEQ ID NO: 1 or a base sequence that differs by one base from the base sequence of SEQ ID NO: 1 and a reverse primer having the base sequence of SEQ ID NO: 2; assessing the possibility of actinomycetes growing in the water based on the results of the detection; a step of taking measures against the proliferation of the actinomycetes before abnormal foaming occurs in the water when it is determined by the evaluation that there is a possibility that the actinomycetes will proliferate in large quantities; and inhibiting abnormal foaming in the water, the abnormal foaming being caused by the actinomycete.

7. Prior to the step of taking the aqueous solution sample, Measuring pH fluctuations and selecting the water The method for suppressing abnormal foaming according to claim 6, further comprising:

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