JP2024066550A - Oligonucleotide for detecting tribolium confusum jacquelin du val - Google Patents

Abstract

To develop an oligonucleotide for detecting Tribolium confusum Jacquelin du Val, family Tenebrionidae, that is a food insect pest to establish a test system that can accurately and quickly detect the Tribolium confusum Jacquelin du Val at low cost.SOLUTION: The present invention provides: an oligonucleotide set for detecting Tribolium confusum Jacquelin du Val, which is composed of two or more types of oligonucleotides containing the characteristic base sequence of the mitochondrial DNA of the Tribolium confusum Jacquelin du Val; a kit for detecting Tribolium confusum Jacquelin du Val containing the oligonucleotide set; and a method for detecting Trifolium confusum Jacquelin du Val using the oligonucleotide set.SELECTED DRAWING: None

Description

The present invention relates to an oligonucleotide for detecting the food pest Tribolium destructor Uyttenboogaart and a method for detecting the Tribolium destructor Uyttenboogaart.

The black-headed flour beetle (Tribolium destructor Uyttenboogaart) was discovered in Sweden in 1934. It is believed to have originated in Africa, but it has been found in Asia, Europe, and North and South America, and its habitat is expanding worldwide. The black-headed flour beetle is known to have a wide range of feeding habits, and is said to form colonies and establish itself in natural environments. In addition, since the black-headed flour beetle is not considered to be flightless, the main factor for its long-distance spread is thought to be human-mediated distribution, such as the transportation of grain from storage and flour milling facilities, and transportation from food and fiber production factories to stores and homes. There is currently no clear evidence that this species has been discovered in Japan, and its establishment has not been confirmed.

The perception of the damage caused by the beetle to stored grain varies from country to country and region to region due to differences in climate and major grain species, but in provinces such as Alberta, Canada, the beetle is considered to be as economically important as other serious pests such as Tribolium confusum and Tribolium castaneum, and the beetle is included as a major stored grain pest (Non-Patent Document 1). In addition, parts or all of Western Australia have declared the beetle a prohibited pest. In addition, there are reports that measures have been taken to eradicate the beetle in Sweden and the UK.

Rice is essential to Japanese cuisine, and Japan has been producing high-quality Japonica rice for many years through tireless efforts, including breeding and improving cultivation methods. Japan has traditionally imported rice from China, the United States, Thailand, and other countries, but with interest in Japanese cuisine growing worldwide, exports of domestically produced agricultural and marine products and processed products are increasing.

Quarantine requirements for exporting polished Japanese rice to China, the world's largest rice-consuming country, require confirmation that the export rice polishing plant and brown rice storage facility are free of the small red rice beetle, the small spotted rice beetle, the small spotted rice beetle, the granarian rice weevil, as well as the black flat-headed flour beetle.

For this reason, when exporting polished rice from Japan to China, as a preventative precaution, it is required to confirm the absence of black flour beetles by using monitoring traps for walking insects in the polishing factories and brown rice storage facilities for export, and to thoroughly clean any areas where pests may be present (Non-Patent Document 2).

As a monitoring trap for the above-mentioned walking insects, a sticky trap (pheromone trap) incorporating an attractant such as a pheromone is generally used. However,
Since the orientation of the insects fixed to the adhesive sheet is not constant, visual identification can be difficult in some cases. In addition, many of the Tenebrionidae family, to which the black flour beetle belongs, are morphologically similar. Furthermore, grain pests are generally small even as adults, and the characteristics of the larvae and eggs are limited, making it extremely difficult to identify them by visual inspection.

The DNA present within cells is divided into nuclear (genomic) DNA, mitochondrial DNA, and chloroplast DNA. Nuclear DNA is used in criminal investigations, to determine blood relationships such as parent-child relationships, and to identify varieties of crops and livestock. Of these, mitochondrial DNA mutates more frequently than nuclear DNA and has a greater number of copies per cell than the genome, making it useful for identifying insect species.

There are various DNA testing methods, such as the traditional PCR method, the LAMP (Loop-Mediated Isothermal Amplification) method, methods that use DNA base sequencing, Southern hybridization, and DNA microarrays. Among these, the LAMP method and the DNA barcoding method that uses DNA base sequencing are used for identification. The LAMP method is highly specific and is useful because it does not require strict and multi-stage temperature changes during DNA reaction like the PCR method, but on the other hand, it has the disadvantage that it is difficult to design primers, and the sequences for which primers can be designed are limited, making it difficult to use. In addition, the DNA barcoding method decodes the DNA sequence and compares the sequence information with known sequence information, so it can identify even the insect name. However, this method has the disadvantage that the operation is complicated, costly, and time-consuming because the base sequence of the analysis sample that was subjected to the PCR method is decoded by a DNA sequencer, and then the sequence of each sample is compared using dedicated software.

Unlike the previously mentioned DNA barcoding, the PCR method is a method in which the detection target is determined in advance and whether or not DNA derived from that target is contained in a sample. The test can determine the presence or absence of the DNA of the target organism simply by subjecting the extracted DNA to PCR, and has the advantage of being able to perform large-scale, rapid determination. One typical example is when the target insect species is roughly known, which is particularly useful for identifying insects that have been captured with a certain degree of selectivity using the aforementioned pheromone trap. In addition, when using a trap that uses pheromones to attract insects, due to its characteristics, there is a possibility that a large number of individuals with similar appearances, such as closely related species of the insect to be attracted, will be captured, but by using appropriately designed primers and probes, it is possible to quickly, accurately, and in large quantities to determine whether or not the insect is the target for identification.

As a method for analyzing the amplification products of the PCR method, a real-time PCR method is also known that enables quantitative analysis by measuring fluorescent signals without electrophoresis, and a set of primers and detection probes for this purpose is provided. For example, Patent Document 1 describes specific and universal probes and amplification primers for rapid detection and identification of bacterial pathogens. The present inventor has already established an oligonucleotide set for detecting major food pests such as the granaria weevil and the red-spotted beetle by the PCR method or real-time PCR method (Patent Document 2). However, the black flat-headed flour beetle is an invasive species of the family Tenebrionidae that has never been confirmed in Japan, and although there is a potential risk of contamination of grains and processed foods, there is a small number of samples and information, and an oligonucleotide set for use in analysis by the PCR method or real-time PCR method has not been established.

JP 2007-125032 A Patent No. 6600831

https://grainscanada.gc.ca/en/grain-quality/manage/identify-an-insect/primary-insect-pests/large-flour-beetle.html https://www.maff.go.jp/j/syouan/syokubo/keneki/k_setumei/

Therefore, in view of the above-mentioned circumstances, the object of the present invention is to develop an oligonucleotide for detecting the black flour beetle, a food pest of the Tenebrionidae family, and to establish a test system capable of detecting the black flour beetle accurately, quickly, and at low cost.

As a result of intensive research to solve the above problems, the inventors have discovered base sequences characteristic of Tribolium confusum, a beetle of the Tenebrionidae family, from mitochondrial DNA sequence information, and by combining oligonucleotides containing these base sequences, they have succeeded in establishing an oligonucleotide set that can accurately and quickly detect Tribolium confusum.
That is, the present invention includes the following inventions.

(1) An oligonucleotide set for detecting Tribolium confusum, which is composed of two or more oligonucleotides containing a characteristic base sequence of the mitochondrial DNA of Tribolium confusum.
(2) The oligonucleotide set for detecting the black flour beetle described in (1), wherein the characteristic base sequence of mitochondrial DNA is a base sequence within the cytochrome c oxidase subunit 1 (CO1) region.
(3) The oligonucleotide set for detecting the black flour beetle according to (1) or (2), wherein the oligonucleotides are used as primers and probes.
(4) The oligonucleotide set for detecting the black flour beetle according to any one of (1) to (3), which is an oligonucleotide set consisting of an oligonucleotide having the base sequence shown in any one of SEQ ID NOs: 1 to 3 below or a complementary sequence thereof, or an oligonucleotide having a base sequence containing at least 15 consecutive bases in the base sequence shown in any one of SEQ ID NOs: 1 to 3 or a complementary sequence thereof.
SEQ ID NO: 1: 5'-CGACCTATAGGAATATCATTCG-3'
SEQ ID NO: 2: 5'-CTCCTCCCCCTGCAGGGTCA-3'
SEQ ID NO: 3: 5'-CCCCTATTCGTTTGAGCTGTAG-3'
(5) A kit for detecting Tribolium confusum, comprising the oligonucleotide set for detecting Tribolium confusum according to any one of (1) to (4).
(6) A method for detecting Tribolium confusum, comprising the steps of: using DNA extracted from a test sample as a template, carrying out PCR amplification using an oligonucleotide set for detecting Tribolium confusum described in any one of (1) to (4), and detecting the obtained amplification product.

According to the present invention, there are provided an oligonucleotide set for detecting the black flour beetle, which is required as a quarantine condition that the black flour be absent from rice milling plants and brown rice storage facilities for export when polished rice produced in Japan is exported, a kit for detecting the black flour beetle containing the oligonucleotide set, and a method for detecting the black flour beetle using the oligonucleotide set. Therefore, according to the present invention, the black flour beetle can be detected accurately, quickly, and at low cost, and the reliability of quality can be strengthened by utilizing it in food management and assurance.

Figure 1 shows the results of detection of the black flour beetle by real-time PCR using a TaqMan ^TM probe. In this method, the black flour beetle and major food pests (22 species) were used as samples, and the nucleotide sequence of SEQ ID NO: 1 was used as the upstream primer, the nucleotide sequence of SEQ ID NO: 2 was used as the downstream primer, and the nucleotide sequence of SEQ ID NO: 3, labeled with a reporter dye (FAM) on the 5' side and a quencher (TAMRA) on the 3' side, was used as the TaqMan ^TM probe. Figure 2 shows the results of detection of Tribolium confusum by real-time PCR using TaqMan ^TM probe. In this method, Tribolium confusum and four major grains were used as samples, and the nucleotide sequence of SEQ ID NO: 1 was used as the upstream primer, the nucleotide sequence of SEQ ID NO: 2 was used as the downstream primer, and the nucleotide sequence of SEQ ID NO: 3, which was labeled with a reporter dye (FAM) on the 5' side and a quencher (TAMRA) on the 3' side, was used as the TaqMan ^TM probe. 3 shows the detection results of the black flour beetle by the Simplex PCR method. In this method, the black flour beetle and major food pests (22 species) were used as samples, and the base sequence of SEQ ID NO: 1 was used as the upstream primer and the base sequence of SEQ ID NO: 2 was used as the downstream primer (P: black flour beetle, M: ladder marker (Gene Ladder 100), N: negative control (no DNA), 1: red flour beetle, 2: Chinese meal beetle, 3: small meal beetle, 4: brown meal beetle, 5: white meal beetle, 6: flat-headed flour beetle, 7: Kashmir red flour beetle, 8: big horned flour beetle, 9: small flour beetle, 10: 10: Tobacco beetle, 11: Bean beetle, 12: Square beetle, 13: Sawtooth beetle, 14: Azuki bean weevil, 15: Rice weevil, 16: Rice weevil, 17: Granaria rice weevil, 18: Indian meal moth, 19: Bacterial moth, 20: Small cut grass beetle, 21: Small red cut grass beetle, 22: Small cut grass beetle). 4 shows the results of detection of Tribolium congenita by Simplex PCR. In this method, Tribolium congenita and four major grains were used as samples, and the base sequence of SEQ ID NO: 1 was used as the upstream primer and the base sequence of SEQ ID NO: 2 was used as the downstream primer (P: Tribolium congenita, M: ladder marker (Gene Ladder 100), N: negative control (no DNA), 23: barley, 24: rice, 25: corn, 26: wheat).

The present invention will be described in detail below.
1. Oligonucleotide set for detecting Tribolium congenita and detection kit The oligonucleotide set for detecting Tribolium congenita of the present invention is composed of two or more oligonucleotides containing a characteristic base sequence of the mitochondrial DNA of Tribolium congenita to be detected. The base length of the oligonucleotide is not limited, but is usually 15 to 30 bases long, preferably 18 to 25 bases long, for a primer, and 10 to 30 bases long for a probe.

The characteristic base sequence of the mitochondrial DNA of the target black flour beetle refers to a base sequence that has low homology with known mitochondrial DNA base sequences of a wide range of food pests and is considered to be characteristic only of the target black flour beetle, and is 10 bases or more in length, preferably 15 bases or more, and more preferably 20 bases or more in length.

In identifying the above-mentioned characteristic base sequence of mitochondrial DNA, first, the base sequence information of the mitochondrial DNA of Tribolium congenita is obtained. The base sequence information may be obtained from a database (NCBI, DDBJ, etc.) or may be obtained by directly analyzing the base sequence as shown in the examples below.

In the present invention, an example of a characteristic base sequence of the mitochondrial DNA of Tribolium confusum is the base sequence of the cytochrome c oxidase subunit 1 (CO1) region.

Next, primers are designed based on the identified characteristic base sequence. Primers are designed taking into consideration the length of the oligonucleotide, GC content, Tm value, complementarity between oligonucleotides, and secondary structures within the oligonucleotide. For example, "The Frontline of PCR - From Basic Technology to Applications" (Protein, Nucleic Acid, Enzyme Special Issue, 1996, Kyoritsu Publishing Co., Ltd.), "Bio Experiment Illustrated 3: Truly Increasing PCR: Cell Engineering Appendix, Visual Experiment Note Series" (by Hiroki Nakayama, Shujunsha Co., Ltd.), and "PCR Technology - Principles and Applications of DNA Amplification" (edited by Henry A. Erlich, supervised by Kuninoshin Kato, Takara Shuzo Co., Ltd.) can be used as a reference.

The specific design standards adopted are as follows:
(a) When the PCR amplified product of the characteristic sequence of the mitochondrial DNA of the black flour beetle was electrophoresed, the amplified product was clearly detected.
(b) The PCR amplification product is 100 to 500 bp, preferably 100 to 250 bp.
(c) The length of the primer should be in the range of 15 to 30 bp to enable specific annealing to the template DNA.
(d) The annealing temperature depends on the melting temperature (Tm), so in order to obtain a highly specific PCR amplification product, primers with similar Tm values should be selected, with a Tm value of 50-70°C, preferably 55-65°C.
(e) The nucleotide sequence at the 3' end of the primer has high homology with the template DNA sequence.
(f) The primers should be free of complementary sequences between each other to prevent the formation of dimers or three-dimensional structures.
(g) In order to ensure stable binding to the template DNA, the GC content should be approximately 50%. Care should be taken to avoid an uneven distribution of GC-rich or AT-rich sequences within the primer.

Probe design can be based on the protocol of the software that comes with commercially available real-time PCR devices such as the ABI Prism 7900HT Real-time PCR System (Life Technologies Japan, Inc.). Probe design criteria generally include that the GC content should be within the range of 20-80%, that the sequence should avoid four or more consecutive G or C bases, that the Tm value should be set about 8-10°C higher than the Tm value of the corresponding primer pair, and that the 5' end of the probe should not be G.

Based on the above criteria, the following oligonucleotide set for detecting Tribolium castaneum of the present invention was established.
[Oligonucleotide set for detecting Tribolium castaneum]
SEQ ID NO: 1: 5'-CGACCTATAGGAATATCATTCG-3'
SEQ ID NO: 2: 5'-CTCCTCCCCCTGCAGGGTCA-3'
SEQ ID NO: 3: 5'-CCCCTATTCGTTTGAGCTGTAG-3'

The oligonucleotides constituting the oligonucleotide set of the present invention may be not only the above-mentioned "oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 1 to 3 or their complementary sequences" but also mutant oligonucleotides thereof as long as they function as primers or probes for detecting Tribolium congenita. For example, the mutant oligonucleotide may be "an oligonucleotide consisting of a base sequence containing at least 15 consecutive bases in the base sequences shown in SEQ ID NOs: 1 to 3 or their complementary sequences". There are no particular restrictions on the type of bases other than the at least 15 consecutive bases, or on the location (3' end side, 5' end side) of the mutant oligonucleotide of at least 15 consecutive bases. In addition, the total length of the mutant oligonucleotide is preferably about 15 to 30 bases. In general, when a probe binds complementarily to a template DNA, the base sequences added to the regions at both ends are of little importance. Therefore, when an oligonucleotide contained in the above-mentioned oligonucleotide set is used as a probe, any 5 or less bases may be added or deleted to both ends of the oligonucleotide having the base sequence of each SEQ ID NO.

The food pest to be detected in this invention is the black flour beetle, a pest that can directly damage or contaminate edible materials, causing a deterioration in the quality of the materials, and includes all of its adults, pupae, larvae, and eggs. In addition, materials that the black flour beetle can contaminate are not limited to food, but also include plant-derived residues, etc.

Oligonucleotides that serve as primers or probes can be synthesized by methods known in the art for synthesizing oligonucleotides, such as the phosphoramidite method or the H-phosphonate method, using a commonly used automated DNA synthesizer (e.g., Model 394 manufactured by Applied Biosystems, Inc.).

The above oligonucleotide set can also be made into a kit. The kit of the present invention may contain the above oligonucleotide set, and may also contain, as necessary, a DNA extraction reagent, a PCR reagent such as a PCR buffer or DNA polymerase, a DNA solution containing a PCR amplified region that serves as a positive control for the reaction, detection reagents such as a stain or an electrophoresis gel, instructions, etc.

2. Method for detecting the black flour beetle The method for detecting a food pest of the present invention comprises the steps of extracting DNA from a test sample, using the DNA as a template to perform a polymerase chain reaction (PCR) with two oligonucleotides selected from the oligonucleotides contained in the oligonucleotide set described above, and detecting the amplification product obtained by the PCR.

As test samples, not only the black flour beetle itself, but also food ingredients, materials in the process of processing, processed food products, etc. that may be eaten or contaminated by the black flour beetle may be used, and are not particularly limited. Specific examples include unprocessed products such as grain grains and their crushed products, and processed products such as cooked rice, chocolate, instant noodles, and natto. The detection results obtained by the method of the present invention can be used for labeling food safety, as well as for confirming the presence or absence of food pest contamination in the production line that was not intended by the producer.

The DNA from the test sample may be extracted by any method known to those skilled in the art as a nucleic acid extraction method, such as the phenol extraction method, the cetyltrimethylammonium bromide (CTAB) method, the alkaline SDS method, etc. These methods may be modified as appropriate, and various DNA extraction kits sold by reagent manufacturers may be used. Depending on the type of sample, filtration with a membrane filter or homogenization may be performed. Various DNA extraction kits sold by reagent manufacturers, such as the DNeasy Plant mini Kit (Qiagen, Inc.), may also be used. The DNA extracted by these methods is preferably kept in a state suitable for use as a PCR template, for example, by dissolving it in an appropriate buffer and storing it at low temperature. After DNA extraction, purification treatments such as chloroform/isoamyl alcohol treatment, isopropanol precipitation, deproteinization with phenol/chloroform, and ethanol precipitation may be performed.

In addition, when the above oligonucleotide is used as a probe, it may be labeled with a labeling substance for detecting a fluorescent signal derived from the amplification product. As a labeled probe, a TaqMan ^TM probe double-labeled with a fluorescent substance and a quencher is preferable. In the TaqMan ^TM probe, the 5' end of a nucleic acid probe is usually modified with a fluorescent substance (reporter fluorescent dye) and the 3' end is modified with a quencher fluorescent dye. Examples of reporter fluorescent dyes include fluorescein-based fluorescent dyes such as 6-FAM (6-carboxyfluorescein), TET (6-carboxy-4,7,2',7'-tetrachlorofluorescein), and HEX (6-carboxy-2',4',7',4,7-hexachlorofluorescein), and examples of quencher fluorescent dyes include rhodamine-based fluorescent dyes such as 6-carboxytetramethylrhodamine (TAMRA) and 6-carboxy-X-rhodamine (ROX). These fluorescent dyes are known and can be used since they are included in commercially available real-time PCR kits.

Next, the extracted DNA is used as a template, and PCR amplification is performed using two oligonucleotides selected from the oligonucleotide set of the present invention. There are no particular limitations on the PCR amplification other than the use of the aforementioned oligonucleotide set, and it may be performed according to a conventional method. Specifically, a cycle including denaturation of the template DNA, annealing of the primer to the template, and extension reaction of the primer using a heat-resistant enzyme (DNA polymerase such as Taq polymerase or Tth DNA polymerase derived from Thermus thermophilus) is repeated to amplify the characteristic base sequence of the mitochondrial DNA of the food pest to be detected. The composition of the PCR solution (amount of template DNA, type of buffer, primer concentration, type and concentration of DNA polymerase, dNTP concentration, magnesium chloride concentration, etc.) and PCR reaction conditions (temperature cycle, number of cycles, etc.) can be appropriately selected and set by a person skilled in the art.

For example, 0.1-100ng of template DNA, 10x PCR reaction buffer, 0.25-1μM of each primer, 0.25-2.5U of DNA polymerase (Taq polymerase, Tth DNA polymerase, etc.), and 250μM of each dNTP are mixed and then diluted to a total volume of 10-100μl. The reaction is carried out at 94-96°C for 5 minutes x 1 cycle, (94-96°C for 30 seconds, 52-58°C for 30 seconds, 70-74°C for 1 minute) x 30 cycles, and 70-74°C for 5 minutes x 1 cycle. This is only one example, and the composition of the PCR solution, reaction temperature, and reaction time can be set appropriately depending on the length and base composition of the oligonucleotide sequence that serves as the primer. This series of PCR operations can be carried out using a commercially available PCR kit or PCR device and following the operating instructions.

The PCR amplification product can be detected by conventional electrophoresis such as agarose gel electrophoresis and capillary electrophoresis, DNA hybridization, real-time PCR, and other methods. For example, in agarose gel electrophoresis, the amplified product is stained with ethidium bromide, SYBR Green solution, etc., and the presence or absence and type of food pests are determined based on the size of the band. The PCR can also be performed using a primer previously labeled with a labeling substance to detect the amplified product. As the labeling substance, fluorescent substances, radioisotopes, chemiluminescent substances, biotin, and other substances well known in the art can be used. In DNA hybridization, the amplified product is bound to an immobilization carrier such as a microarray, and the amplified product is confirmed by fluorescence or enzyme reaction, etc. The immobilization carrier for detection is a support on which an oligonucleotide having a sequence that can hybridize with a characteristic sequence of the mitochondrial DNA of the black flat-headed flour beetle to be detected is immobilized. As the support, for example, a nylon membrane, a nitrocellulose membrane, glass, a silicon chip, and the like can be used.

The real-time PCR method includes the intercalator method, in which an intercalator such as SYBR Green I, a compound that emits fluorescence by binding to double-stranded DNA together with a primer pair, is added to the PCR reaction system, and the TaqMan method, in which a probe (TaqMan ^TM probe) labeled with a fluorescent substance called a reporter at the 5' end and a quenching substance called a quencher at the 3' end is added to the PCR reaction system. Either method can be used in the present invention, but the TaqMan method is preferred. In the TaqMan method, the TaqMan ^TM probe specifically hybridizes to the template DNA under the conditions used for the extension reaction of polymerase in the PCR amplification reaction, and is decomposed as the DNA chain is extended, i.e., the template DNA is amplified, and the amount of fluorescence in the PCR solution increases as the fluorescent substance is released. This increase in the amount of fluorescence serves as an indicator of the amplification of the template DNA, and the state of amplification in PCR can be easily detected in real time. The real-time PCR method can be performed using a commercially available real-time PCR kit or real-time PCR device based on a normal method known to those skilled in the art, except for using the above-mentioned oligonucleotide set, and can be performed according to the operating instructions attached to the device. As a real-time PCR device, for example, ABI Prism 7900HT Real-time PCR System or the like can be used.

Detecting the black flour beetle in a test sample is carried out as follows. First, real-time PCR is performed on the test sample to obtain an amplification curve. Next, an appropriate threshold for the increase in fluorescence (ΔRn) is set in the region where the increase in the fluorescent signal is exponentially related to the cycle number, and the presence or absence of food pest DNA in the test sample is determined based on whether or not the amplification curve intersects with the threshold. The cycle number at which this amplification curve intersects with the threshold is called the Ct value (Threshold Cycle).

The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

Example 1: Design of oligonucleotides (primer/probe) for detecting Tribolium congenita (1) Obtaining Tribolium congenita Since Tribolium congenita does not exist in Japan, the Yokohama Plant Protection Station of the Ministry of Agriculture, Forestry and Fisheries obtained them from the Czech Republic with import permission from the Minister of Agriculture, Forestry and Fisheries, and individuals were subjected to insecticide treatment in accordance with the import permission conditions set by the Minister of Agriculture, Forestry and Fisheries and were used for the following tests.

(2) DNA extraction DNA was extracted from the above-mentioned Tribolium confusum individuals using the DNeasy Blood & Tissue Kit (Qiagen, Inc.). DNA was extracted by placing 25 mg of the specimen in a 1.5 ml tube, adding it to 200 μL of Buffer ATL containing 20 μL of Proteinase K included in the DNeasy Blood & Tissue Kit, mixing while grinding with a pestle, and incubating at 56°C overnight. 4 μl of ribonuclease (RNase) A was added and mixed, and the mixture was incubated at room temperature for 5 minutes. 200 μL of Buffer AL was then added and mixed thoroughly, followed by adding 200 μl of ethanol (96-100%) and mixing thoroughly again.

The mixture was added to the DNeasy Spin Column provided with the DNeasy Blood & Tissue Kit and centrifuged to adsorb the DNA onto the filter. Next, 200 μl of Buffer AW1 was added and centrifuged to wash the filter with the adsorbed DNA. After that, 200 μl of Buffer AW2 was added and centrifuged to elute the total DNA, including mitochondrial DNA, from the filter.

(3) DNA sequence analysis of the cytochrome C oxidase subunit (CO1) region of mitochondrial DNA and design of oligonucleotides for detection of Tribolium congenita
The nucleotide sequence information (Accession No. FJ743723) of a part of the CO1 region of the mitochondrial DNA of Tribolium congenita was obtained from the NCBI database, primers were designed from this nucleotide sequence information, and PCR amplification was performed using the total DNA including the mitochondrial DNA extracted in (2) as a template, resulting in a clear amplified DNA whose length matched the length expected from the nucleotide sequence information. The nucleotide sequence of this PCR amplified product was then determined by direct sequencing, and was compared with the nucleotide sequence information in the NCBI database, which matched. Therefore, as determined by the expert's appraisal, the above specimen was identified as Tribolium congenita.

Therefore, based on the above base sequence information (Accession No. FJ743723), we compared the CO1 sequence information of other Tenebrionidae pests, and designed the following DNA sequence for the portion where the target sequence length was determined to be relatively short and high specificity was obtained, and used it as an oligonucleotide for detecting the black flour beetle.
SEQ ID NO: 1: 5'-CGACCTATAGGAATATCATTCG-3'
SEQ ID NO: 2: 5'-CTCCTCCCCCTGCAGGGTCA-3'
SEQ ID NO: 3: 5'-CCCCTATTCGTTTGAGCTGTAG-3'

(Example 2) Confirmation of the performance of oligonucleotides for detecting Tribolium castaneum (real-time PCR method using TaqMan ^TM probe)
Detection of Tribolium congenita was carried out by real-time PCR (TaqMan ^TM probe method) using the oligonucleotide set for detecting Tribolium congenita designed in Example 1. The nucleotide sequence of SEQ ID NO: 1 was used as the upstream primer, the nucleotide sequence of SEQ ID NO: 2 was used as the downstream primer, and the nucleotide sequence of SEQ ID NO: 3, labeled with a reporter dye (FAM) on the 5' side and a quencher (TAMRA) on the 3' side, was used as the TaqMan ^TM probe.

The test samples used were the black flour beetle, as well as the following major food pests (22 species) and major grains (4 species) that may be contaminated by food pests.
was used.
<Curculionidae>
Rice weevil, rice weevil, granaria rice weevil
<Anobiidae>
Cigarette Beetles
<Beetle family>
Breastmilk Beetle
<Family: Platyceridae>
Stag beetle, sawtooth beetle
<Bruchidae>
Azuki bean weevil
<Gelechiidae>
Bakuga
<Dermestidae>
Himemarukatsuobushimushi, Himekatsuobushimushi, Himeakakatsuobushimushi
<Tenebrionidae>
Red flour beetle, Chinese meal beetle, dwarf meal beetle, brown meal beetle, broad-leaved meal beetle, Kashmir red flour beetle, large-horned red flour beetle, dwarf red flour beetle
<Pyralidae>
Indian Meal Moth
<Poaceae>
Barley, rice, corn, wheat

Mitochondrial DNA was extracted from each sample according to the method of Example 1 (2), and using these as template DNA, detection of Tribolium congenita was performed by real-time PCR (TaqMan ^TM probe method) as follows: A reaction solution (total volume 10 μl) containing 5 μl of GeneAce Probe qPCR Mix α No ROX (manufactured by Nippon Gene Co., Ltd.), 0.2 μM TaqMan ^TM probe labeled with FAM at the 5' end and TAMRA at the 3' end of SEQ ID NO: 3, 0.5 μM of each primer pair (SEQ ID NO: 1 and 2), and 1 ng total DNA per well was prepared, and each was incubated at 95°C for 10 minutes using a CFX96 Touch ^TM real-time PCR analysis system (manufactured by Bio-Rad Laboratories, Inc.), and then amplified by repeating 30 cycles of 95°C for 30 seconds and 59°C for 1 minute to detect.

The test results were judged as positive or negative based on the Ct value when the threshold value (Th value) was fixed at 500 in the CFX96 Touch ^TM real-time PCR analysis system.

The results are shown in Figures 1 and 2. As shown in Figure 1, the amount of fluorescence derived from the amplification product of mitochondrial DNA of Tribolium confusum was clearly detected from around the 16th cycle. On the other hand, no fluorescence from the amplification products of mitochondrial DNA was detected in the reaction solutions of major food pests other than the black flour beetle used in this experiment, including the red flour beetle, cockroach meal beetle, dwarf meal beetle, brown meal beetle, common meal beetle, flat-headed flour beetle, Kashmir flour beetle, large horned flour beetle, lesser flour beetle, cigarette beetle, grain beetle, mealybug beetle, square-headed flat beetle, sawtooth flat beetle, adzuki bean weevil, rice weevil, rice weevil, granarian rice weevil, Indian meal moth, box moth, lesser cut grass beetle, lesser cut grass beetle, and lesser cut grass beetle (22 species in total). In addition, the Ct value from Tribolium congenita calculated when the Th value was fixed at 500 was obtained as an actual value of 19.40, but other stored grain pests were N/A (not detectable), and the presence or absence of fluorescence detection was clearly distinguished. Similarly, as shown in FIG. 2, no fluorescence from the amplification product of mitochondrial DNA was observed in the major grains of barley, rice, corn, and wheat (a total of four types). From the above judgment results, the real-time PCR method using the TaqMan ^TM probe according to this embodiment was able to specifically detect Tribolium congenita among many stored grain pests and major grains.

(Example 3) Confirmation of the performance of oligonucleotides for detecting Tribolium castaneum (Simplex PCR method)
Detection of Tribolium confusum was carried out by Simplex PCR using the oligonucleotides for detecting Tribolium confusum designed in Example 1. The base sequence of SEQ ID NO: 1 was used as the upstream primer, and the base sequence of SEQ ID NO: 2 was used as the downstream primer.

Mitochondrial DNA was extracted from each sample according to the method of Example 1 (2), and using these as template DNA, food pest detection was performed by the Simplex PCR method as follows: A reaction solution (total volume 10 μl) containing 0.5 U of AmpliTaq Gold DNA Polymerase (Mg ²⁺ free buffer) (Thermo Fisher Scientific), 1×PCR Buffer II (Mg ²⁺ free), 200 μM of each dNTP, 1.5 mM MgCl ₂ , 0.5 μM of each primer pair (base sequence of SEQ ID NO: 1 and base sequence of SEQ ID NO: 2), and 1 ng of total DNA per well was prepared, and each was incubated at 95°C for 10 minutes in an S1000 ^TM thermal cycler, and then amplified by repeating 40 cycles of 95°C for 30 seconds and 59°C for 1 minute.

To determine whether the test was positive or negative, 8 μl of each 10 μl reaction solution was subjected to electrophoresis on a 3% agarose gel together with the DNA marker Gene Ladder 100 (0.1-2 kbp), stained with ethidium bromide, and then the presence or absence of stained DNA bands was confirmed under ultraviolet light irradiation using a UV transilluminator.

The results are shown in Figures 3 and 4. In Figure 3, a band of about 200 bp indicating an amplification product derived from the mitochondrial DNA of the black flour beetle was clearly detected in lane number P. On the other hand, the amplification products of other grain pests besides the black flour beetle, such as the red flour beetle (lane number 1), the Chinese meal beetle (lane number 2), the lesser meal beetle (lane number 3), the brown meal beetle (lane number 4), the common meal beetle (lane number 5), the flat-headed flour beetle (lane number 6), the Kashmir flour beetle (lane number 7), the large-horned flour beetle (lane number 8), the lesser flour beetle (lane number 9), the cigarette beetle (lane number 10), and the grain long bostrich beetle (lane number 11). No bands derived from the amplification products of mitochondrial DNA were observed in the reaction mixtures of 11), 12), 13), 14), 15), 16), 17), 18), 19), 19), 20), 21), 22). M is the Gene Ladder 100 DNA marker, and N is the negative control (no DNA). In addition, no bands derived from the amplification products of mitochondrial DNA were observed in the reaction mixtures of 11), 12), 13), 14), 15), 23), 24), 25), 26). From the above judgment results, the qualitative PCR method of this example was able to specifically detect the black flour beetle, among many stored grain pests and major grains.

The present invention can be used to confirm the presence or absence of the black flour beetle in the food manufacturing and food processing fields.

Claims (6)

An oligonucleotide set for detecting the black flour beetle, consisting of two or more oligonucleotides containing the characteristic base sequence of the mitochondrial DNA of the black flour beetle. The oligonucleotide set for detecting Tribolium confusum according to claim 1, wherein the characteristic base sequence of mitochondrial DNA is a base sequence within the cytochrome c oxidase subunit 1 (CO1) region. The oligonucleotide set for detecting Tribolium confusum according to claim 1 or 2, wherein the oligonucleotides are used as primers and probes. The oligonucleotide set for detecting the black flour beetle according to claim 1, wherein the oligonucleotide set is an oligonucleotide set consisting of an oligonucleotide having the base sequence shown in any one of SEQ ID NOs: 1 to 3 below or a complementary sequence thereof, or an oligonucleotide having a base sequence containing at least 15 consecutive bases in the base sequence shown in any one of SEQ ID NOs: 1 to 3 or a complementary sequence thereof.
SEQ ID NO: 1: 5'-CGACCTATAGGAATATCATTCG-3'
SEQ ID NO: 2: 5'-CTCCTCCCCCTGCAGGGTCA-3'
SEQ ID NO: 3: 5'-CCCCTATTCGTTTGAGCTGTAG-3' A kit for detecting Tribolium congenita, comprising the oligonucleotide set for detecting Tribolium congenita according to claim 1 or 4. A method for detecting Tribolium congenita, comprising the steps of: using DNA extracted from a test sample as a template, carrying out PCR amplification using the oligonucleotide set for detecting Tribolium congenita described in claim 1 or 4, and detecting the resulting amplification product.

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