Oligonucleotide for detecting Acamthochiton rubrolineatus

Abstract

To establish an inspection system that can accurately, quickly and inexpensively detect Trogoderma varium in a distinguished manner from other main food pests or other Trogoderma, in a trap survey of pests to be quarantined.SOLUTION: Provided are an oligonucleotide set for detecting Trogoderma varium which includes a characteristic base sequence of a mitochondrion DNA of Trogoderma varium, and is constituted of two kinds or more of oligonucleotide, a Trogoderma varium detection kit including the oligonucleotide set, and a method for detecting Trogoderma varium using the oligonucleotide set.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an oligonucleotide for detecting Trogoderma varium, a kit for detecting Trogoderma varium, and a method for detecting Trogoderma varium.

Background Art

[0002] Trogoderma varium (Matsumura et Yokoyama) is classified in the genus Trogoderma of the family Dermestidae in the order Coleoptera, and is the most commonly inhabiting Trogoderma in Japan. Insects belonging to the genus Trogoderma (referred to as Trogoderma insects) are known in Japan, in addition to Trogoderma varium, there are seven species: Trogoderma glabrum, Trogoderma chinensis, Trogoderma parvulum, Trogoderma exiguum, Trogoderma inclusum, Trogoderma versicolor, and Trogoderma kibonis (Non-Patent Documents 1 and 2). Some of these Trogoderma insects are problematic as important pests in plant quarantine.

[0003] A typical stored grain that is vulnerable to damage by pests in Japan is rice. Rice is positioned as one of the main export items in Japan, and the Ministry of Agriculture, Forestry and Fisheries, etc. are promoting the international spread of Japanese cuisine, and along with that, promoting the consumption of Japanese-produced rice and Japanese sake, which is a processed product, for Japanese restaurants overseas, and are also expanding the cultivation of rice for export.

[0004] On the one hand, in various foreign countries, respective plant quarantine systems are established to prevent the invasion and spread of pests and diseases. Agricultural products exported from Japan need to comply with the plant quarantine conditions of the importing country. Therefore, the Ministry of Agriculture, Forestry and Fisheries is making requests for lifting the export ban to foreign countries in order to improve the export environment of Japanese agricultural products. For example, when exporting polished rice to China, the "Regulations on the Implementation of Quarantine for the Export of Polished Rice to the People's Republic of China (Notice of the Director of the Consumer Safety Bureau No. 3741, 20th June 2008)" is stipulated. According to these implementation regulations, only polished rice that has been polished and fumigated, etc. at designated registered facilities approved by the Chinese side can be exported. In the approval procedure for designated registered facilities, it is necessary to install traps using the attractant pheromone in the facility to confirm the absence of the moths of the genus Anomala (trap survey). The moths of the genus Anomala targeted by the Chinese side for quarantine are Anomala rufocuprea, Anomala exoleta and Anomala aenea. If these are found in the facility, export suspension measures will be taken, and management such as taking measures to exterminate the target species by fumigation, etc. and confirming the effectiveness will be required, resulting in an additional troublesome process that is time-consuming and costly (Non-Patent Document 3).

[0005] For phytosanitary purposes, it is extremely important to first identify whether the seeds are subject to quarantine. If they are, measures such as fumigation will be taken, so species identification is crucial. So far, in the pheromone trap surveys of the mackerel scale insects, it has been reported that the red mackerel scale insect, the black mackerel scale insect, and the small mackerel scale insect were captured, and among them, the red mackerel scale insect accounted for the majority (Non-Patent Documents 4 and 5). When exporting to China, it is necessary to identify three species: the small red mackerel scale insect, the variegated mackerel scale insect, and the small mackerel scale insect. However, the mackerel scale insects are very similar in form to each other, and their adult bodies are densely covered with scales on the body surface. Although characteristic markings can be recognized, many of these markings are very similar, and for those collected at the habitat site, the scales themselves have fallen off due to abrasion caused by physical contact, etc. Even if the markings of the individuals immersed in the trap (trap) using an oil-type attractant (pheromone) remain undamaged, they turn black due to the oil and the contrast of the markings becomes unclear. Therefore, it is extremely difficult to distinguish species by visual inspection.

[0006] In recent years, DNA analysis has also been widely used in insects and is particularly useful when visual identification is very difficult. The "White Paper on the Environmentally Recirculating Society" compiled by the Ministry of the Environment in fiscal year 2008 states that the total number of known biological species in the world is approximately 1.75 million. Among them, there are about 6,000 species of mammals, about 9,000 species of birds, about 950,000 species of insects, and about 270,000 species of vascular plants. According to this, insects account for about 55% of living organisms, and it is reasonable to classify and identify a very diverse range of insects using DNA.

[0007] The DNA present in cells can be divided into mitochondrial DNA and chloroplast DNA in addition to nuclear (genomic) DNA. Nuclear DNA is used in criminal investigations, determining blood relationships such as parentage, and variety identification of crops and livestock. Among these, mitochondrial DNA has a higher mutation frequency than nuclear DNA and a greater number of copies per cell than the genome, so it is considered useful for determining the species of insects.

[0008] In DNA-based testing methods, in addition to the conventionally used PCR method, there are various methods such as the LAMP (Loop-Mediated Isothermal Amplification) method, those using DNA sequencing, Southern hybridization, DNA microarray, etc. Among these, the methods used for identification include the LAMP method, the DNA barcoding method using DNA sequencing, etc. The LAMP method has high specificity and is useful because it does not have the strict and multi-step temperature change steps during DNA reaction like the PCR method. However, on the other hand, the difficulty of primer design is too high. Although examples of using it for the detection of genetically modified corn strains have been reported (Patent Document 1), there are drawbacks that the sequences for which primer design can be done are limited and it is difficult to use. Also, in the DNA barcoding method, since the DNA sequence is decoded and the sequence information is compared with known sequence information for utilization, it is possible to identify up to the insect species name. However, in this method, after the base sequence of the analysis sample subjected to the PCR method is decoded by a DNA sequencer, the sequences are compared one by one using dedicated software. Therefore, the operation is frequent, the cost is high, and it takes time, which is a drawback, especially when analyzing a large number of samples, the efficiency is low.

[0009] Unlike the DNA barcoding described above, the PCR method is a technique for determining in advance the detection target and examining whether the DNA derived from it is contained in the sample. The test can determine the presence or absence of the DNA of the organism to be detected only by subjecting the extracted DNA to PCR, and has the feature that it can be determined in large quantities and at high speed. One typical example is particularly useful for identifying insects that have been selectively captured to some extent by, for example, the aforementioned pheromone trap when the target insect species is roughly known. Also, when a pheromone trap is used for attracting 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 attracted insects, will be captured. However, if appropriately designed primers and probes are used, it is possible to quickly, in large quantities, and accurately confirm whether it is the object to be identified.

[0010] 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 2 describes specific and universal probes and amplification primers for rapid detection and identification of bacterial pathogens. The present inventor has successively established oligonucleotide sets for detecting major food pests such as the granaria weevil and the red-legged cutworm beetle by PCR or real-time PCR (Patent Document 3). As described above, the morphology of the spotted cutworm beetles, which are also subject to export quarantine, is very similar, and identification by the markings on their scales is also uncertain in the field, making it extremely difficult to distinguish the species by visual inspection, and therefore there is a demand for establishing an inspection system using the PCR method or real-time PCR method. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Patent No. 4899180 [Patent Document 2] JP 2007-125032 A [Patent Document 3] Patent No. 6600831 [Non-patent literature]

[0012] [Non-Patent Document 1] Nobuo Obayashi, Family: Bucconidae, Colored Illustrated Guide to Japanese Beetles III, pp.125-130 (1985) [Non-Patent Document 2] Toshiharu Yoshida, Nao Watanabe, Nobuyuki Sonda, Illustrated Guide to Pests of Stored Foods: From Practical Identification Methods to Control Methods, 268 pages, October 26, 1989, National Rural Education Association [Non-Patent Document 3] Ministry of Agriculture, Forestry and Fisheries, Plant Protection Station, Pest Information, "Export Quarantine of Milled Rice to China" No. 85 (2008) [Non-Patent Document 4] Soichi Hirao, Kansei Sugimoto, "Capture Results of Trogoderma beetles (Trogoderma genus) using Pheromone Traps", Journal of the Pest Society of Japan, Vol. 7, No. 1, pp. 4-8 (1992)

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0013] Therefore, in view of the above circumstances, an object of the present invention is to develop oligonucleotides for detecting Tribolium castaneum, a species of Trogoderma beetles that is particularly difficult to visually distinguish among the insect groups captured in large quantities in trap surveys of quarantine target pests in rice milling facilities and export-related warehouses, by PCR method or real-time PCR method, and to establish an inspection system that can accurately and quickly detect Tribolium castaneum at low cost, distinguishing it from other Trogoderma beetles.

Means for Solving the Problems

[0014] As a result of intensive research to solve the above problems, the present inventors obtained a DNA region encoding an unknown cytochrome c oxidase subunit 1 (CO1) from the mitochondria of Tribolium castaneum, and then analyzed the sequence information. Subsequently, characteristic base sequences of Tribolium castaneum were found from this sequence information, and an oligonucleotide set capable of accurately and quickly detecting Tribolium castaneum was successfully established by combining oligonucleotides containing these base sequences. That is, the present invention includes the following inventions.

[0015] An oligonucleotide set for detecting the red seabream isopod, composed of two or more oligonucleotides, containing a characteristic base sequence of the mitochondrial DNA of the red seabream isopod. (2) The oligonucleotide set for detecting the red seabream isopod according to (1), wherein the characteristic base sequence of the mitochondrial DNA is a base sequence within the cytochrome c oxidase subunit 1 (CO1) region. (3) The oligonucleotide set for detecting the red seabream isopod according to (1) or (2), wherein the oligonucleotide is used as a primer and a probe. (4) The oligonucleotide set for detecting the red seabream isopod according to any one of (1) to (3), wherein the oligonucleotide set is composed of an oligonucleotide having a base sequence shown in SEQ ID NOs: 1 to 3 below or its complementary sequence, or an oligonucleotide having a base sequence containing at least 15 consecutive bases in the base sequence shown in SEQ ID NOs: 1 to 3 or its complementary sequence. SEQ ID NO: 1: 5'-CCTAGCTGTACGATTGACAGC-3' SEQ ID NO: 2: 5'-GGACTGGATTATTGCGACAGC-3' SEQ ID NO: 3: 5'-CACAGGAACTCACTTATCACTAGCC-3' (5) A kit for detecting the red seabream isopod, containing the oligonucleotide set for detecting the red seabream isopod according to any one of (1) to (4). (6) A method for detecting the red seabream isopod, including the steps of using the DNA extracted from the test sample as a template, performing PCR amplification using the oligonucleotide set for detecting the red seabream isopod according to any one of (1) to (4), and detecting the obtained amplification product.

Advantages of the Invention

[0016] According to the present invention, there are provided an oligonucleotide set for detecting the red flour beetle, Tribolium castaneum Herbst, which is a food pest, a kit for detecting Tribolium castaneum Herbst containing the oligonucleotide set, and a method for detecting Tribolium castaneum Herbst using the oligonucleotide set. Therefore, according to the present invention, since Tribolium castaneum Herbst can be detected accurately, quickly, and at low cost, the reliability of quality can be enhanced by utilizing it for food management and assurance.

Brief Description of the Drawings

[0017]

Figure 1

Figure 2

Mode for Carrying Out the Invention

[0018] Hereinafter, the present invention will be described in detail. 1. Oligonucleotide Set and Detection Kit for Detecting Japanese Common Cockchafer The oligonucleotide set for detecting the Japanese common cockchafer of the present invention is composed of two or more kinds of oligonucleotides containing a characteristic nucleotide sequence of the mitochondrial DNA of the Japanese common cockchafer to be detected. The base length of the oligonucleotide is not limited, but in the case of a primer, it is usually 15 to 30 bases long, preferably 18 to 25 bases long, and in the case of a probe, it is 10 to 30 bases long.

[0019] The characteristic base sequence of the mitochondrial DNA of the target spotted gizzard shad larvae refers to a base sequence that has low homology with the known base sequences of mitochondrial DNA of a wide range of food pests and is considered characteristic only of the target spotted gizzard shad larvae, with a length of 10 bases or more, preferably 15 bases or more, and more preferably 20 bases or more.

[0020] In identifying the characteristic base sequence of the above mitochondrial DNA, first, the base sequence information of the mitochondrial DNA of the spotted gizzard shad larvae is obtained. Since the base sequence information of the spotted gizzard shad larvae is not registered in databases (such as NCBI, DDBJ, etc.), as shown in the following examples, it can be obtained by directly analyzing the base sequence.

[0021] In the present invention, examples of the characteristic base sequence of the mitochondrial DNA of the spotted gizzard shad larvae include the base sequence of the cytochrome c oxidase subunit 1 (CO1) region.

[0022] Next, based on the identified characteristic base sequence, primers are designed. The design of the primers is carried out considering factors such as the length of the oligonucleotide, GC content, Tm value, complementarity between oligonucleotides, and secondary structure within the oligonucleotide. For example, references such as "The Frontiers of PCR - From Basic Techniques to Applications" (Protein, Nucleic Acid, Enzyme Special Issue 1996, Kyoritsu Shuppan Co., Ltd.), "Illustrated Biotechnology Experiment 3 Truly Enhanced PCR: Cell Engineering Separate Sheet, Experimental Notebook Series You Can See with Your Own Eyes" (written by Hiroki Nakayama, Shujunsha Co., Ltd.), "PCR Technology - Principles and Applications of DNA Amplification -" (edited by Henry A Erlich, supervised by Kuniyuki Kato, Takara Shuzo Co., Ltd.) can be referred to.

[0023] The specific design criteria adopted are as follows. (a) When the PCR amplification product of the characteristic sequence of the mitochondrial DNA of the spotted gizzard shad larvae is electrophoresed, the amplification product can be clearly detected. (b) The PCR amplification product is 100 - 500 bp, preferably 100 - 250 bp. (c) The primer length ranges from 15 - 30 bp to enable specific annealing with the template DNA. (d) Since the annealing temperature depends on the Tm (melting temperature), in order to obtain a highly specific PCR amplification product, the Tm value is 50 - 70 °C, preferably 55 - 65 °C, and primers with similar Tm values are selected. (e) The base sequence at the 3'-end of the primer has a high homology with the template DNA sequence. (f) To avoid the formation of dimers or three-dimensional structures, the primers should avoid complementary sequences between the two primers. (g) To ensure a stable binding with the template DNA, the GC content is preferably about 50%, and care is taken to avoid uneven distribution of GC-rich or AT-rich regions within the primer.

[0024] Also, the probe design can be carried out based on the protocol of the software attached to commercially available real-time PCR devices such as the ABI Prism 7900HT Real-time PCR System (Life Technologies Japan Co., Ltd.). As the probe design criteria, the GC content is within the range of 20 - 80%, avoid 4 or more consecutive G or C bases within the sequence, set it about 8 - 10 °C higher than the Tm value of the corresponding primer pair, and generally, the 5'-terminal of the probe should not be G.

[0025] Based on the above criteria, the following oligonucleotide sets for detecting Acmaeodera sp. were established for the present invention. [Oligonucleotide Set for Detecting Acmaeodera sp.] SEQ ID NO: 1: 5'-CCTAGCTGTACGATTGACAGC-3' SEQ ID NO: 2: 5'-GGACTGGATTATTGCGACAGC-3' Sequence number 3: 5'-CACAGGAACTCACTTATCACTAGCC-3'

[0026] The oligonucleotides constituting the oligonucleotide set of the present invention are not only the oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 1 to 3 or their complementary sequences, but also mutant oligonucleotides as long as they can function as primers or probes for detecting Acalolepta luxuriosa. For example, as mutant oligonucleotides, there may be mentioned "oligonucleotides 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 types of bases other than the at least 15 consecutive bases, or the sites (3'-terminal side, 5'-terminal side) where the mutant oligonucleotides of at least 15 consecutive bases are present. Also, the total length of the mutant oligonucleotides is preferably about 15 to 30 bases. Generally, when a probe binds complementarily to template DNA, the importance of the base sequences added to the regions at both ends is low. Therefore, when using the oligonucleotides contained in the above oligonucleotide set as probes, arbitrary bases of 5 or less may be added or deleted at both ends of the oligonucleotides having the base sequences of each sequence number.

[0027] The food pest to be detected in the present invention is Acalolepta luxuriosa, which directly damages substances that can be eaten as food grains or causes a decrease in the quality of substances by mixing in, and includes any of its adults, pupae, larvae, and eggs. Also, the substances in which Acalolepta luxuriosa is mixed are not limited to foods, but also include plant-derived residues and the like.

[0028] The oligonucleotides serving as primers or probes can be synthesized using a method known in the art as a method for synthesizing oligonucleotides, for example, the phosphoramidite method, the H-phosphonate method, etc., using a commonly used DNA automatic synthesizer (for example, Model 394 manufactured by Applied Biosystems).

[0029] The above oligonucleotide set can also be made into a kit. The kit of the present invention only needs to contain the above oligonucleotide set. If necessary, it may contain reagents for DNA extraction, PCR reagents such as PCR buffer and DNA polymerase, a DNA solution containing a PCR amplification region as a positive control for the reaction, detection reagents such as staining agents and electrophoresis gels, and an instruction manual, etc.

[0030] 2. Method for detecting Acalymma vittatum The method for detecting food pests of the present invention comprises extracting DNA from a test sample, using the DNA as a template, performing a polymerase chain reaction (PCR) using two kinds of oligonucleotides selected from the oligonucleotides contained in the above oligonucleotide set, and detecting the amplification product obtained by the PCR.

[0031] As the test sample, not only Acalymma vittatum itself but also food raw materials, materials in the processing process, products such as processed foods that may be damaged or contaminated by Acalymma vittatum can be used, and there is no particular limitation. Specifically, those in an unprocessed state such as cereal grains and their ground products, and processed products such as cooked rice, chocolate, cup noodles, natto, etc. can be mentioned. The detection result obtained by the method of the present invention can be used for indicating the safety of food, and can also be used for confirming the presence or absence of food pest contamination in a production line unintended by the producer.

[0032] DNA can be extracted from the test sample using any method known to those skilled in the art as a nucleic acid extraction method. For example, phenol extraction method, cetyltrimethylammonium bromide (CTAB) method, alkaline SDS method, etc. can be mentioned. Also, these methods may be appropriately modified, or various DNA extraction kits sold by reagent manufacturers may be used. Depending on the type of sample, filtration through a membrane filter or homogenization is performed. Also, various DNA extraction kits such as the DNeasy Plant mini Kit (manufactured by Qiagen Co., Ltd.) sold by reagent manufacturers may be used. The DNA extracted by these methods is preferably kept in a state suitable for use as a template for PCR. For example, it is preferably dissolved in an appropriate buffer and stored at a low temperature. Also, after DNA extraction, purification treatments such as chloroform / isoamyl alcohol treatment, isopropanol precipitation, protein removal with phenol / chloroform, and ethanol precipitation may be performed.

[0033] Also, when the above oligonucleotide is used as a probe, it may be labeled with a labeling substance for detecting a fluorescence signal derived from the amplification product. As the labeled probe, a TaqMan TM probe is preferred. The TaqMan TM probe usually modifies the 5' end of the nucleic acid probe with a fluorescent substance (reporter fluorescent dye) and the 3' end with a quenching substance (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). 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 are included in commercially available real-time PCR kits, so they can be used.

[0034] Next, using the extracted DNA as a template, PCR amplification is performed using two types selected from the oligonucleotides included in the above-described oligonucleotide set of the present invention. The PCR amplification is not particularly limited except for using the above-described oligonucleotide set, and may be carried out according to a conventional method. Specifically, by repeating a cycle including denaturation of template DNA, annealing of the template to the primer, and extension reaction of the primer using a heat-resistant enzyme (such as DNA polymerase such as Taq polymerase and Tth DNA polymerase derived from Thermus thermophilus), the characteristic base sequence of the mitochondrial DNA of the target red sea bream is amplified. 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.), PCR reaction conditions (temperature cycle, number of cycles, etc.) can be appropriately selected and set by those skilled in the art.

[0035] For example, after mixing 0.1 to 100 ng of DNA as a template, 10× PCR reaction buffer, 0.25 to 1 μM of each primer, 0.25 to 2.5 U of DNA polymerase (such as Taq polymerase, Tth DNA polymerase, etc.), and 250 μM of each dNTP, the reaction is carried out on a dilution such that the total liquid volume becomes 10 to 100 μl at 94 to 96°C for 5 minutes × 1 cycle, (94 to 96°C for 30 seconds, 52 to 58°C for 30 seconds, 70 to 74°C for 1 minute) × 30 cycles, 70 to 74°C for 5 minutes × 1 cycle. This is only an example, and the composition of the PCR solution, reaction temperature, and time can be appropriately set according to the length and base composition of the oligonucleotide sequence serving as the primer. These series of PCR operations can be carried out using a commercially available PCR kit or PCR apparatus according to the operation manual.

[0036] The detection of PCR amplification products can be confirmed using conventional electrophoresis methods such as agarose gel electrophoresis or capillary electrophoresis, and methods such as DNA hybridization and real-time PCR. For example, in agarose gel electrophoresis, it is stained with ethidium bromide, SYBR Green solution, etc., and the amplification product is detected as a single band, and the presence or absence and type of food pests are determined based on its size. In addition, the PCR can also be performed using primers previously labeled with a labeling substance to detect the amplification product. As the labeling substance, fluorescent substances, radioisotopes, chemiluminescent substances, biotin, etc., which are well-known in the technical field, can be used. In DNA hybridization, the amplification product is bound to an immobilized carrier such as a microarray, and the amplification product is confirmed by fluorescence or enzyme reaction, etc. The immobilized carrier for detection is a support on which an oligonucleotide having a sequence capable of hybridizing with a characteristic sequence of mitochondrial DNA of the spotted lady beetle to be detected is immobilized. As the support, for example, nylon membrane, nitrocellulose membrane, glass, silicon chip, etc. can be used.

[0037] In the real-time PCR method, an intercalator method in which an intercalator such as SYBR Green I, which is a compound that emits fluorescence by binding to double-stranded DNA, is added to the PCR reaction system together with a primer pair, or 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, namely the TaqMan method. Both can be used in the present invention, but the TaqMan method is preferred. In the TaqMan method, TaqMan TMThe probe specifically hybridizes to the template DNA under the conditions used for the polymerase extension reaction in the amplification reaction by PCR, is decomposed as the DNA strand elongates, i.e., as the template DNA is amplified, and releases a fluorescent substance, thereby increasing the fluorescence amount in the PCR solution. This increase in the fluorescence amount serves as an indicator of the amplification of the template DNA, and the amplification state in PCR can be simply detected in real time. The real-time PCR method can be carried out based on the usual methods known to those skilled in the art, using a commercially available real-time PCR kit or a real-time PCR apparatus, and following the operation manuals attached thereto, except for using the above oligonucleotide set. As the real-time PCR apparatus, for example, ABI Prism 7900HT Real-time PCR System etc. are used.

[0038] When detecting the red flour beetle in a test sample, it is carried out as follows. First, perform the real-time PCR method on the test sample to obtain an amplification curve. Next, in the region where the increase in the fluorescence signal has an exponential relationship with the number of cycles, set an appropriate threshold (Threshold) for the increase in fluorescence amount (ΔRn), and determine the presence or absence of food pest DNA in the test sample based on whether the amplification curve intersects with the threshold (Threshold). The cycle number at which this amplification curve intersects with the threshold (Threshold) is called the Ct value (Threshold Cycle).

Example

[0039] Hereinafter, the present invention will be described in more detail using examples, but the present invention is not limited to these examples.

[0040] (Example 1) Oligonucleotide for detecting the red flour beetle (Design of primer and probe) (1) Extraction of DNA DNA extraction was performed on the specimens identified as *Hemigrapsus sanguineus* by morphological identification by experts. For DNA extraction, the DNeasy Blood & Tissue Kit (manufactured by Qiagen Co., Ltd.) was used. Approximately 25 mg of the specimen pest was placed in a 1.5-ml tube, added to 200 μl of Buffer ATL containing 20 μl of Proteinase K attached to the DNeasy Blood & Tissue Kit, and mixed while grinding with a pestle, and incubated at 56°C overnight. 4 μl of Ribonuclease (RNase) A was added and mixed, and incubated at room temperature for 5 minutes. Then, 200 μl of Buffer AL was added, and after thorough mixing, 200 μl of ethanol (96 - 100%) was added and mixed thoroughly again.

[0041] The mixture was added to the DNeasy Spin Column attached to the DNeasy Blood & Tissue Kit and centrifuged to adsorb DNA to the filter. Subsequently, 200 μl of Buffer AW1 was added and centrifuged to wash the filter to which DNA was adsorbed. Then, after adding 200 μl of Buffer AW2 and centrifuging, total DNA containing mitochondrial DNA was eluted from the filter.

[0042] (2) DNA sequence analysis of the cytochrome C oxidase subunit (CO1) region of mitochondrial DNA and design of oligonucleotides for detecting *Hemigrapsus sanguineus* Since the base sequence information of the mitochondrial DNA of Acmaea scabra was not registered in databases (NCBI, DDBJ, etc.), after amplification using the common primers LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3' (SEQ ID NO: 4) and HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' (SEQ ID NO: 5) for amplifying the region of the cytochrome c oxidase subunit 1 gene (CO1) of animals, which are commonly used in the standard DNA barcoding method [0. Folmer et al., DNA primers for amplification of mitochondrial Cytochrome C oxidase subunit I from diverse metazoan invertebrates Molecular Marine Biology and Biotechnology 3(5), 294-299(1994)], the vicinity was further analyzed to newly obtain the base sequence information.

[0043] Next, the obtained base sequence information and the known CO1 sequence information of related species were carefully examined and compared, and the following DNA sequences were designed for the locations where it was determined that the target sequence length was relatively short and high specificity could be obtained, and used as oligonucleotides for detecting Acmaea scabra. SEQ ID NO: 1: 5'-CCTAGCTGTACGATTGACAGC-3' SEQ ID NO: 2: 5'-GGACTGGATTATTGCGACAGC-3' SEQ ID NO: 3: 5'-CACAGGAACTCACTTATCACTAGCC-3'

[0044] (Example 2) Performance confirmation of oligonucleotides for detecting Acmaea scabra (Real-time PCR method using TaqMan TM probe) Using the oligonucleotide set for detecting Acmaea scabra designed in Example 1, the real-time PCR method (TaqMan TMDetection of *Callosobruchus chinensis* was carried out by the probe method. The base sequence of SEQ ID NO: 1 was used as the upstream primer, the base sequence of SEQ ID NO: 2 was used as the downstream primer, and the base sequence with the 5'-side of the base sequence of SEQ ID NO: 3 labeled with a reporter dye (FAM) and the 3'-side labeled with a quencher (TAMRA) was used as a TaqMan TM probe.

[0045] As test samples, the following stored grain pests (23 species) containing *Callosobruchus chinensis*, and grains (4 species) damaged by stored grain pests were used. The DNA extraction method was the same as the DNA extraction in Example 1(1). For grain DNA, GM Quicker2 (manufactured by Nippon Gene Co., Ltd.) was used, and DNA was extracted according to the method attached to the kit.

[0046] <Stored grain pests> *Ephestia elutella*, *Rhyzopertha dominica*, *Cryptolestes ferrugineus*, *Cryptolestes pusillus*, *Tribolium castaneum*, *Oryzaephilus surinamensis*, *Silvanus surinamensis*, *Typhaea stercorea*, *Tribolium confusum*, *Tribolium destructor*, *Tribolium freemani*, *Callosobruchus analis*, *Callosobruchus chinensis*, *Callosobruchus maculatus*, *Callosobruchus phaseoli*, *Plodia interpunctella*, *Lasioderma serricorne*, *Cryptolestes pusillus*, *Callosobruchus subinnotatus*, *Callosobruchus chinensis*, *Callosobruchus analis*, *Callosobruchus maculatus* and *Callosobruchus chinensis*

[0047] <Grains damaged by stored grain pests> Barley, rice, corn, wheat

[0048] Total DNA containing mitochondrial DNA was extracted from each sample according to the method of Example 1(1), and using these as template DNA, real-time PCR method (TaqMan TMThe detection of Anoplophora malasiaca by the probe method was performed as follows. 5 μl of SsoAdvanced Universal Probes Supermix (manufactured by Bio-Rad Laboratories, Inc.) per well, 0.2 μM TaqMan 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 SEQ ID NO: 2), and a reaction solution (total volume 10 μl) containing 1 ng of total DNA were prepared. Each reaction solution was incubated at 95°C for 30 seconds in a CFX96 Touch TM Real-Time PCR Analysis System (manufactured by Bio-Rad Laboratories, Inc.), and then amplified by repeating 95°C for 5 seconds → 60°C for 5 seconds for 30 cycles for detection. TM Positive / negative test determination was performed by judging based on the Ct value when the threshold value (Th value) was fixed at 500 in the CFX96 Touch

[0049] Real-Time PCR Analysis System. TM

[0050] The results are shown in Fig. 1. As shown in Fig. 1, the fluorescence amount derived from the amplification product of mitochondrial DNA of *Cryptolestes ferrugineus* was clearly detected from around 16 cycles. On the other hand, for the reaction solution not containing the template DNA used in this experiment, the reaction solutions of stored-product pests other than *Cryptolestes ferrugineus*, such as *Ephestia elutella*, *Rhyzopertha dominica*, *Tribolium castaneum*, *Tribolium confusum*, *Sitophilus oryzae*, *Sitophilus zeamais*, *Sitophilus granarius*, *Oryzaephilus surinamensis*, *Gibbium aequinoctiale*, *Lasioderma serricorne*, *Cathartus quadricollis*, *Sitophilus linearis*, *Sitophilus rugicollis*, *Sitophilus oryzae*, *Callosobruchus chinensis*, *Callosobruchus maculatus*, *Callosobruchus analis*, *Callosobruchus phaseoli*, *Plodia interpunctella*, *Ephestia cautella*, *Cryptolestes pusillus*, *Cryptolestes ferrugineus*, *Cryptolestes turcicus*, *Cryptolestes pusillus* (a total of 22 species), and the reaction solutions of grains such as *Hordeum vulgare*, *Oryza sativa*, *Zea mays*, *Triticum aestivum* (a total of 4 species), fluorescence derived from the amplification product of mitochondrial DNA was not observed. Also, when the Th value was fixed at 500, the Ct value derived from *Cryptolestes ferrugineus* calculated was 20.01, which is a real number, while the other stored-product pests were N / A (not detectable), and the presence or absence of fluorescence detection could be clearly discriminated. From the above determination results, it was shown that the real-time PCR method using the oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 1 to 3 can specifically detect *Cryptolestes ferrugineus* from among a number of stored-product pests and grains.

[0051] (Example 3) Performance confirmation of oligonucleotides for detecting *Cryptolestes ferrugineus* (Simplex PCR method) Using the oligonucleotides for detecting *Cryptolestes ferrugineus* designed in Example 1, *Cryptolestes ferrugineus* was detected by the Simplex PCR method. 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.

[0052] Mitochondrial DNA was extracted from each sample according to the method of Example 1(1), and using these as template DNAs, detection of *Callosobruchus chinensis* by the Simplex PCR method was carried out as follows. That is, 0.25 U of AmpliTaq Gold DNA Polymerase (manufactured by Thermo Fisher Scientific) per well, 1×PCR BufferII (Mg 2+ free), 200 μM of each dNTP, 1.5 mM MgCl 2、 A reaction solution (total volume 10 μl) containing 0.5 μM of each primer pair (SEQ ID NO: 1 and SEQ ID NO: 2) and 1 ng of total DNA 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 → 60°C for 30 seconds → 72°C for 30 seconds.

[0053] Judgment of test positive / negative was performed by subjecting 10 μl of each reaction solution together with Gene Ladder 100 (0.1 - 2 kbp), which is a DNA marker, to electrophoresis on a 3% agarose gel, passing through gel staining with ethidium bromide, and confirming the presence or absence of a DNA band stained under ultraviolet irradiation by a UV transilluminator.

[0054] The results are shown in Fig. 2. A band of approximately 161 bp indicating an amplification product derived from the mitochondrial DNA of *Trogoderma granarium* was clearly detected at lane number P. On the other hand, for the reaction solutions of stored-product pests other than *Trogoderma granarium*, such as the tobacco cutworm (*Spodoptera litura*) (lane number 1), the rice weevil (*Sitophilus oryzae*) (lane number 2), the flat grain beetle (*Cryptolestes ferrugineus*) (lane number 3), the sawtoothed grain beetle (*Oryzaephilus surinamensis*) (lane number 4), the lesser grain borer (*Rhyzopertha dominica*) (lane number 5), the red flour beetle (*Tribolium castaneum*) (lane number 6), the confused flour beetle (*Tribolium confusum*) (lane number 7), the drugstore beetle (*Stegobium paniceum*) (lane number 8), the broad-horned flour beetle (*Gnatocerus cornutus*) (lane number 9), the Kashmir flour beetle (*Gnathocerus kasmirensis*) (lane number 10), the large-horned flour beetle (*Gnathocerus maxillosus*) (lane number 11), the lesser flour beetle (*Gnathocerus pusillus*) (lane number 12), the azuki bean weevil (*Callosobruchus chinensis*) (lane number 13), the cowpea weevil (*Callosobruchus maculatus*) (lane number 14), the bean weevil (*Acanthoscelides obtectus*) (lane number 15), the granary weevil (*Sitophilus granarius*) (lane number 16), the rice moth (*Corcyra cephalonica*) (lane number 17), the rice stem borer (*Chilo suppressalis*) (lane number 18), the lesser grain moth (*Ephestia elutella*) (lane number 19), the lesser red flour beetle (*Tribolium freemani*) (lane number 20), the lesser flour moth (*Ephestia kuehniella*) (lane number 21), and the lesser confused flour beetle (*Tribolium confusum*) (lane number 22), and for the grains barley (lane number 23), rice (lane number 24), corn (lane number 25), wheat (lane number 26), and the reaction solution without the template DNA used in this experiment (lane number N), no band derived from the amplification product of mitochondrial DNA was observed. Mk is a Gene Ladder 100 DNA marker. From the above determination results, it was shown that the Simplex PCR method using the oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2 can specifically detect *Trogoderma granarium* from among a number of stored-product pests and grains.

Industrial Applicability

[0055] The present invention can be used to confirm the presence or absence of food pests such as *Trogoderma granarium* in the fields of food production and food processing.

Claims

1. An oligonucleotide set for detecting Acetes erythraeus larvae, which is composed of a combination of two or more oligonucleotides selected from the base sequences shown in SEQ ID NOs: 1 to 3 below, SEQ ID NO: 1: 5'-CCTAGCTGTACGATTGACAGC-3' SEQ ID NO: 2: 5'-GGACTGGATTATTGCGACAGC-3' SEQ ID NO: 3: 5'-CACAGGAACTCACTTATCACTAGCC-3' The oligonucleotide set for detecting Acetes erythraeus larvae, which is any one of the following (1) or (2). (1) An oligonucleotide set using the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 and the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 as primers, and the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3 as a probe (2) An oligonucleotide set using the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 and the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 as primers

2. A kit for detecting Acetes erythraeus larvae, which contains the oligonucleotide set for detecting Acetes erythraeus larvae according to Claim 1.

3. A method for detecting Acetes erythraeus larvae, which includes a step of performing PCR amplification using the DNA extracted from a test sample as a template and the oligonucleotide set for detecting Acetes erythraeus larvae according to Claim 1, and detecting the obtained amplification product.

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