JP2022173818A - Oligonucleotides for detection of red spotted deer beetle - Google Patents

Abstract

An object of the present invention is to establish an inspection system capable of accurately and quickly detecting red spotted depot beetle, distinguishing it from other major food pests and other spotted depot beetles, at a low cost in trap surveys of quarantine pests. [Means for Solving Problem] An oligonucleotide set for detecting the red spotted deer, comprising two or more types of oligonucleotides containing a characteristic base sequence of the mitochondrial DNA of the red spotted depot, a kit for detecting the red spotted depot, and the same A detection method for red spotted deer beetle using an oligonucleotide set. [Selection figure] None

Description

TECHNICAL FIELD The present invention relates to an oligonucleotide for detecting red spotted depot beetle, a kit for detecting red spotted depot beetle, and a method for detecting red spotted depot beetle.

Trogoderma varium (Matsumura et Yokoyama) is classified in the genus Trogoderma in the order Coleoptera, family Dermestidae, and is the most commonly inhabited species in Japan. Insects belonging to the genus Trogoderma (referred to as Trogoderma) include, in Japan, in addition to the red madara bonito, the black madara bonito, the chamadara bonito, the yellow madara bonito, the lesser red bonito, the Kazari madara bonito, and the kimadara bonito. Seven types are known (Non-Patent Documents 1 and 2). Among these spotted deer beetles, there are some that pose a problem as important pests in terms of plant quarantine.

Rice is a typical stored grain that is susceptible to pest damage in Japan. Rice is positioned as one of Japan's major export items, and the Ministry of Agriculture, Forestry and Fisheries is promoting activities to promote the spread of Japanese cuisine internationally, and along with this, is promoting the use of Japanese rice and processed products for overseas Japanese restaurants. In addition to promoting the consumption of a certain type of sake, we are promoting the expansion of rice cultivation for export.

On the other hand, other countries have their own plant quarantine systems to prevent the invasion and spread of pests, and agricultural products exported from Japan must comply with the plant quarantine requirements of the destination country. Therefore, the Ministry of Agriculture, Forestry and Fisheries is requesting foreign countries to lift the export ban in order to improve the export environment for Japan's agricultural products. For example, when exporting polished rice to China, the ``Implementation Procedures for Export Quarantine of Polished Rice to the People's Republic of China (Notification No. 20 Shoan No. 3741 of the Director-General of the Consumer Affairs Bureau dated June 20, 2008)'' is stipulated. According to the implementation guidelines, only rice milled and fumigated at designated registered facilities approved by the Chinese side can be exported. In the authorization procedure for designated registered facilities, it is necessary to install traps using pheromones as attractants in the facilities and to confirm that there are no outbreaks of the deer beetles (trap survey). The species of the spotted deer that are subject to quarantine by the Chinese side are the lesser red beetle, the red beetle, and the lesser red beetle. If any of these are found at the facility, exports will be suspended and the target species will be eradicated by fumigation, etc. And management such as confirmation of the effect is necessary, and a troublesome process that is costly in terms of time and labor is additionally generated (Non-Patent Document 3).

In plant quarantine, it is first clarified whether the species is subject to quarantine, and if it is subject to quarantine, measures such as fumigation are taken. So far, it has been reported that in pheromone-trap surveys of the genus Spermidae, Spermum beetle, Spermum beetle, and Spermum beetle were captured, and among these, Spermum beetle accounted for the majority (Non-Patent Document 4, 5). When exporting to China, it is necessary to identify three species, namely, the lesser reddish beetle, the reddish red beetle, and the lesser reddish beetle. The scales are densely planted, and although characteristic spots can be seen, many of these spots are very similar, and the scales themselves are worn away due to physical contact etc. in the specimens collected at the site where they live. Even if the spots remain undamaged, the spots will turn black due to the oil on the individuals that have been immersed in a trap that uses a type of attractant (pheromone) that captures them with oil. It is extremely difficult to visually identify the species because the contrast between the two is unclear.

In recent years, DNA analysis has also been widely used for insects, and is particularly useful when visual identification is extremely difficult. According to the Environmental Recycling Society White Paper compiled by the Ministry of the Environment in 2008, the total number of known species in the world is about 1.75 million, of which about 6,000 are mammals, about 9,000 are birds, and There are about 950,000 species and about 270,000 species of vascular plants. Insects account for about 55% of living things, and it makes sense to classify and identify an extremely diverse range of insects using DNA.

DNA present in cells is divided into nuclear (genomic) DNA, mitochondrial DNA and chloroplast DNA. Nuclear DNA is used for criminal investigations, kinship relationships such as parentage, and breed identification in crops and livestock. Of these, mitochondrial DNA has a higher mutation frequency than nuclear DNA and has a higher copy number per cell than the genome, and is considered useful for determining the species of insects.

In addition to the conventionally used PCR method, there are various DNA testing methods such as the LAMP (Loop-Mediated Isothermal Amplification) method, DNA sequencing, Southern hybridization, and DNA microarray. . Among these, methods used for appraisal include the LAMP method, the DNA barcoding method using base sequencing of DNA, and the like. The LAMP method is useful because it has high specificity and does not require the strict and multi-step change steps of the temperature during the DNA reaction like the PCR method. An example of its use for detecting strains has been reported (Patent Document 1), but it has the drawback of being difficult to use because the sequences that can be used for primer design are limited. In addition, the DNA barcoding method decodes the DNA sequence and compares the sequence information with known sequence information for use, so even the name of the insect can be identified. However, this method uses a DNA sequencer to decipher the base sequences of analysis samples that have been subjected to the PCR method, and then compares the sequences one by one using dedicated software. It is a drawback and inefficient especially when analyzing a large number of samples.

The PCR method, unlike the above-mentioned DNA barcoding, is a method in which a target to be detected is determined in advance, and whether or not the sample contains DNA derived from the target is determined. The test can determine the presence or absence of the DNA of the organism to be detected simply by subjecting the extracted DNA to PCR. One typical example is when the target insect species is roughly known, such as identification of insects that have been more or less selectively captured by the aforementioned pheromone traps. In addition, when pheromone traps are used to attract insects, due to their characteristics, there is a possibility that many individuals with similar appearances, such as closely related species of the insects to be attracted, may be captured. By using such primers and probes, it is possible to quickly, in large quantities, and accurately confirm whether or not the object is the subject of appraisal.

Real-time PCR, which enables quantitative analysis by measuring fluorescence signals without electrophoresis, is also known as a method for analyzing amplification products of the PCR method, and primers and detection probes for that purpose are provided as a set. ing. For example, US Pat. No. 6,200,009 describes specific and universal probes and amplification primers for rapid detection and identification of bacterial pathogens. The present inventors have successively established oligonucleotide sets for detecting major food pests such as granaria magnolia weevil and red-eyed beetle by PCR and real-time PCR (Patent Document 3). As mentioned above, spotted deer beetles, which are also subject to export quarantine, have a very similar morphology, and identification by the markings on their scales is uncertain at the site, so it is extremely difficult to distinguish the species by visual inspection. Therefore, it is required to establish a testing system using PCR or real-time PCR.

Japanese Patent No. 4899180 JP-A-2007-125032 Japanese Patent No. 6600831

Nobuo Obayashi, Beetle family, Primary color Japanese beetle encyclopedia III, pp.125-130 (1985) Toshiharu Yoshida, Naoshi Watanabe, Nobuyuki Sonda, Illustrated "Pests of Stored Food" From Practical Identification Methods to Control Methods, 268 pages, October 26, 1989, National Rural Education Association Ministry of Agriculture, Forestry and Fisheries, Plant Protection Station, Pest Information, "Export Quarantine of Polished Rice to China" No.85 (2008) Souichi Hirao, Masaru Sugimoto, ``Trapping performance of spotted deer beetles (genus Trogoderma) by pheromone trap'', Journal of Pestology, Vol.7, No.1, pp.4-8 (1992) Souichi Hirao, Catch results of trogoderma deer by pheromone traps in facilities related to food ingredients, Kandoukun, Vol.6, No.4, pp.182-186 (1995)

Therefore, in view of the above-mentioned circumstances, the object of the present invention is, among insects caught in large quantities in trap surveys of quarantine pests in rice milling facilities and export-related warehouses, the spotted deer, which is particularly difficult to distinguish visually. We have developed oligonucleotides for the detection of the red spotted depot beetle, which is one of the species, by PCR and real-time PCR methods, and a test that can distinguish the red spotted depot beetle from other spotted depot beetles accurately, quickly, and at a low cost. to establish a system.

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 the dermestidae, red spotted depot, and obtained the sequence information. Analyzed. Then, from this sequence information, we found nucleotide sequences characteristic of the red spotted depot beetle, and combined oligonucleotides containing these nucleotide sequences to successfully establish an oligonucleotide set that can accurately and rapidly detect the red spotted depot beetle.
That is, the present invention includes the following inventions.

(1) An oligonucleotide set for detecting the red spotted depot, comprising two or more oligonucleotides containing the characteristic base sequences of the mitochondrial DNA of the red spotted depot.
(2) The oligonucleotide set for detecting red spotted deer beetle according to (1), wherein the characteristic nucleotide sequence of mitochondrial DNA is the nucleotide sequence in the cytochrome c oxidase subunit 1 (CO1) region.
(3) The oligonucleotide set for detecting red spotted deer beetle according to (1) or (2), wherein the oligonucleotides are used as primers and probes.
(4) the oligonucleotide set comprises an oligonucleotide consisting of the base sequences shown in SEQ ID NOS: 1 to 3 below or complementary sequences thereof, or at least 15 contiguous bases in the base sequences shown in SEQ ID NOS: 1 to 3 or complementary sequences thereof The oligonucleotide set for detecting red spotted deer beetle according to any one of (1) to (3), which is an oligonucleotide set composed of an oligonucleotide consisting of a base sequence containing a base 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 red spotted depot, comprising the oligonucleotide set for detecting red spotted depot beetle according to any one of (1) to (4).
(6) Using the DNA extracted from the test sample as a template, PCR amplification is performed using the oligonucleotide set for detection of red spotted deer beetle according to any one of (1) to (4), and the resulting amplified product is detected. A method for detecting red spotted deer beetle, comprising steps.

INDUSTRIAL APPLICABILITY According to the present invention, there are provided an oligonucleotide set for detecting the food pest of red spotted depot beetle, a kit for detecting red spotted depot beetle containing the oligonucleotide set, and a method for detecting red spotted depot beetle using the oligonucleotide set. Therefore, according to the present invention, it is possible to detect red spotted deer beetle accurately and quickly at a low cost, so that the reliability of quality can be enhanced by utilizing it for food management and assurance.

FIG. 1 shows the results of detection of Red spotted depot beetle by real-time PCR using TaqMan ^™ probes. In this method, the nucleotide sequence of SEQ ID NO: 1 is the upstream primer, the nucleotide sequence of SEQ ID NO: 2 is the downstream primer, the 5' side of the nucleotide sequence of SEQ ID NO: 3 is the reporter dye (FAM), and the 3' side is the quencher ( TAMRA) were used as TaqMan ^TM probes (typical grain storage pests other than red spotted beetle: Tobacco beetle, diamondback beetle, beetle beetle, saw beetle, red flour beetle, gypsy beetle, Brown beetle, brown rice beetle, flatfish beetle, Kashmir beetle beetle, big bean beetle beetle, lesser bean beetle, adzuki bean weevil, bean weevil, bean weevil, beetle weevil, granaria beetle, Noshimemadarameiga, tap moth, Himemarukatsuobushimushi, Lesser red beetle , red-eyed beetle, red spotted beetle, barley, rice, maize, wheat). FIG. 2 shows the detection results of the red spotted deer beetle by the Simplex PCR method. In this method, the nucleotide sequence of SEQ ID NO: 1 was used as the upstream primer, and the nucleotide sequence of SEQ ID NO: 2 was used as the downstream primer (1: Nicotiana beetle, 2: Rhinoceros beetle, 3: Rhinoceros beetle, 4: Saw beetle Hirata beetle, 5: Red flour beetle, 6: Germ beetle, 7: Iron meal beetle, 8: Brown rice beetle, 9: Hirata meal beetle, 10: Kashmir beetle, 11: Bighorn meal beetle, 12: Red beetle Modoki, 13: adzuki bean weevil, 14: maize weevil, 15: maize weevil, 16: granaria maize weevil, 17: Noshime Madarameiga, 18: Bakuga, 19: Himemarukatsuobushimushi, 20: Lesser red beetle, 21: Himekatsuobushimushi, P: 22: Red spotted deer, 23: Barley, 24: Rice, 25: Maize, 26: Wheat, N: No template, M: Gene Ladder 100 DNA marker).

The present invention will be described in detail below.
1. Oligonucleotide set for detection of red spotted depot and detection kit The oligonucleotide set for detection of the red spotted depot beetle of the present invention is composed of two or more types of oligonucleotides containing the characteristic base sequences of the mitochondrial DNA of the red spotted depot beetle to be detected. . Although the base length of the oligonucleotide is not limited, it is usually 15 to 30 base lengths, preferably 18 to 25 base lengths, for primers, and 10 to 30 base lengths for probes.

The characteristic base sequence of the mitochondrial DNA of the red spotted depot beetle to be detected has low homology with known mitochondrial DNA base sequences of a wide range of food pests, and is considered to be characteristic only to the target red spotted depot beetle to be detected. , a base sequence of 10 bases or more, preferably 15 bases or more, more preferably 20 bases or more.

In order to identify the characteristic base sequences of the above-mentioned mitochondrial DNA, first, the base sequence information of the mitochondrial DNA of the red spotted deer beetle is obtained. Since the base sequence information of the red spotted deer beetle is not registered in databases (NCBI, DDBJ, etc.), it can be obtained by directly analyzing the base sequence as shown in Examples below.

In the present invention, the characteristic base sequence of the mitochondrial DNA of the red spotted depot beetle includes, for example, the base sequence of the cytochrome c oxidase subunit 1 (CO1) region.

Next, primers are designed based on the identified characteristic nucleotide sequences. Primer design takes into consideration oligonucleotide length, GC content, Tm value, complementarity between oligonucleotides, secondary structure within oligonucleotides, etc. Until Application" (Protein, Nucleic Acid, Enzyme Special Issue, 1996, Kyoritsu Shuppan Co., Ltd.), and "Bio Experiment Illustrated 3 PCR that Really Increases: Cell Engineering Attachment Visual Experiment Notebook Series" (Hiroki Nakayama, Shujun Co., Ltd.) Publishing Co., Ltd.), "PCR Technology - Principles and Applications of DNA Amplification" (edited by Henry A. Erlich, supervised by Kuninoshin Kato, Takara Shuzo Co., Ltd.).

The design criteria specifically adopted are as follows.
(a) The amplification product is clearly detected when the PCR amplification product of the characteristic sequence of the mitochondrial DNA of the red spotted depot beetle is electrophoresed.
(b) the PCR amplification product is 100-500bp, preferably 100-250bp;
(c) Primer length should be in the range of 15-30 bp to allow specific annealing with the template DNA.
(d) Since the annealing temperature depends on the Tm (melting temperature), select primers with a Tm value of 50-70°C, preferably 55-65°C and close to each other in order to obtain a highly specific PCR amplification product. to do.
(e) High homology between the base sequence at the 3' end of the primer and the template DNA sequence.
(f) Avoid complementary sequences between the primers so that they do not form dimers or conformations.
(g) In order to ensure stable binding to the template DNA, the GC content should be approximately 50%, and consideration should be given to avoid uneven distribution of GC-rich or AT-rich primers as much as possible.

Moreover, probes may be designed based on a software protocol attached to a commercially available real-time PCR device such as ABI Prism 7900HT Real-time PCR System (Life Technologies Japan Co., Ltd.). The design criteria for the probe are that the GC content is within the range of 20-80%, avoiding 4 or more consecutive G or C bases in the sequence, and 8-10°C higher than the Tm value of the corresponding primer pair. As a general rule, it should be set to a high degree and the 5' end of the probe should not be G.

Based on the above criteria, the following was established as an oligonucleotide set for detection of the red spotted deer beetle of the present invention.
[Oligonucleotide set for detecting red spotted deer beetle]
SEQ ID NO: 1: 5'-CCTAGCTGTACGATTGACAGC-3'
SEQ ID NO: 2: 5'-GGACTGGATTATTGCGACAGC-3'
SEQ ID NO: 3: 5'-CACAGGAACTCACTTATCACTAGCC-3'

Oligonucleotides constituting the oligonucleotide set of the present invention can function not only as the above-mentioned "oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 3 or complementary sequences thereof" but also as primers or probes for detecting the red spotted deer beetle. As long as it is the mutated oligonucleotide. For example, mutant oligonucleotides include "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 complementary sequences thereof". There are no particular restrictions on the types of bases other than at least 15 consecutive bases, and the site (3' terminal side, 5' terminal side) of the mutated oligonucleotide with at least 15 consecutive bases. Moreover, the total length of the mutated oligonucleotide is preferably about 15 to 30 bases. In general, when a probe complementarily binds to a template DNA, the base sequences added to both terminal regions are of little importance. Therefore, when the oligonucleotides contained in the above oligonucleotide set are used as probes, any 5 or less bases may be added or deleted at both ends of the oligonucleotide having the base sequence of each SEQ ID NO. .

The food pest to be detected in the present invention is the red spotted deer beetle, which is a pest that directly damages edible substances as food, or that can lead to deterioration of the quality of substances by mixing them, and its adults, pupae, larvae, and eggs. including any of Substances mixed with red spotted deer beetles are not limited to foods, but include plant-derived residues and the like.

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

The above oligonucleotide set can also be made into a kit. The kit of the present invention may contain any of the oligonucleotide sets described above, and may optionally contain DNA extraction reagents, PCR buffers, PCR reagents such as DNA polymerase, and PCR amplification as a positive control for the reaction. DNA solutions containing the regions, detection reagents such as stains and electrophoresis gels, instructions, and the like may be included.

2. Method for detecting red spotted depot beetle The method for detecting food pests of the present invention comprises extracting DNA from a test sample, using the DNA as a template, and using two types of oligonucleotides selected from the oligonucleotides contained in the above oligonucleotide set. performing polymerase chain reaction (PCR) and detecting the amplified product obtained by the PCR.

The sample to be tested is not limited to the Red Madara depot beetle itself, but is not particularly limited and includes food raw materials, materials in the process of processing, and products such as processed foods that may be damaged or contaminated by the Red Madara depot beetle. Specifically, non-processed products such as cereal grains and pulverized products thereof, and processed products such as cooked rice, chocolate, cup noodles, and natto (fermented soybeans) can be used. The detection results obtained by the method of the present invention can be used for food safety labeling, and can also be used to confirm the presence or absence of contamination of food pests in production lines unintended by producers.

Any nucleic acid extraction method known to those skilled in the art may be used for extracting DNA from a test sample. Examples thereof include phenol extraction, cetyltrimethylammonium bromide (CTAB), and alkaline SDS. . Moreover, these methods may be modified as appropriate, and various DNA extraction kits sold by reagent manufacturers may be used. Filtration with a membrane filter or homogenization is performed depending on the type of sample. In addition, various DNA extraction kits such as DNeasy Plant mini Kit (manufactured by Qiagen Co., Ltd.) sold by reagent manufacturers may be used. DNA extracted by these methods is preferably maintained in a state suitable for use as a template for PCR, for example, preferably dissolved in an appropriate buffer and stored at a low temperature. Further, after DNA extraction, purification treatment such as chloroform/isoamyl alcohol treatment, isopropanol precipitation, deproteinization with phenol/chloroform, and ethanol precipitation may be performed.

In addition, when the above oligonucleotides are used as probes, they may be labeled with a labeling substance for detecting fluorescence signals derived from amplification products. Preferred labeled probes are TaqMan ^™ probes that are doubly labeled with a fluorescent substance and a quencher. TaqMan ^TM probes are usually modified with a fluorescent substance (reporter fluorescent dye) at the 5′ end of the nucleic acid probe and a quencher (quencher fluorescent dye) at the 3′ end. Examples of reporter fluorochromes include 6-FAM (6-carboxyfluorescein), TET (6-carboxy-4,7,2',7'-tetrachlorofluorescein), HEX (6-carboxy-2',4', Fluorescein-based fluorescent dyes such as 7',4,7-hexachlorofluorescein), and examples of quencher fluorescent dyes include 6-carboxytetramethylrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), and the like. and rhodamine-based fluorescent dyes. These fluorescent dyes are known and are included in commercially available kits for real-time PCR, so they can be used.

Next, using the extracted DNA as a template, PCR amplification is performed using two oligonucleotides selected from the above-described oligonucleotide set of the present invention. PCR amplification is not particularly limited except for using the oligonucleotide set described above, and may be performed according to a conventional method. Specifically, a cycle involving denaturation of the template DNA, annealing of the primer to the template, and extension reaction of the primer using a thermostable enzyme (Taq polymerase or DNA polymerase such as Tth DNA polymerase derived from Thermus thermophilus) is repeated. Thereby, the characteristic base sequence of the mitochondrial DNA of the red spotted deer beetle to be detected is amplified. The composition of the PCR solution (amount of template DNA, type of buffer solution, 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 determined by those skilled in the art. can be selected and set appropriately.

For example, after mixing 0.1 to 100 ng of template DNA, 10x PCR reaction buffer, 0.25 to 1 μM each of primers, 0.25 to 2.5 U of DNA polymerase (Taq polymerase, Tth DNA polymerase, etc.), and 250 μM each of dNTPs, 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 , 70-74°C for 5 minutes x 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 sequences that serve as primers. A series of these PCR operations can be performed using a commercially available PCR kit or PCR device according to the operating instructions.

Detection of PCR amplification products can be confirmed using conventional electrophoresis such as agarose gel electrophoresis and capillary electrophoresis, DNA hybridization, real-time PCR, and the like. For example, in agarose gel electrophoresis, the product is stained with ethidium bromide, SYBR Green solution, or the like, and the amplified product is detected as a single band. Alternatively, the PCR can be performed using primers that have been labeled with a labeling substance in advance, and the amplified product can be detected. As the labeling substance, fluorescent substances, radioisotopes, chemiluminescent substances, biotin, etc., which are well known in the art can be used. In addition, in DNA hybridization, an amplification product is bound to an immobilizing carrier such as a microarray, and the amplification product is confirmed by fluorescence, enzymatic reaction, or the like. The immobilizing carrier for detection is a support on which an oligonucleotide having a sequence capable of hybridizing with the characteristic sequence of the mitochondrial DNA of the red spotted depot beetle to be detected is immobilized. Examples of supports that can be used include nylon membranes, nitrocellulose membranes, glass, and silicon chips.

The real-time PCR method includes 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 together with a primer pair, is added to the PCR reaction system, or the 5' end is called a reporter. There is a TaqMan method in which a probe (TaqMan ^TM probe) labeled with a fluorescent substance and a quenching substance called a quencher at the 3′ end is added to the PCR reaction system. In the TaqMan method, the TaqMan ^TM probe specifically hybridizes to the template DNA under the conditions used for the polymerase elongation reaction in the PCR amplification reaction, and is degraded as the DNA strand elongates, that is, as the template DNA is amplified. Liberation of the fluorescent substance increases the amount of fluorescence in the PCR solution. This increase in the amount of fluorescence serves as an indicator of template DNA amplification, and the state of amplification in PCR can be easily detected in real time. The real-time PCR method is based on a conventional method known to those skilled in the art, using a commercially available real-time PCR kit or a real-time PCR device, and following the operating instructions attached thereto, except for using the above oligonucleotide set. You can do it. As a real-time PCR device, for example, ABI Prism 7900HT Real-time PCR System or the like is used.

When detecting red spotted deer beetle in a test sample, the procedure is as follows. First, a test sample is subjected to real-time PCR to obtain an amplification curve. Next, in a region where the increase in fluorescence signal has an exponential relationship with the number of cycles, an appropriate threshold for the increase in fluorescence amount (ΔRn) is set, and depending on whether the amplification curve and the threshold intersect, Determine the presence or absence of food pest DNA in the test sample. The number of cycles at which this amplification curve crosses the threshold (Threshold) is called a Ct value (Threshold Cycle).

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

(Example 1) Oligonucleotides for detection of red spotted deer beetle (design of primers and probes)
(1) Extraction of DNA DNA was extracted from specimens identified as red spotted deer beetles by morphological appraisal by experts. For DNA extraction, DNeasy Blood & Tissue Kit (manufactured by Qiagen Co., Ltd.) was used. Approximately 25 mg of the pest sample was placed in a 1.5 ml tube, and 200 µl containing 20 µl of Proteinase K included in the DNeasy Blood & Tissue Kit was added. Buffer ATL was added to the mixture and mixed while grinding with a pestle, and incubated at 56°C overnight. 4 μl of ribonuclease (RNase) A was added, mixed, and incubated at room temperature for 5 minutes. After that, 200 μl of Buffer AL was added, and after thorough mixing, 200 μl of ethanol (96-100%) was added and thoroughly mixed again.

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 centrifugation was performed to wash the filter with adsorbed DNA. After that, 200 µl of Buffer AW2 was added, and after centrifugation, total DNA containing mitochondrial DNA was eluted from the filter.

(2) DNA sequence analysis of the cytochrome C oxidase subunit (CO1) region of mitochondrial DNA and design of oligonucleotides for detection of the red spotted depot beetle The base sequence information of the red spotted depot beetle mitochondrial DNA is not registered in databases (NCBI, DDBJ, etc.). LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3' (SEQ ID NO: 4), which is a common primer for amplifying the animal cytochrome c oxidase subunit 1 gene (CO1) region, which is newly used in the DNA barcoding method. and HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' (SEQ ID NO: 5) [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)], we further analyzed the vicinity and obtained new nucleotide sequence information.

Next, we carefully examined and compared the obtained base sequence information with the known CO1 sequence information of related species, and found that the target sequence length was relatively short and high specificity could be obtained. The following DNA sequence was designed and used as an oligonucleotide for detecting red spotted deer beetle.
SEQ ID NO: 1: 5'-CCTAGCTGTACGATTGACAGC-3'
SEQ ID NO: 2: 5'-GGACTGGATTATTGCGACAGC-3'
SEQ ID NO: 3: 5'-CACAGGAACTCACTTATCACTAGCC-3'

(Example 2) Confirmation of performance of oligonucleotides for detecting red spotted deer beetle (real-time PCR method using TaqMan ^TM probe)
Using the oligonucleotide set designed in Example 1 for detection of the red spotted depot beetle, the red spotted depot beetle was detected by real-time PCR (TaqMan ^TM probe method). The base sequence of SEQ ID NO: 1 is an upstream primer, the base sequence of SEQ ID NO: 2 is a downstream primer, the 5' side of the base sequence of SEQ ID NO: 3 is a reporter dye (FAM), and the 3' side is a quencher (TAMRA). The labeled base sequences were used as TaqMan ^™ probes.

As test samples, the following grain storage pests (23 species) including the red spotted depot beetle and grains (4 species) that were damaged by the storage pests were used. The DNA extraction method was the extraction of DNA in Example 1 (1). Grain DNA was extracted using GM Quicker2 (manufactured by Nippon Gene Co., Ltd.) according to the method attached to the kit.

<Grain storage pests>
Tobacco beetle, long-spinned beetle, beetle beetle, sawtooth beetle, flour beetle, snail beetle, beetle beetle, beetle beetle, brown beetle beetle, flat beetle beetle, Kashmir beetle, beetle beetle, red bean beetle Weevil, maize weevil, maize weevil, granaria maize weevil, Noshime mackerel moth, Baku moth, Himemarukatsuobushimushi, Lesser red beetle, Lesser beetle beetle, Himemadarakatsuobushimushi and Akamadarakatsuobushimushi

<Grains damaged by stored grain pests>
barley, rice, maize, wheat

Total DNA containing mitochondrial DNA was extracted from each sample according to the method of Example 1(1), and using these as template DNA, detection of red spotted deer beetle by real-time PCR method (TaqMan ^TM 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 ^TM probe of SEQ ID NO: 3 labeled with FAM at the 5′ end and TAMRA at the 3′ end, 0.5 μM of each primer pair (sequence No. 1 and SEQ ID No. 2) and 1 ng of total DNA were prepared (total volume: 10 μl), and each was incubated at 95° C. for 30 seconds using a CFX96 Touch ^TM real-time PCR analysis system (manufactured by Bio-Rad Laboratories, Inc.). , 95°C for 5 seconds → 60°C for 5 seconds, and amplified by repeating 30 cycles.

The test positive/negative judgment was performed by judging 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 FIG. As shown in FIG. 1, the amount of fluorescence derived from the amplification product of the mitochondrial DNA of the red spotted deer beetle was clearly detected from around the 16th cycle. On the other hand, a reaction solution containing no template DNA that was tested in this experiment, tobacco beetles that are grain storage pests other than red spotted beetle, diamondback beetle, red beetle, saw beetle, flour beetle, gypsy beetle, beetle beetle, Brown rice meal beetle, Hirata flour beetle, Kashmir beetle beetle, Bighorn beetle beetle, Red bean meal beetle, Adzuki bean weevil, bean weevil, bean weevil, beetle weevil, granaria beetle, Noshime madarameiga, tap moth, Himemaru beetle, Lesser red beetle, Hime beetle beetle Fluorescence derived from the amplification product of mitochondrial DNA was not observed in the reaction mixtures of the genus S. depot (22 species in total) and the reaction mixtures of cereals such as barley, rice, corn, and wheat (4 species in total). In addition, the Ct value derived from the red spotted deer beetle calculated when the Th value was fixed at 500 was obtained as a real number of 20.01, but other grain storage pests were N/A (not detectable), and fluorescence was clearly detected. It was possible to determine the presence or absence of From the above determination results, the real-time PCR method using the oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOS: 1 to 3 can specifically detect the red spotted deer beetle from a large number of storage insect pests and grains.

(Example 3) Confirmation of performance of oligonucleotide for detection of red spotted deer beetle (Simplex PCR method)
Using the oligonucleotides designed in Example 1 for detection of the red spotted depot, the simplex PCR method was used to detect the red spotted depot. The nucleotide sequence of SEQ ID NO: 1 was used as an upstream primer, and the nucleotide sequence of SEQ ID NO: 2 was used as a downstream primer.

Mitochondrial DNA was extracted from each sample according to the method of Example 1(1), and using these DNAs as template DNA, detection of red spotted deer beetle by Simplex PCR was carried out as follows. That is, AmpliTaq Gold DNA Polymerase (manufactured by Thermo Fisher Scientific) 0.25U, 1×PCR BufferII (Mg ²⁺ free), 200 μM each dNTP, 1.5 mM MgCl _{2 ,} 0.5 μM each primer pair (SEQ ID NO: 1 and sequence No. 2), prepare a reaction mixture containing 1 ng of total DNA (total volume 10 μl), incubate at 95°C for 10 minutes in an S1000 ^TM thermal cycler, then incubate at 95°C for 30 seconds → 60°C for 30 seconds → 72°C for 30 seconds. Amplification was repeated for 40 cycles.

Positive/negative determination was made by subjecting 10 μl of each reaction solution to 3% agarose gel electrophoresis together with a DNA marker, Gene Ladder 100 (0.1-2 kbp), staining the gel with ethidium bromide, followed by UV transillumination. The presence or absence of a stained DNA band was confirmed under ultraviolet irradiation by a tar.

The results are shown in FIG. In lane number P, a band of about 161 bp representing an amplification product derived from mitochondrial DNA of red spotted depot beetle was clearly detected. On the other hand, tobacco beetles (lane number 1), long-nosed beetles (lane number 2), beetles (lane number 3), saw beetles (lane number 4), red beetles (lane No. 5), gypsy beetle (lane number 6), brown rice beetle (lane number 7), brown rice meal beetle (lane number 8), flatfish red beetle (lane number 9), Kashmir red beetle (lane number 10) , Bighorn flour beetle (lane number 11), lesser bean flour beetle (lane number 12), adzuki bean weevil (lane number 13), maize weevil (lane number 14), beetle weevil (lane number 15), granaria maize weevil (lane number 16) , Noshime Madara Meiga (Lane No. 17), Bakuga (Lane No. 18), Himemarukatsuobushimushi (Lane No. 19), Himea Red Katsuobushimushi (Lane No. 20), Himemadara Katsuobushimushi (Lane No. 21) and Himemadara Katsuobushimushi (Lane No. 22) of barley (lane number 23), rice (lane number 24), maize (lane number 25), wheat (lane number 26), and a reaction solution containing no template DNA (lane No band derived from the mitochondrial DNA amplification product was observed for No. N). Mk is the Gene Ladder 100 DNA marker. From the above determination results, it was shown that the Simplex PCR method using oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2 can specifically detect the red spotted deer beetle from a large number of storage insect pests and grains.

INDUSTRIAL APPLICABILITY The present invention can be used to confirm the presence or absence of food pests such as red spotted deer beetles in the fields of food manufacturing and food processing.

Claims (6)

An oligonucleotide set for detecting the red spotted depot, comprising two or more types of oligonucleotides containing the characteristic base sequences of the mitochondrial DNA of the red spotted depot. 2. The oligonucleotide set for detecting red spotted depot beetle according to claim 1, wherein the characteristic base sequence of mitochondrial DNA is the base sequence in the cytochrome c oxidase subunit 1 (CO1) region. 3. The oligonucleotide set for detecting red spotted deer beetle according to claim 1 or 2, wherein said oligonucleotides are used as primers and probes. The oligonucleotide set comprises an oligonucleotide consisting of the base sequences shown in SEQ ID NOS: 1 to 3 below or complementary sequences thereof, or a base sequence containing at least 15 consecutive bases in the base sequences shown in SEQ ID NOS: 1 to 3 or complementary sequences thereof The oligonucleotide set for detecting red spotted deer beetle according to any one of claims 1 to 3, which is an oligonucleotide set composed of oligonucleotides consisting of.
SEQ ID NO: 1: 5'-CCTAGCTGTACGATTGACAGC-3'
SEQ ID NO: 2: 5'-GGACTGGATTATTGCGACAGC-3'
SEQ ID NO: 3: 5'-CACAGGAACTCACTTATCACTAGCC-3' A kit for detecting red spotted depot, comprising the oligonucleotide set for detecting red spotted depot beetle according to any one of claims 1 to 4. Using the DNA extracted from the test sample as a template, performing PCR amplification using the oligonucleotide set for detecting red spotted deer beetle according to any one of claims 1 to 4, and detecting the resulting amplified product. , a method for detecting red spotted deer beetle.

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