JP2007061061A - Method for detecting listeria monocytogenes - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting Listeria monocytogenes specifically, quickly and in a high sensitivity. <P>SOLUTION: This oligonucleotide primer specifically hybridizing with an optional base sequence designed from a base sequence derived from the Listeria monocytogenes, the method for detecting the L. monocytogenes by the detection of nucleic acid amplification and a kit for detecting the L. monocytogenes are provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for detecting Listeria monocytogenes, and more specifically, contamination of food and infection of Listeria monocytogenes using a highly sensitive detection method for genes. It relates to a diagnostic method.

  Listeriosis is an infection caused by Listeria monocytogenes (L. monocytogenes), causing serious symptoms such as sepsis and meningitis in both humans and animals, and progressing to severe cases. In that case, the fatality rate is high (20-30%). The rate of seriousness of compromised hosts such as the elderly and infants is high, and it is known that miscarriages occur at high rates when infected with pregnant women.

  This infection began to gain attention in food hygiene since the 1980s, and in Europe and the United States, mass infections in humans (such as the deaths of several hundred people) via food such as vegetable salads, dairy products, and processed meat products This is due to the fact that a large scale (including examples) was reported one after another. An outbreak of milk in 1983 and soft cheese in 1985 was reported in the United States, and contamination of milk and dairy products became an international issue. Therefore, in January 1988, the International Dairy Federation (IDF) presented a test method (IDF143) based on a provisional culture method, and in Japan a test method based on this method was also issued in 1993 by the former Ministry of Health and Welfare in L. monocytogenes in cheese. (Introduction of contamination prevention of milk and dairy product listeria, Hygiene No. 169)

  The detection / separation method by the culture method has not yet been standardized worldwide, and there are roughly three methods in which the medium to be used and the culture time are slightly different. The above-mentioned methods proposed by IDF, the ISO (International Standard) method, and the FDA method (food in general) and USDA-FSIS method (meat, eggs and their products) in the US Proposed. Both methods currently require more than a week for the final separation and identification.

  Listeria is a Gram-positive, facultative anaerobic non-spore-short bacillus that grows flagella and shows motility when cultured at 30 ° C or below. The optimal growth temperature is 30 to 37 ° C, but it can grow in a wide temperature range of -0.4 to 45 ° C. Heat resistance is slightly higher than other non-spore-forming bacteria such as Salmonella and E. coli, has a wide growth pH range (pH 4.4-9.4), and resists high concentrations of salt (10%). Therefore, even if the amount of contaminating bacteria is not a problem immediately after processing, it can be propagated to a sufficient amount of bacteria for the onset in the process of distribution at low temperature and storage at the end (consumer). It can be said that it is a very troublesome fungus causing food poisoning.

  Listeria are classified into 6 species, of which only L. monocytogenes is known to be pathogenic to humans. Listeria spp. Other than L. monocytogenes are five species, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, L. grayi, but contamination to food is caused by Listeria other than L. monocytogenes. Since they are often contaminated with bacteria, it is important to distinguish them.

  L. monocytogenes is a combination of 12 types of cell antigens (O antigen) and 4 types of flagellar antigens (H antigens), resulting in 13 serotypes (1 / 2a, 1 / 2b, 1 / 2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, 7). Studies from various countries have shown that the majority of human listeriosis-derived strains (clinical isolates) are limited to specific serotypes, with three serotypes: 1 / 2a, 1 / 2b, and 4b. Total accounted for over 90% of the total. In Japan, about 60% is 4b. On the other hand, most of the food-derived strains are 1 / 2a, 1 / 2b, 1 / 2c, and then 4b is isolated. The serotype distribution of strains isolated from individuals is not always consistent. The cause is still unknown, but there is some selectivity related to the pathogenesis from infection (resistance to serotypes 1 / 2a, 1 / 2b, 1 / 2c, etc.), and antibiotics in each serotype Differences in resistance are speculated. As serotypes to be separated from food, it is considered to be important in the order of 1 / 2b and 1 / 2a starting from 4b, and it is essential to detect these serotypes in food inspection.

  In Japan, no large-scale food poisoning accidents have occurred, and it is said that there is less listeriosis than in the West. However, in the “Research on Health Damage of Food-Derived Listeria Bacteria” by the Food Safety Committee of the Cabinet Office (leader: Shingo Isumi / National Institute of Health Sciences), about 80 cases of severe listeriosis occurred annually. It is shown that the number of cases is almost the same as in Europe and the United States, although it is slightly smaller. According to the study, the contamination of Japanese foods is almost the same as in Europe and the United States, mainly meat and Ready-to-eat foods, and bacteria are isolated from raw meat with a high probability of 30 to 40%, especially high in chicken. A tendency to become a rate is recognized. The contamination rate of imported natural cheese is as low as about 3%, but most of the contaminated items show an unusually high number of bacteria and are at high risk. It is estimated that the rate of accidents is low due to the widespread use of cooking methods that heat enough in Japan, but it is not unusual for large-scale food poisoning accidents to occur in Japan due to diversification of life and culture. It can be said that there is.

At present, many types of kits used as detection methods other than the culture method are commercially available, and their sensitivity is about 10 ⁵ to 10 ⁶ CFU / mL. Since the amount of contaminating bacteria in food is usually less than 10 CFU per gram, the enrichment procedure is essential, and any kit requires 30-52 hours of pre-culture before obtaining a sample. The selective enrichment medium used for the pre-enrichment culture is specified by each kit, and some of them employ a two-stage enrichment method.

  Many of the kits are ELISA methods using antibodies and require 2 to 4 hours of operation after pre-enrichment culture. Immunochromatography kits and immunofluorescence kits that are easier to operate and can be judged in a short time have been developed. However, specificity problems such as the inability to specifically detect only L. monocytogenes, and expensive dedicated The situation has not reached widespread use, such as the need for automatic measuring devices.

  As a test reagent targeting a gene, there are those using a DNA probe method or a real-time PCR method, which have higher detection sensitivity and specificity than the detection method using the above-mentioned antibody. The US FDA-FSIS (US Department of Agriculture Food Safety Inspection) detects specific temperature of the PCR product (melting temperature) and performs specific detection without gel electrophoresis analysis. "L. monocytogenes screening method" has been introduced and seems to be accepted as a reliable test method. However, this method also requires complicated operation and time, and an expensive dedicated measuring device.

  In addition, there is a PCR primer set that has not been made into a kit, but has high academic reliability and is used as a confirmation aid in combination with a culture method. PCR (Mono A / B, amplification product: 371 bp, non-patent document 1) targeting iap gene (invasive gene, hereinafter referred to as iap gene) and PCR targeting Hly gene encoding hemolytic toxin Listeriolysin O ( Hly A / B, amplification product: 730 bp, non-patent document 2) and the like have been introduced in many academic literatures and are used in a wide range. However, these methods also require complicated operations and time.

Thus, although the PCR method is recognized as a highly reliable method in the L. monocytogenes test, a further improvement in sensitivity and a quick and simple gene detection method are required.
The present inventors can achieve the object of the present invention by using a detection method that is more sensitive, specific, and requires a shorter time than the currently known methods, immunological measurement methods and PCR methods, that is, the LAMP method. It was.

Bubert A, Kohler S, Goebel W. The homologous and heterologous regions within the iap gene allow genus- and species-specific identification of Listeria spp. By polymerase chain reaction.Appl Environ Microbiol 1992; 58: 2625−2632. Johnson, WM, SD Tyler, EP Ewan, FE Ashton, G. Wang, and KR Rozee.Detection of genes coding for listeriolysin and Listeria monocytogenes antigen A (ImaA) in Listeria spp.by the polymerase chain reaction.Microb.Path. 1992 12: 79-86.

  The object of the present invention is to allow L. monocytogenes, which is a causative agent of listeriosis, to be detected rapidly, simply, with high sensitivity and specifically from food samples and clinical specimens (eg, culture solutions).

  As a result of intensive studies to solve the above problems, the present inventors have produced oligonucleotide primers that selectively hybridize with the base sequence derived from the iap gene of L. monocytogenes, and L. The inventors have found that L. monocytogenes can be detected with high sensitivity by amplifying a base sequence specific to monocytogenes, thereby completing the present invention.

That is, the present invention has the following configuration.
(1) Oligonucleotide primers designed to specifically amplify and detect serotypes of L. monocytogenes, the nucleotide sequence derived from the iap gene represented by SEQ ID NO: 1, and the 721 to 1100 An oligonucleotide primer designed from an arbitrary base sequence selected from these base sequences or a base sequence complementary thereto.
(2) including at least 15 consecutive bases selected from the base sequences represented by SEQ ID NOs: 2 to 9 selected from the base sequences derived from the iap gene of L. monocytogenes or the base sequences complementary thereto (1 ) Oligonucleotide primers.
(3) Select a base sequence region of F3c, F2c, F1c from the 3 ′ end side on the target nucleic acid of L. monocytogenes iap gene, and select a base sequence region of R3, R2, R1 from the 5 ′ end side. When the complementary base sequence is F3, F2, F1, and R3c, R2c, R1c, it is characterized by comprising at least one base sequence selected from the following (a) to (d) (1 The oligonucleotide primer according to (2).
(a) A base sequence having the F2 region of the target nucleic acid on the 3 ′ end side and the F1c region of the target nucleic acid on the 5 ′ end side.
(b) A base sequence having the F3 region of the target nucleic acid.
(c) A base sequence having the R2 region of the target nucleic acid on the 3 ′ end side and the R1c region of the target nucleic acid on the 5 ′ end side.
(d) A base sequence having the R3 region of the target nucleic acid.
(4) The iap gene base sequence specific to L. monocytogenes can be amplified and consists of the base sequence selected from the following (a) and / or (b) from the 5 ′ end to the 3 ′ end: The oligonucleotide primer according to (1) to (2).
(a) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 2)-(any base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 3) -3'
(b) 5 '-(base sequence of SEQ ID NO: 6)-(arbitrary base sequence having 0 to 50 bases)-(base sequence complementary to the base sequence of SEQ ID NO: 7) -3'
(5) A method for detecting L. monocytogenes, comprising performing an amplification reaction of a target nucleic acid region of an iap gene of L. monocytogenes using the oligonucleotide primers according to (1) to (4).
(6) The method for detecting L. monocytogenes according to (5), wherein the amplification reaction of the target nucleic acid region of the iap gene of L. monocytogenes is the LAMP method.
(7)
The presence or absence of L. monocytogenes is detected by detecting amplification of the target nucleic acid region of the iap gene of L. monocytogenes using the oligonucleotide primers described in (1) to (4). Method for detecting monocytogenes.
(8) In the L. monocytogenes detection method, a kit comprising the oligonucleotide primer according to (1) to (4).

  According to the present invention, L. monocytogenes can be detected specifically, sensitively and rapidly. Hereinafter, the present invention will be described in detail.

  Samples used in the present invention include primary enrichment culture solutions and secondary enrichment culture solutions obtained from food specimens and appropriate media, and bacterial suspensions prepared from growing colonies on agar media in separate culture, etc. Examples include samples at each culture stage containing cultured cells derived from L. monocytogenes contaminated and adhered to food. The culture method of L. monocytogenes is different depending on the type of ingredients to be tested and the country, and the medium used is different. In Japan, EB medium (milk and dairy products) and UVM medium, in Europe Half-Fraser medium and Fraser Medium (food and livestock feed), LEB medium (general food) and UVM medium (meat, eggs and their products) are used for each enrichment culture in the United States. At present, a method using Half-Fraser medium and Fraser medium used mainly in Europe has been proposed as ISO / DIS 11290-1 (1996) to shift to this method in the future. As the separation medium, Oxford agar medium, PALCAM agar medium, or the like is used.

  When using primary and secondary enrichment culture solutions containing test foods as samples, after digesting food-derived proteins with proteolytic enzymes, etc., of course, general DNA extraction and purification methods such as methods using phenol and chloroform have already been used. An extracted nucleic acid obtained using a commercially available extraction kit (for example, DNeasy Tissue Kit from Qiagen or Extragen II from Kainos) is used as a specimen. Alternatively, an unpurified sample processing solution may be used as a specimen for rapid detection.

  In order to amplify such nucleic acids derived from living organisms, a new nucleic acid amplification method recently developed by Natomi et al. That does not require temperature control, which is essential for PCR, is called the LAMP (Loop-mediated Isothermal Amplification) method. This can be achieved by a loop-mediated isothermal amplification method (Patent Publication WO 00/28082 pamphlet). This method anneals its 3 'end to the template nucleotide to serve as the starting point for complementary strand synthesis, and by combining the primer that anneals with the loop formed at this time, isothermal complementary strand synthesis reaction is performed. This is a possible nucleic acid amplification method. In the LAMP method, the 3 'end of the primer always anneals to the region derived from the sample, so the check mechanism based on complementary binding of the base sequence functions repeatedly, resulting in high sensitivity and specificity. Highly efficient nucleic acid amplification reaction is possible.

  Therefore, at least 4 types of primers (2 types of inner primers F and R; IPF and IPR, 2 types of outer primers; OPF and OPR) that recognize 6 regions of the template nucleotide are used. A loop primer which is a primer of the above can be used. The loop primer is a primer having a base sequence complementary to the base sequence of the single-stranded part of the loop structure on the 5 ′ end side of the dumbbell structure. When this primer is used, the starting point of nucleic acid synthesis is increased, and the reaction time can be shortened and the detection sensitivity can be increased (Patent Document WO 02/24902 pamphlet). As long as the base sequence of the loop primer is complementary to the base sequence of the single-stranded portion of the loop structure on the 5 ′ end side of the dumbbell structure described above, it may be selected from the base sequence of the target gene or its complementary strand. The base sequence may be used. Further, one or two kinds of loop primers may be used. In addition, F in each primer is a primer display that complementarily binds to the sense strand of the target base sequence and provides a starting point for synthesis, while R is complementary to the antisense strand of the target base sequence. A primer display that provides a starting point for synthesis. Here, the length of the oligonucleotide used as a primer is 10 bases or more, preferably 15 bases or more, either chemically synthesized or natural, and each primer may be a single oligonucleotide, It may be a mixture of oligonucleotides.

The present inventors obtained the nucleotide sequence of a LAMP primer and its combination capable of rapidly amplifying a specific nucleotide sequence from the nucleotide sequence represented by SEQ ID NO: 1 from the nucleotide sequence derived from the L. monocytogenes iap gene. As a result of intensive research, the following primer sets were selected so that all serotypes could be detected from the nucleotide sequences 721 to 1100 of the nucleotide sequence derived from the iap gene.
IPF: 5'-CAGGTGCAGCTTGTTGAGTAGGCTGGCACAAAATTACTTACAACG-3 '(SEQ ID NO: 10)
OPF: 5'-GGCGCTGGTGTTGATAACAG-3 '(SEQ ID NO: 4)
IPR: 5'-CAAGCAACTACACCTGCACCGCTTTTAACAGCGTGTGTAGTAGC-3 '(SEQ ID NO: 11)
OPR: 5'-CCGTATTTTACGGATAAAGCCC-3 '(SEQ ID NO: 12)
LPF: 5'-GCTACTTTGTCAGTTAAGTATTTACCG-3 '(SEQ ID NO: 13)
LPR: 5'-AGCAGAAACGAAAGAAACTCCAG-3 '(SEQ ID NO: 9)

The enzyme used in nucleic acid synthesis is not particularly limited as long as it is a template-dependent nucleic acid synthase having strand displacement activity. Examples of such enzymes include Bst DNA polymerase (large fragment), Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNA polymerase I, and preferably Bst DNA polymerase (large fragment).

  Known techniques can be applied to the detection of nucleic acid amplification products by the LAMP reaction. For example, a labeled oligonucleotide that specifically recognizes the amplified base sequence, a fluorescent intercalator method (Patent Document JP-A-2001-242169), or a reaction solution after completion of the reaction is directly subjected to agarose gel electrophoresis. Can be easily detected. In agarose gel electrophoresis, a large number of bands with different base lengths are detected in a ladder shape from the LAMP amplification product. In addition, in the LAMP method, a large amount of substrate is consumed by nucleic acid synthesis, and pyrophosphate as a by-product reacts with coexisting magnesium to become magnesium pyrophosphate, and the reaction solution becomes cloudy to the extent that it can be confirmed with the naked eye. Therefore, it is also possible to confirm the white turbidity by using a measuring device capable of optically observing the increase in turbidity after the reaction or during the reaction, for example, a change in absorbance at 400 nm using a normal spectrophotometer ( (Patent Literature International Publication No. 01/83817 pamphlet).

  Various reagents necessary for detecting nucleic acid amplification using the primer of the present invention can be packaged in advance to form a kit. Specifically, various oligonucleotides necessary as a primer or loop primer of the present invention, four types of dNTPs serving as a substrate for nucleic acid synthesis, a template-dependent nucleic acid synthase having a strand displacement activity, and conditions suitable for enzymatic reaction Provided buffers and salts, protective agents for stabilizing enzymes and templates, and reagents necessary for detection of reaction products as necessary are provided as kits.

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

Example 1: Confirmation of detection sensitivity
The detection sensitivity of LAMP method and PCR method was compared.
1. Preparation of samples and reagents
1) L. monocytogenes cultured cells and purified genomic DNA
For the examination, L. monocytogenes of serotype 4b (separated from raw ham) provided by the Tokyo Health and Safety Research Center was used. Purified genomic DNA was prepared from bacterial cells cultured overnight on sheep blood agar medium (manufactured by Eiken Chemical Co., Ltd.) using Qiagen DNeasy Tissue Kit. The copy number concentration was determined from the nucleic acid concentration obtained by absorbance measurement and the known total genome size (2.9 Mbp, GenBank No: AE017262), and used for the following tests. As the template DNA, a purified genomic DNA stock solution was diluted 10-fold with TE buffer (manufactured by Nippon Gene Co., Ltd.) stepwise and heat-treated at 95 ° C. for 5 minutes.

2) Primers used for PCR
Primers used in the PCR method include the upstream primer (SEQ ID NO: 14) and downstream primer (SEQ ID NO: 15) of the Mono A / B set developed by Bubert et al., And the Hly A / B set developed by Johnson et al. An upstream primer (SEQ ID NO: 16) and a downstream primer (SEQ ID NO: 17) were used. The PCR reaction solution composition and reaction conditions faithfully reproduced the conditions described in Non-Patent Documents 1 and 2.
3) Primers used in the LAMP method As primers, IPF (SEQ ID NO: 10), OPF (SEQ ID NO: 4), IPR (SEQ ID NO: 11), OPR (SEQ ID NO: 12), LPF (SEQ ID NO: 13), LPR (SEQ ID NO: 9) Was used.

2. Reaction by nucleic acid amplification method
1) Reaction by PCR
Each PCR reaction of Mono A / B and Hly A / B sets was performed by the method described in the above-mentioned non-patent literature.
<Mono A / B set reaction solution composition and reaction conditions>
Each reagent was prepared as follows per reaction.
・ 10 x Ex Taq buffer (+ Mg) 5.0μL
・ 2.5mM dNTPs mixture 4.0μL
・ DDW (sterilized ultrapure water) 34.5μL
・ 100pmol / μL Mono A (SEQ ID NO: 14) 0.5μL
・ 100pmol / μL Mono B (SEQ ID NO: 15) 0.5μL
・ 5U / μL TaKaRa Ex Taq HS 0.5μL
To the reaction solution, 5.0 μL of the sample at each dilution stage was added, and each PCR reaction was performed with a final reaction volume of 50.0 μL. The temperature cycle conditions for the PCR reaction were 94 ° C for 3 minutes, followed by heat denaturation at 94 ° C for 1 minute, annealing at 56 ° C for 45 seconds, and polymerase extension reaction at 72 ° C for 45 seconds for a total of 30 cycles. After standing for 8 minutes, the reaction was completed. The time required was about 2 hours. After completion of the reaction, 10 μL of the reaction solution was electrophoresed on a 2% agarose gel and stained with ethidium bromide. The molecular size of the specific amplification product in Mono A / B PCR is 371 bp.

<Hly A / B set reaction solution composition and reaction conditions>
・ 10 x Taq buffer (-Mg) 5.0μL
・ 25mM MgCl ₂ 4.0μL
・ 2.5mM dNTPs mixture4.0μL
・ DDW (sterilized ultrapure water) 30.9μL
・ 100pmol / μL 9/5 F (SEQ ID NO: 16) 0.5μL
・ 100pmol / μL 9/5 R (SEQ ID NO: 17) 0.5μL
・ 5 U / μL TaKaRa Ex Taq 0.1 μL
To the reaction solution, 5.0 μL of the sample at each dilution stage was added, and each PCR reaction was performed with a final reaction volume of 50.0 μL. The temperature cycle conditions for the PCR reaction were set at 94 ° C for 3 minutes, then heat-denatured at 94 ° C for 1 minute, annealed at 55 ° C for 1 minute, and polymerase extension reaction at 72 ° C for 1 minute for a total of 30 cycles. The reaction was terminated after standing for a minute. The travel time was approximately 2 hours and 30 minutes. After completion of the reaction, 10 μL of the reaction solution was electrophoresed on a 2% agarose gel and stained with ethidium bromide. The molecular size of the specific amplification product in Hly A / B PCR is 730 bp.

2) Reaction by LAMP method
For amplification by LAMP method, each reagent concentration in 25 μL of the final reaction solution was prepared as follows. In addition, the synthesis | combination of the primer requested the operon biotechnology company, and used what was purified by HPLC (high performance liquid chromatography).
Reaction solution composition20 mM Tris-HCl pH8.8
・ 10 mM KCl
・ 6 mM MgSO ₄
・ 1.0 mM dNTPs
・ 10 mM (NH ₄ ) ₂ SO ₄
・ 0.2M Betaine (SIGMA)
・ 1.0% Tween20
・ 1.6μM IPF (SEQ ID NO: 10)
・ 1.6μM IPR (SEQ ID NO: 11)
・ 0.4μM OPF (SEQ ID NO: 4)
・ 0.4μM OPR (SEQ ID NO: 12)
・ 0.8μM LPF (SEQ ID NO: 13)
・ 0.8μM LPR (SEQ ID NO: 9)
・ 8U Bst DNA polymerase (NEB)

  For the LAMP reaction, add 5 μL of the sample solution of each concentration to 20 μL of the above reagent, and use the real-time turbidimeter LA-200 (Eiken Chemical) as a final reaction solution of 25 μL at 65 ° C. for 60 minutes in a dedicated 0.2 mL tube. Used to detect the reaction in real time. As shown in FIG. 1, the LAMP method was able to detect 6 copies of L. monocytogenes genomic DNA per reaction within 60 minutes. As a comparison, two PCRs were detected up to 6,000 copies per reaction with Mono A / B (Figure 2) and up to 60 copies per reaction with Hly A / B (Figure 3). Compared with the two PCR methods used as controls, the LAMP method was able to detect with higher sensitivity. In addition, the LAMP method was superior in terms of rapidity as compared with the PCR method requiring an amplification reaction of about 2 hours and further electrophoresis analysis.

Example 2: Confirmation of LAMP amplification products
Using genomic DNA purified from cultured cells of L. monocytogenes serotype 4b as a template, the LAMP product amplified with the above primer set was confirmed by electrophoresis and restriction enzyme PstI. The restriction enzyme PstI selectively recognizes and cleaves a part of the target base sequence derived from the iap gene of L. monocytogenes as a template, and does not recognize each base sequence of the primer set. FIG. 4 is a diagram showing the results of electrophoresis. 1 μL of the reaction solution after completion of the LAMP reaction was electrophoresed on a 2% agarose gel and stained with ethidium bromide. As is clear from lane 2 of FIG. 4, a ladder pattern peculiar to the LAMP product was confirmed. In addition, digestion was confirmed in the sample treated with PstI (lane 3). From the above results, it was revealed that the target base sequence was specifically amplified.

Example 3: Specificity of primers (each serotype)
In order to confirm the reactivity of L. monocytogenes of serotypes other than 4b, each serotype strain of 1 / 2a, 1 / 2b, 1 / 2c, 3a, 3b, 4a, 4c, 4d, 4e was also used. Example 1-1) Purified genomic DNA of each serotype strain was prepared by the same operation as described in “L. monocytogenes cultured cells and purified genomic DNA”, and the copy number concentration was determined. For all serotypes including serotype 4b, the number of copies per reaction was fixed at 60 copies, and the reaction was performed by the LAMP method in the same manner as described above. As shown in FIG. 5, 60 copies of genomic DNA of all tested serotypes could be amplified as templates, and all could be detected within 60 minutes simultaneously. Moreover, as shown in FIG. 6, the same ladder pattern peculiar to the LAMP product was confirmed in all the amplification products obtained from all the serotypes tested. Moreover, as shown in FIG. 7, digestion was also confirmed in the samples obtained by treating amplification products obtained from all the serotypes tested with PstI, and it was confirmed that the same digestion pattern was obtained. From the above results, it was revealed that the target nucleotide sequences of L. monocytogenes of all serotypes tested were amplified.

Example 4: Specificity of primers (other Listeria species and other species)
In order to confirm the specificity, Listeria spp. Other than L. monocytogenes were combined with general food poisoning causal bacteria and enteric bacteria, and the reactivity was examined for a total of 128 strains of 66 species. Listeria species other than L. monocytogenes include Listeria innocua, Listeria ivanovii, Listeria grayi, Listeria seeligeri, Listeria welshimeri. It is known that Listeria innocua is genetically highly homologous to L. monocytogenes. In addition to important food poisoning bacteria such as enterohemorrhagic Escherichia coli, Salmonella, Vibrio parahaemolyticus, Shigella, and other common food-causing bacteria and enterobacteria, bacteria such as Pseudomonas aeruginosa and Bacillus subtilis are widely present in the environment. Many species were selected. Add genomic DNA extracted / purified from these cells or heat-treated cultured cells to 60,000-600,000 copies (or CFU) in sufficient excess as a DNA sample and check if they react. went. As shown in Table 1, all the 128 strains of 66 strains tested did not react at all, confirming good specificity. As described above, it was revealed that the designed primer set specifically recognizes L. monocytogenes, including related bacterial species, and does not show cross-reactivity with other bacterial species.

According to the present invention, an oligonucleotide primer that selectively hybridizes with a base sequence specific to L. monocytogenes is prepared, and by amplifying the base sequence specific to L. monocytogenes by the LAMP method, L. monocytogenes Can be detected specifically, sensitively and rapidly.

The detection sensitivity of LAMP method by real-time turbidity method is shown. The horizontal axis represents time (minutes), and the vertical axis represents absorbance at 400 nm (turbidity). Electrophoresis diagram showing detection sensitivity by Mono A / B PCR method. From left: Lane 1: Marker (TaKaRa 100bp Ladder Marker), Lane 2: 600,000 copies, Lane 3: 60,000 copies, Lane 4: 6,000 copies, Lane 5: 600 copies, Lane 6: 60 copies, Lane 7: 6 copies, Lane 8: No mold Electrophoretic diagram showing detection sensitivity by Hly A / B PCR method. The same reference numerals as those in FIG. Electrophoretic diagram of digested product with LAMP amplification product and restriction enzyme PstI. From left: Lane 1: Marker (TaKaRa 100bp Ladder Marker), Lane 2: LAMP product (PstI undigested), Lane 3: LAMP product (PstI digested) Specificity of LAMP method by real-time turbidity method is shown. The horizontal axis represents time (minutes), and the vertical axis represents absorbance at 400 nm (turbidity). Electrophoretic diagram of each serotype LAMP amplification product (PstI undigested). From left to right, lane 1: marker (TaKaRa 100bp Ladder Marker), lane 2: serotype 1 / 2a-derived LAMP product, lane 3: serotype 1 / 2b-derived LAMP product, lane 4: serotype 1 / 2c-derived LAMP product, lane 5: LAMP product derived from serotype 3a, lane 6: LAMP product derived from serotype 3b, lane 7: LAMP product derived from serotype 4a, lane 8: LAMP product derived from serotype 4b, lane 9: LAMP product derived from serotype 4c, lane 10: LAMP product derived from serotype 4d, Lane 11: LAMP product derived from serotype 4e, Lane 12: Marker (TaKaRa 100bp Ladder Marker) The electropherogram (PstI digestion) of the digested product of each serotype LAMP amplification product with the restriction enzyme PstI is the same as the code in FIG.

Claims (8)

Oligonucleotide primer designed to specifically amplify and detect Listeria monocytogenes serotypes, derived from the iap gene (invasion-related gene) shown in SEQ ID NO: 1. An oligonucleotide primer designed from an arbitrary base sequence selected from base sequences 721 to 1100 of the base sequence, or a base sequence complementary thereto. Selected from the nucleotide sequences shown in SEQ ID NOs: 2 to 9 selected from the nucleotide sequences derived from the iap gene (invasive gene) of Listeria monocytogenes (isteria monocytogenes), or a complementary nucleotide sequence thereto, 2. The oligonucleotide primer according to claim 1, comprising at least 15 consecutive bases. The base sequence region of F3c, F2c, F1c from the 3 ′ end side of the target nucleic acid of the Listeria monocytogenes iap gene (invasive gene) is called R3, R2, R1 from the 5 ′ end side. When a base sequence region is selected and each complementary base sequence is F3, F2, F1, and R3c, R2c, R1c, at least one base sequence selected from the following (a) to (d) The oligonucleotide primer according to claim 1, which comprises:
(a) A base sequence having the F2 region of the target nucleic acid on the 3 ′ end side and the F1c region of the target nucleic acid on the 5 ′ end side.
(b) A base sequence having the F3 region of the target nucleic acid.
(c) A base sequence having the R2 region of the target nucleic acid on the 3 ′ end side and the R1c region of the target nucleic acid on the 5 ′ end side.
(d) A base sequence having the R3 region of the target nucleic acid. It can amplify the base sequence of iap gene (invasive gene) specific to Listeria monocytogenes, and is selected from the following (a) and / or (b) from 5 'end to 3' end The oligonucleotide primer according to claim 1 or 2, which comprises a base sequence.
(a) 5 '-(base sequence complementary to the base sequence of SEQ ID NO: 2)-(any base sequence having 0 to 50 bases)-(base sequence of SEQ ID NO: 3) -3'
(b) 5 '-(base sequence of SEQ ID NO: 6)-(arbitrary base sequence having 0 to 50 bases)-(base sequence complementary to the base sequence of SEQ ID NO: 7) -3' A listeria monosite characterized by performing an amplification reaction of a target nucleic acid region of an iap gene (invasion-related gene) of Listeria monocytogenes using the oligonucleotide primer according to claims 1 to 4. Genesis (Listeria monocytogenes) detection method. 6. The Listeria monocytogenes (Listeria monocytogenes) iap gene (invasive gene) amplification reaction of the target nucleic acid region is LAMP method according to claim 5, Detection method. By detecting amplification of a target nucleic acid region of an iap gene (invasion-related gene) of Listeria monocytogenes using the oligonucleotide primers according to claims 1 to 4, Listeria monocytogenes (Listeria monocytogenes) A method for detecting Listeria monocytogenes, comprising detecting the presence or absence of monocytogenes). In the detection method of Listeria monocytogenes (Listeria monocytogenes), the kit characterized by including the oligonucleotide primer of Claims 1-4.