JP5818587B2 - Method for detecting C. perfringens and kit for C. perfringens detection - Google Patents

Description

  The present invention relates to a rapid and simple detection method and detection kit for detecting Clostridium perfringens, and more specifically, amplifying using a transcription product derived from the cpe gene of Clostridium perfringens as a template, and detecting the amplified product And a kit for detection.

Clostridium perfringens, an obligate anaerobic gram-positive bacillus, is widely distributed in the intestinal tract of humans and animals, or in soils such as sewage, rivers, seas, and arable land, and has a high contamination rate of foods. Food poisoning caused by C. perfringens is caused by enterotoxin, a protein with a molecular weight of about 35,000. Generally, enterotoxin is easily heat-resistant, and it is weak against acids and many of them are inactivated by gastric juice. It is an infectious type of food poisoning that occurs when a large amount of live enterotoxin-producing Clostridium perfringens is ingested.

By the way, food poisoning occurs when food is contaminated with about 10 ⁶ cfu enterotoxin-producing Clostridium perfringens per gram of food. As a method for detecting Clostridium perfringens, serological tests, enterotoxin production tests, biochemical property tests, and the like have been carried out after enrichment culture and bacterial isolation culture under anaerobic conditions. These detection methods require a very long time of about 3-5 days and a great deal of labor before obtaining results.

In addition, the nucleotide sequence of the enterotoxin gene (cpe) of Clostridium perfringens has already been released, and a method for detecting enterotoxin-producing Clostridium perfringens using genetic techniques has been developed. Use of genetic techniques, particularly nucleic acid amplification methods, can be a highly sensitive test method, and thus is highly beneficial for the detection of infectious bacteria including food poisoning bacteria.

One of these nucleic acid amplification methods is PCR (Polymerase
There is an inspection method using the Chain Reaction method, and suitable primers have been discovered. This method requires not only a special device that can raise and lower the temperature at intervals of 1 minute because the cycle from thermal denaturation of the template to annealing of the template and primer to the primer extension reaction is one cycle. The time required for the inspection is about 3 hours.

Further, as described in, for example, Japanese Patent No. 3419299, when the PCR product is electrophoresed on an agarose gel, it takes about 30 minutes. Carcinogenicity is required for the final detection of the PCR product. It is necessary to use a substance such as ethidium bromide.

  Furthermore, as another one, a method using a LAMP (Loop-Mediated Isothermal Amplification) method (see JP 2007-159533 A) has been developed, and a primer suitable for the method has been discovered. This method is a method in which template and primer annealing and primer extension reaction are carried out at an isothermal temperature of 65 ° C., and pyrophosphoric acid produced during the primer extension reaction reacts with magnesium in the reaction solution to cause white turbidity. Is used to know that the primer extension reaction has occurred due to the production of turbidity, and the amplification time can be as short as about 1 hour compared with the PCR method described above. .

  In this case, whether or not the amplification reaction has occurred is to know that the primer extension reaction has occurred due to the production of turbidity associated with the reaction turbidity of pyrophosphate produced during the primer extension reaction. However, when any sample is used as a sample, it is necessary to consider the possibility of non-specific amplification. However, in the detection method using magnesium pyrophosphate, even if a non-specific amplification reaction occurs, it becomes cloudy and is false positive. I can't wipe out the concerns.

  Fluorescently labeled oligonucleotide for detection that does not function as a primer in amplification reactions typified by TaqMan probes and Beacon probes used in real-time detection systems such as quantitative PCR (Japanese Patent No. 3419299), or benzo-1 By using a chemiluminescent compound having a 3,3-thiazole skeleton (see Japanese Patent Application Laid-Open No. 7-89959), it is possible to avoid detection of non-specific amplification products by the sequence-specific hybrid reaction. is there.

  However, if fluorescent probes are used, a special device for detecting those fluorescent probes is additionally required. Traditionally, there is a Southern hybridization method or a Northern hybridization method to detect using a sequence-specific hybrid reaction without using a fluorescent probe, but it takes a lot of time and labor to detect. Not realistic.

Japanese Patent No. 3419299 JP 2007-159533 A JP-A-7-89959

  In order to prevent the occurrence of food poisoning in a wide variety of facilities, it is not possible to accurately test the various foods as samples at each site before ingesting food contaminated with bacteria. That is, the accurate inspection must be quick and simple. In addition, inspection methods using carcinogenic substances should be avoided if testing at the food site is assumed. In order to ensure effectiveness in a wide variety of facilities, special equipment should be unnecessary.

  In general, when testing through enrichment culture or isolation culture of Clostridium perfringens, which is a causative agent of food poisoning, usually a very long time of about 3-5 days and a great deal of labor are spent. It is virtually impossible to do the inspection before. Therefore, if a method for detecting enterotoxin-producing Clostridium perfringens using a genetic technique is used, it is possible to carry out a test during the day.

  However, in the case of a test method using primers optimized for the PCR method, special equipment is required and it is not effective in implementation in a wide variety of facilities. Moreover, the time until detection is long, and it is inaccurate because it is not in time for the inspection before food intake. Furthermore, the use of carcinogenic substances at the detection stage cannot be envisaged for inspection at food sites.

  In the case of a test method using primers optimized for the LAMP method, it can be determined whether or not an amplification reaction has occurred, but in the detection method using magnesium pyrophosphate, whether the obtained amplification product is derived from the target nucleic acid or not. Cannot determine whether it is derived from specific amplification. Moreover, about 1 hour is necessary for amplification time, and lacks quickness as a test before food intake.

  An object of the present invention is to provide a method for accurately detecting enterotoxin-producing Clostridium perfringens using a system that avoids the detection of non-specific amplification products by a quick and simple operation without using a special instrument, and the method used for the same. It is to provide a detection kit.

In order to solve the above-mentioned problems as a whole, the present inventors, first, enterotoxin gene, which is a toxin of Clostridium perfringens, in particular, mRNA, which is a transcription product thereof, as a target for capturing live bacteria of enterotoxin-producing Clostridium perfringens that cause food poisoning. did. NASBA (Nucleic Acid Sequence-based) as a means of detecting C. perfringens
In the case of using the (Amplification) method, it is not necessary to use a special device, and the target nucleic acid can be easily amplified in one step using RNA as a template.

  This NASBA method is a nucleic acid amplification method. NASBA (Nucleic Acid Sequence-based Amplification) method is used as a template, using three types of enzymes (reverse transcriptase, T7 RNA polymerase, RNase H) as a template. On the other hand, a single-stranded RNA having a reverse strand can be suitably produced as an amplification product. In this NASBA method, the amplification reaction proceeds isothermally, so if there is a heat block, the amplification reaction can be carried out, and no special equipment is required.

  Furthermore, because the amplification product of the NASBA method is a single-stranded RNA, when a single-stranded oligonucleotide that is not used as a primer for amplification reaction is used as a probe for sequence-specific hybrid binding, a double-stranded nucleic acid is amplified. Compared to the conventional amplification method, the amplification product can be detected not only by the hybrid binding reaction between the probe and the amplification product under the condition that no competing strand exists, but also the step of denaturing the double strand into a single strand can be omitted. There are advantages.

  Furthermore, examples of the NASBA product detection method include a plate method using a hybrid reaction using a sequence-specific probe and a nucleic acid chromatography method. In particular, in the nucleic acid chromatography method, a target-specific NASBA product that flows using capillary action is sequence-specifically sandwiched between an oligonucleotide probe immobilized on a specific position on a membrane strip and an oligonucleotide probe labeled with colored particles. It can be said that it is a system that can be easily detected without preparing and using any other special equipment because it is captured by hybridization and is visible through colored particles only when the target NASBA product is captured by both probes.

  The plate method and nucleic acid chromatography method using the hybrid reaction using the above sequence-specific probe are very suitable for detecting the target NASBA product, and even a very small amount of target can be detected in a short time. It has characteristics.

  In addition, the detection of amplification products by amplification of the target NASBA product has a detection principle that does not detect non-specific amplification products, is very easy to operate during detection, has a short detection time, and uses special equipment. It was decided to use a nucleic acid chromatography method that could be detected without any problems.

  Furthermore, the present inventors can specifically hybridize with a sequence that is in the amplification region and is different from the primer used in the amplification reaction, and further have characteristics suitable for a detection method using a nucleic acid chromatography method. Completion of the present invention to find two types of probes and accurately detect enterotoxin-producing Clostridium perfringens using a system that avoids the detection of non-specific amplification products in a quick and simple operation without using a special instrument. It came to.

That is, the first invention is a method for detecting Clostridium perfringens having Clostridium perfringens having an enterotoxin (cpe) gene, which comprises subjecting a target nucleic acid arbitrarily extracted from a specimen sample to a NASBA method. By using a primer pair consisting of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 and the RNA polymerase promoter sequence added to the 5 ′ end side thereof A first step of amplifying as a single-stranded nucleic acid; a first probe comprising a nucleotide sequence represented by SEQ ID NO: 3; and a nucleotide represented by SEQ ID NO: 4 by subjecting the amplification product of the first step to nucleic acid chromatography. Detect using a probe pair consisting of a second probe consisting of a sequence And 2 step method for detecting Clostridium perfringens, characterized in that Ru comprising a.

The second invention relates to a method for detecting Clostridium perfringens according to the first invention, wherein the RNA polymerase promoter sequence is the T7 RNA polymerase promoter sequence shown in SEQ ID NO: 14 .

Furthermore, the third invention is the first or second invention, wherein one of the first probe and the second probe is carried on a development support, and the other is bound to a labeled polymer carrier. The development support is modified with a carboxyl group, the labeled polymer carrier is modified with a carboxyl group, and the first probe is 3 ′ of the nucleotide sequence shown in SEQ ID NO: 3. Alternatively, an amino group is added to the 5 ′ end side, and the second probe has an amino group added to the 3 ′ or 5 ′ end side of the nucleotide sequence shown in SEQ ID NO: 4. The present invention relates to a method for detecting Clostridium perfringens.

Furthermore , the fourth invention is a kit for the detection of Clostridium perfringens having Clostridium perfringens having an enterotoxin (cpe) gene, the forward primer comprising the nucleotide sequence shown in SEQ ID NO: 1, A target nucleic acid consisting of a nucleotide sequence shown in SEQ ID NO: 2 and a reverse primer consisting of an RNA polymerase promoter sequence added to the 5 ′ end thereof, and a target nucleic acid arbitrarily extracted from a sample sample is converted into a single-stranded nucleic acid by the NASBA method. As a primer pair, a first probe consisting of the nucleotide sequence shown in SEQ ID NO: 3, and a second probe consisting of the nucleotide sequence shown in SEQ ID NO: 4. A probe pair to be detected by chromatography. It relates to a kit for Clostridium perfringens detection and butterflies.

According to the present invention, the enterotoxin-producing Clostridium perfringens can be specifically detected without using a special instrument. In addition, cpe RNA can be detected with high sensitivity in a very short time, and enterotoxin-producing Clostridium perfringens of about 10 ⁶ cfu per gram of food, which is the standard for developing food poisoning, can be detected quickly and reliably from food samples. It was possible.

A table showing the results of examination when selecting the optimal probe pair for nucleic acid chromatography detection. A table showing the results of examination when selecting the optimal primer pair for the NASBA amplification method. A table showing the results of examining the performance of the inspection method that combines the primer pair suitable for the NASBA method and the probe pair suitable for the nucleic acid chromatograph, particularly the speed of time to reach detection sensitivity. A table showing the results of examining the performance, especially the specificity, of a test method that combines a primer pair suitable for NASBA and a probe pair suitable for nucleic acid chromatography. Nucleic acid chromatographic measurement using nucleotide sequences that indicate the performance of a test method that combines a primer pair suitable for NASBA and a probe pair suitable for nucleic acid chromatography, especially that it can detect enterotoxin-producing Clostridium perfringens in food samples Test piece. Nucleic acid chromatographic measurement using nucleotide sequences that indicate the performance of a test method that combines a primer pair suitable for NASBA and a probe pair suitable for nucleic acid chromatograph, especially that it can detect enterotoxin-producing Clostridium perfringens in plasma preparations Test piece.

[Step (a)] — Amplification of target nucleic acid extracted from the sample Target nucleic acid arbitrarily extracted from the sample during the detection of food poisoning causal organisms that target the C. perfringens having the enterotoxin (cpe) gene As a target nucleic acid specific to the enterotoxin-producing Clostridium perfringens, a nucleic acid in a region contained in the cpe gene, in particular, mRNA which is a transcription product thereof can be exemplified.

  Moreover, from the forward primer which consists of the nucleotide sequence shown by sequence number 1 used at the time of amplification of a target nucleic acid by the step (a), the nucleotide sequence shown by sequence number 2, and the RNA polymerase promoter sequence added to the 5 'terminal side The reverse primer pair is a primer pair that can amplify the cpe gene, in particular, its transcription product, mRNA, as a target nucleic acid, and is a nucleotide sequence shown in SEQ ID NOs: 1 and 2.

  More specifically, a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 (5'-GAATAACTATAGGAGAACAA-3 '), the nucleotide sequence shown in SEQ ID NO: 2 (5'-CACATCTTTCACCAGTTTCA-3') and 5 ' A combination of primer pairs of reverse primers consisting of an RNA polymerase promoter sequence added to the terminal side is used.

  Examples of the RNA polymerase promoter sequence described above include T7 RNA polymerase promoter sequence, T3 RNA polymerase promoter sequence, SP6 RNA polymerase promoter sequence, etc. Among them, the T7 RNA polymerase promoter sequence exhibits high RNA amplification efficiency. It is preferable at the point which can do.

  An example of the T7 RNA polymerase promoter sequence is 5'-AATTCTAATACGACTCACTATAGGGAG-3 '(SEQ ID NO: 14). In the nucleotide sequences shown in SEQ ID NOs: 1 and 2, one base or several bases (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) are missing. Even if it is lost, substituted or added, any primer can be used as long as it can amplify the target nucleic acid.

  The forward primer and reverse primer can be synthesized by a conventional method using a DNA synthesizer or the like. As described above, these primer pairs are the nucleotide sequences shown in SEQ ID NOs: 1 and 2, which can amplify the cpe gene, in particular, its transcription product, mRNA, as a target nucleic acid.

  That is, more specifically, a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 (5'-GAATAACTATAGGAGAACAA-3 '), the nucleotide sequence shown in SEQ ID NO: 2 (5'-CACATCTTTCACCAGTTTCA-3') and its A combination of primer pairs of reverse primers consisting of an RNA polymerase promoter sequence added to the 5 ′ end side is used.

  Examples of the RNA polymerase promoter sequence include a T7 RNA polymerase promoter sequence, a T3 RNA polymerase promoter sequence, an SP6 RNA polymerase promoter sequence, and the like. Among them, the T7 RNA polymerase promoter sequence is preferable in terms of high RNA amplification efficiency. . In this case, examples of the promoter sequence of T7 RNA polymerase include 5'-AATTCTAATACGACTCACTATAGGGAG-3 '(SEQ ID NO: 14).

  In the nucleotide sequences shown in SEQ ID NOs: 1 and 2, one base or several bases (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) are missing. Even if it is lost, substituted or added, any primer can be used as long as it can amplify the target nucleic acid. The forward primer and reverse primer can be synthesized by a conventional method using a DNA synthesizer or the like.

Examples of test samples that may contain nucleic acids of food poisoning bacteria to be detected in the step (a) include feces, urine, blood, tissue homogenate, food materials and microorganisms contained in them, and cultures of such microorganisms. Examples include cell lysates. As a method for extracting a nucleic acid from such a test sample, in addition to a known method such as an RNA extraction method using guanidine thiocyanate or a nucleic acid extraction method using EDTA-SDS-phenol-ethanol, Extractagen II (manufactured by Tosoh Corporation) ) And Mora-Extract (Advanced
Commercial products such as Microorganism Research) can also be used.

  When the target nucleic acid is a target RNA, the amplification method of the target nucleic acid used in the above step (a) includes NASBA method, transcription-mediated amplification method (TMA method), RNA amplification by concerted reaction of transcriptase and reverse transcriptase, There are known RNA amplification methods such as a detection method (Transcription Reverse Transcription Concerted Reaction, TRC method).

  For example, in the case of the NASBA method, the TMA method, and the TRC method, there is a common point that an amplification product is obtained by using a transcription reaction using RNA polymerase as a principle, but there is also a difference. In the case of the TRC method, unlike the NASBA method and the TMA method, the amplification reaction is not started in principle only by adding the forward primer and the reverse primer to the amplification reagent. This is because a target mRNA cleavage step with a Caesar probe is necessary as a pre-stage of the nucleic acid amplification step.

  That is, the TRC method can be said to be an amplification method having a large number of steps in the amplification reaction and having complexity compared to the NASBA method and the TMA method. The NASBA method and the TMA method have almost the same amplification principle, but the number of enzymes used in the amplification reaction is two types of reverse transcriptase and RNA polymerase in the TMA method, whereas the reverse transcription is performed in the NASBA method. There are three types, enzyme, RNA polymerase and RNaseH, and this is the difference between the two methods. In the TMA method, the RNaseH activity contained in reverse transcriptase is used to carry out a reaction that degrades the double-stranded RNA strand of DNA and RNA.

  In other words, the TMA method uses a single enzyme (reverse transcriptase) to carry out the reverse transcription reaction that synthesizes cDNA using RNA as a template and the reaction that breaks down the double-stranded RNA strand of DNA and RNA. In contrast, in the NASBA method, these two reactions (reverse transcription reaction and reaction that degrades the double-stranded RNA strand of DNA and RNA) do not compete with one enzyme, and a dedicated enzyme is used for each reaction. It has a feature of performing an amplification reaction efficiently. Considering these differences in nucleic acid amplification methods, among others, the NASBA method, for example, the NASBA method described in Japanese Patent No. 2650159 can be advantageously used.

  The NASBA method described above is a method of amplifying a target RNA in a sample via RNA polymerase or reverse transcriptase. For example, in the NASBA method described in Japanese Patent No. 2650159, the following steps are performed. It is disclosed that.

(A) a step in which a target nucleic acid (first template) and a reverse primer having an RNA polymerase promoter sequence are hybridized in a sequence-specific manner;
(B) a step of synthesizing DNA from the 3 ′ end of the reverse primer using a target nucleic acid as a template by reverse transcriptase to form a double-stranded nucleic acid consisting of an RNA strand and a DNA strand;
(C) a step of degrading only RNA of RNaseH from double-stranded nucleic acid consisting of RNA strand and DNA strand to form single-stranded DNA;
(D) Next, the step of hybridizing the single-stranded DNA (second template) and the forward primer in a sequence-specific manner;
(E) Using reverse transcriptase to synthesize DNA from the 3 ′ end of the forward primer using the single-stranded DNA as a template (second template) to form double-stranded DNA having an RNA polymerase promoter ;
(F) a step of synthesizing a single-stranded RNA (third template) from the double-stranded DNA by RNA polymerase;
(G) a step of hybridizing the single-stranded RNA (third template) and a forward primer in a sequence-specific manner;
(H) Using reverse transcriptase, DNA is synthesized from the 3 ′ end of the forward primer using the single-stranded RNA (third template) as a template to form a double-stranded nucleic acid consisting of an RNA strand and a DNA strand. Process;
(I) A step of forming single-stranded DNA (fourth template) by degrading only RNA of double-stranded nucleic acid consisting of RNA strand and DNA strand with RNaseH;
(J) Next, the step of hybridizing the single-stranded DNA (fourth template) and the reverse primer in a sequence-specific manner;
(K) A step of synthesizing DNA from the 3 ′ end of the reverse primer using reverse transcriptase from the single-stranded DNA as a template (fourth template) to form a double-stranded DNA having an RNA polymerase promoter;
(L) a step of synthesizing a single-stranded RNA (third template) from the double-stranded DNA by RNA polymerase;
(M) Repeat steps (G) to (L).

[Step (b)] — A step of detecting an amplification product using a nucleic acid chromatography method The amplification product of the step (a) amplified by the NASBA method is carried on a development support using a nucleic acid chromatography method. The second probe consisting of the nucleotide sequence shown in SEQ ID NO: 3 and the first probe consisting of the nucleotide sequence shown in SEQ ID NO: 4 bound to the labeled polymer carrier are detected by hybridization.

  The probe pair used in the step (b) is a probe pair that is complementary to the amplification product in the step (a) and selected from the nucleotide sequence excluding the nucleotide sequence used in the primer. And the nucleotide sequence shown in 4. More specifically, it consists of a first probe consisting of the nucleotide sequence shown in SEQ ID NO: 3 (5'-CATGGTAATATCTCTGATGATGGATC-3 ') and a nucleotide sequence shown in SEQ ID NO: 4 (5'-TAACAGGAATATGGCTTAGTAAAAC-3'). A second probe pair combination is used.

  In the sequence listing, the DNA sequence is shown for convenience, but it may be a probe pair consisting of a nucleotide sequence of DNA or a probe pair consisting of a nucleotide sequence of RNA. However, a probe pair consisting of a nucleotide sequence of DNA is preferable because of its excellent stability as a probe.

  Which of the probe pairs is used as the first probe or the second probe can be appropriately selected and determined. In the nucleotide sequence shown in SEQ ID NO: 3 or 4, one base or several bases (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, further preferably 1) bases. Can be used as a probe in the present invention as long as a specific target nucleic acid can be detected even if is deleted, substituted or added.

  As a method for solid-phase bonding the first probe to a labeled polymer carrier, a DNA having the nucleotide sequence shown in SEQ ID NO: 3 having an additional group introduced at the 5 ′ or 3 ′ end is labeled with an additional group introduced Examples thereof include a method of binding to a polymer carrier. The end of the DNA into which the additional group is introduced is preferably a 5 'end. In addition, examples of the additional group include an amino group, a carboxyl group, a hydroxyl group, and a thiol group. For example, when the labeled polymer carrier is modified with a carboxyl group, an amino group is preferable.

  Examples of the polymer carrier in the labeled polymer carrier include carboxymethyl cellulose (CMC), hydrophilic resins such as polyacrylate having a carboxyl group, acrylic latex, polyester latex, polystyrene latex, polyurethane latex, and polyacetic acid. Examples of the latex include vinyl latex, SBR resin, NBR resin, polyamide latex, and carboxy-modified polystyrene latex.

  In this case, when an amino group is introduced into the nucleotide sequence shown in SEQ ID NO: 3, the first probe is attached to the labeled polymer carrier by a reaction that forms a covalent bond via the amino group and the carboxyalkyl group. Since it becomes easy to bind, a carboxy-modified polystyrene latex in which a carboxyl group is introduced into a polystyrene latex is preferable. Specifically, a carboxyl group-containing polystyrene latex (solid content 4% (w / w)) ( Duke Scientific) or carboxyl group-containing polystyrene latex (solid content 10% (w / w)) (Bangs) can be used.

As a specific method for preparing the first probe-bound labeled polymer carrier to which the first probe is bound, for example, an amino group is added to the 5 ′ end of the nucleotide sequence shown in SEQ ID NO: 3. The introduced first probe, carboxyl group-containing polystyrene latex (Bangs), and water-soluble carbodiimide are mixed in 50 mM MES (2-Morpholinoethanesulfonic acid, monohydrate) (Dojindo Laboratories) buffer solution, latex After binding the carboxyl group in the amino group and the amino group of the first probe, monoethanolamine (manufactured by Wako Pure Chemical Industries, Ltd.) was added and reacted, and the reaction solution was centrifuged and the supernatant was removed. HEPES (2- [4- (2-Hydroxyethyl) -1- piperazinyl] containing a nonionic surfactant in the resulting precipitate
ethanesulfonic acid) (manufactured by Saikyo Kasei Co., Ltd.) and a method of washing and resuspending with a buffer solution.

  Further, the size of the particle diameter of the polymer carrier can be appropriately selected, but is preferably selected from particle diameters smaller than the membrane pore diameter. For example, the particle diameter of 500 nm or less can be selected. Further, as the above-described labeled polymer carrier, a polymer carrier exhibiting a color distinguishable from the color of the development support may be used, or a polymer carrier colored with a pigment or the like may be used.

  Furthermore, examples of the method for preparing the support carrying and carrying the second probe include a method in which the second probe is immobilized on a predetermined position on the development support. Specific examples include a method in which a probe having the nucleotide sequence shown in SEQ ID NO: 4 having an additional group introduced at the 5 'or 3' end is immobilized on a development support.

  The end of the DNA into which the additional group is introduced is preferably the 3 'end. Examples of the additional group include an amino group, a carboxyl group, a hydroxyl group, and a thiol group. For example, when the development support is modified with a carboxyl group, an amino group is preferable. Regardless of the presence or absence of addition, it is possible to produce the second probe using a known method such as a chemical synthesis method.

  Examples of the development support include nylon membrane derivatives such as nylon membranes and carboxyl group-modified nylon membranes, cellulose membrane derivatives such as cellulose membranes and nitrocellulose membranes, and the like. When an amino group is introduced, the second probe can be immobilized on a predetermined position on the development support by a reaction that causes a covalent bond via the amino group and the carboxyalkyl group. Since it becomes easy, a carboxyl group-modified nylon membrane is more preferable.

  As a specific method for preparing the second probe-carrying development support, for example, a carboxyl group-modified nylon membrane is treated with water-soluble carbodiimide, washed with deionized water and activated, and the activated carboxyl group-modified nylon is used. A solid phase is appropriately immobilized on a membrane at a predetermined position so that nucleotides having the second probe sequence can be identified.

  The air-dried state is air-dried for about 15 minutes, and then the carboxyl group-modified nylon membrane on which the nucleotide having the second probe sequence is immobilized is treated with a Tris base buffer to eliminate the active group. An example is a method of preparing a membrane on which is immobilized by washing with deionized water. The form of immobilizing the nucleotide having the second probe sequence is not particularly limited, and may be a line or a circular spot.

  As a method for detecting the amplification product in the above step (b), the amplified single-stranded nucleic acid is hybridized to the second probe and the labeled polymer carrier-bound first probe that are immobilized on a development support. The method is not particularly limited as long as it is a method of detecting by soybean, but it is preferable to hold the first probe-bound labeled polymer carrier in which the first probe is bound to the labeled polymer carrier in advance in the holding unit. An absorption pad made by ADVANTEC can be preferably exemplified.

  Such a holding part composed of a development pad holding the first probe-bound labeled polymer carrier or the like is sequentially connected to the other end of the second probe-carrying development support so that it can be advantageously used in the detection method of the present invention. It can be used as a test piece for nucleic acid chromatography. In addition, as a manufacturing method of the above-mentioned holding | maintenance part, the method of apply | coating the labeling polymer support | carrier which the nucleotide which has a 1st probe arrangement | sequence has couple | bonded to a holding | maintenance part, and can dry can be illustrated.

In the step (b), the amplification product in the step (a) described above is detected by hybridizing to the second probe supported on the development support and the first probe bound to the labeled polymer carrier, for example, By immersing the nucleic acid chromatograph test piece in a solution containing an amplification product, the first probe-bound labeled polymer carrier is leached from the holding part to the development support, and the first probe on the development support Can be captured by sandwich hybridization of the amplification product at the predetermined position.

[Step (c)] — The step of determining and evaluating the detection image, followed by the labeled polymer carrier to which the nucleotide having the first probe sequence is bound, the nucleotide having the second probe sequence being the solid phase on the development support The presence or absence of a colored line or colored spot appearing at a predetermined position by accumulating at a predetermined position that has reached the position that has been converted to the presence or absence of Clostridium perfringens by determining directly or with a fluorescence visualization device as a detection image, The amount of food poisoning to be detected (enterotoxin-producing Clostridium perfringens) can be detected by such evaluation. In addition, it is preferable to make the said determination into visual determination from the point of simplicity.

[Certificate for Clostridium perfringens]
The kit for detecting enterotoxin-producing Clostridium perfringens of the present invention can be used for the detection of the enterotoxin-producing Clostridium perfringens of the present invention, and is a target nucleic acid specific for enterotoxin-producing Clostridium perfringens arbitrarily extracted from a sample. A forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a reverse primer pair consisting of the nucleotide sequence shown in SEQ ID NO: 2 and an RNA polymerase promoter sequence added to the 5 ′ end thereof. It is a thing.

  In addition to the primer pair, the kit may include various reagents used in the RNA amplification method in the step (a) described above and various reagents used in the amplification product detection method in the step (b). Good.

  Or an amplification set that amplifies as a single-stranded nucleic acid using a primer from a target nucleic acid specific to enterotoxin-producing Clostridium perfringens arbitrarily extracted from the sample; a nucleotide sequence complementary to the amplification product, eg, SEQ ID NO: 3 A first probe-bound labeled polymer carrier in which a first probe comprising the nucleotide sequence shown is bound to a labeled polymer carrier; a second probe paired with the first probe, such as the nucleotide shown in SEQ ID NO: 4 A kit including a second probe-carrying development support on which a second probe having an array is solid-phased may be used, and a kit including the amplification set and the test piece for nucleic acid chromatography is also suitable. It can be illustrated.

Such a kit can be suitably used for detecting the enterotoxin-producing Clostridium perfringens of the present invention.

[ Production of synthetic RNA ]
In order to confirm the primer and probe performance, a synthetic RNA of the cpe gene region was prepared by DNA cloning using a plasmid vector. Specifically, first, a DNA fragment of the target gene cpe of Clostridium perfringens was inserted into a plasmid vector, and then transformed into E. coli serving as a host to prepare a recombinant in which the target gene region was cloned. After extracting the recombinant plasmid from E. coli, RNA was synthesized using MEGAscript Kit (Ambion) according to the protocol. The copy number of the synthetic RNA was calculated from the absorbance at a wavelength of 260 nm using a spectrophotometer.

[ Preparation of probe-bound labeled polymer carrier ]
In order to bind the oligonucleotide probe and the carboxyl group-containing polystyrene latex, a 5 ′ terminal amino group-introduced oligonucleotide having an amino group introduced at the 5 ′ terminal of the probe sequence was synthesized by a DNA synthesizer. A nucleotide having a probe sequence of SEQ ID NOs: 3 and 10-13, a carboxyl group-containing polystyrene latex (manufactured by Bangs), a water-soluble carbodiimide and 50 mM MES (2-Morpholinoethanesulfonic).
acid, monohydrate) (manufactured by Dojindo Laboratories) in a buffer solution to combine the carboxyl group in the latex with the amino group of the probe, then add monoethanolamine (manufactured by Wako Pure Chemical Industries, Ltd.) Further reaction.

  The supernatant is removed after centrifugation of the above reaction solution, and HEPES (2- [4- (2-Hydroxyethyl) -1- piperazinyl] ethanesulfonic acid) containing a nonionic surfactant in the resulting precipitate (Saikyo Kasei Co., Ltd.) (Manufactured) Washed and resuspended in a buffer solution, a labeled polymer carrier to which a nucleotide having a probe sequence was bound was prepared and stored at 4 ° C. until use.

[ Preparation of a development support in which an oligonucleotide having a probe sequence is immobilized on a predetermined position where it can be identified ]
In order to bind the oligonucleotide probe and a carboxyl group-modified nylon membrane (Pole), a 3 ′ terminal amino group-introduced oligonucleotide having an amino group introduced at the 3 ′ end of the probe sequence was synthesized by a DNA synthesizer. The membrane was treated with water-soluble carbodiimide, washed with deionized water and activated.

  One kind of the above 3'-terminal amino group-introduced oligonucleotides (SEQ ID NOs: 3, 4 and 11 to 13) was bound to the activated carboxyl group-modified nylon membrane and air-dried for 15 minutes. After treating the air-dried membrane with Tris base buffer to remove the remaining active groups, the membrane is washed with deionized water and air-dried again, whereby the oligonucleotide having the probe sequence can be identified. A development support solidified in a line at the position could be produced.

[ Preparation of nucleic acid chromatographic test pieces ]
Furthermore, the test piece for nucleic acid chromatograph used for this invention was produced as follows. Specifically, a labeled polymer carrier to which a nucleotide having a probe sequence is bound is dissolved in a buffer solution, applied to a development pad (manufactured by ADVANTEC), and then dried to form a holding part. Furthermore, a holding portion was connected to one end of the development support at a portion that did not overlap the development support. Further, an absorption pad (manufactured by ADVANTEC) was connected to the other end of the development support to obtain a test piece for nucleic acid chromatography measurement.

[ Determination of probe pair ]
A test piece for nucleic acid chromatographic measurement is prepared using a development support on which the probes of SEQ ID NOs: 3, 4, 11-13 are solid-phased and a labeled polymer carrier bonded to the probes of SEQ ID NOs: 3, 10-13. Using 10 ¹⁰ copies of in vitro synthesized RNA, probe pairs that could be detected by nucleic acid chromatography most efficiently were investigated.

[ Result ]
It was found that the target nucleic acid can be detected most efficiently when the nucleotide of SEQ ID NO: 3 is used as the first probe and the nucleotide of SEQ ID NO: 4 is used as the second probe (see FIG. 1).

[ Determination of forward primer ]
As the forward primer, the nucleotide sequences shown in SEQ ID NOs: 1, 5, and 6 were used. As a template, use the cpe RNA prepared in [Preparation of synthetic RNA] in Example 1 and convert the in vitro synthetic RNA to RNase.
Serial dilution with free water was made to 100 copies / test. In addition, as a reagent necessary for carrying out the NASBA method, a NASBA Amplification kit (manufactured by Kainos) was used, and the NASBA method was carried out according to the operation procedure of the kit.

However, the amplification time was shortened to 60 minutes instead of 90 minutes described in the instruction manual. The detection of the amplification product could be confirmed by agarose gel electrophoresis. Specifically, electrophoresis was performed at a constant voltage of 100 V for 40 minutes using a 2% agarose gel. For other operations, etc., Molecular
Clonig was conducted according to the second edition.

[ Result ]
It was found that when SEQ ID NO: 1 was used as the forward primer, an amplification product of the desired length was obtained by the NASBA method with any reverse primer (see FIG. 2 (A)).

[ Determination of reverse primer ]
Next, SEQ ID NO: 1 is used as the forward primer, and the nucleotide sequence shown in SEQ ID NOs: 2, 7, 8, 9 with the RNA polymerase promoter sequence added to the 5 ′ end side is used as the reverse primer, NASBA amplification time was shortened to 30 minutes using 10 copies / test in vitro synthetic RNA as a template, and other operations were performed in the same manner as in [Determination of Forward Primer] in Example 2 above, but with higher efficiency. Possible primer pairs were investigated.

[ Result ]
As a result, it was proved that the amplification efficiency was most excellent in the NASBA method when SEQ ID NO: 2 was used as the reverse primer to be paired with the forward primer of SEQ ID NO: 1 (see FIG. 2B).

[ Confirmation of usefulness of primer pair and probe pair ]
As the forward primer, the nucleotide sequence shown in SEQ ID NO: 1 was used, and as the reverse primer, the nucleotide sequence shown in SEQ ID NO: 2 in which an RNA polymerase promoter sequence was added to the 5 ′ end side, 10 copies / NASBA amplification was performed using the test in vitro synthetic RNA as a template. As a negative control, RNase free water was used instead of in vitro synthesized RNA.

  In this case, as a reagent necessary for the NASBA method, a NASBA Amplification kit (manufactured by Kainos Co., Ltd.) was used, and the NASBA method was performed according to the operation procedure of the kit. However, the amplification time was shortened to 15 minutes or 30 minutes instead of 90 minutes described in the instruction manual. For detection of the amplification product, a nucleic acid chromatographic test strip using the nucleotide sequence shown in SEQ ID NO: 3 as the first probe and the nucleotide sequence shown in SEQ ID NO: 4 as the second probe was used. The detection line was performed visually or with an immunochromatographic reader (manufactured by Hamamatsu Photonics).

[ Result ]
As a result, the amplification product can be detected visually and with an immunochromatographic reader (see FIG. 3), and a primer that amplifies only 10 copies of the template by the NASBA method in an extremely short time of 15 minutes must be found. succeeded in.

[ Specificity test ]
In order to confirm whether only the Clostridium perfringens having the enterotoxin gene can be detected in the respective results obtained in Examples 1 to 3 described above, the NCTC8239 strain was used as the Clostridium perfringens having the enterotoxin gene, and the enterotoxin was used. As a Welsh strain having no gene, the ACTT13124 strain was used, and the specificity was examined using the primer set and probe set of the present invention.

  C. perfringens was cultured using Bacto ™ Brain Heart Infusion (Becton Dicknson) at 37 ° C. Nucleic acid extraction from the cultured cells was performed using Extragen II (manufactured by Tosoh Corporation) according to the instructions for use. The amount of extracted nucleic acid was calculated from the absorbance at a wavelength of 260 nm using a spectrophotometer.

  In this case, the nucleotide sequence shown in SEQ ID NO: 1 is used as the forward primer, and the nucleotide sequence shown in SEQ ID NO: 2 in which an RNA polymerase promoter sequence is added to the 5 ′ end side as the reverse primer. Then, amplification by NASBA method was performed using 1.4 μg of nucleic acid extracted from NCTC8239 strain or ACTT13124 strain of Clostridium perfringens.

  As a reagent necessary for carrying out the NASBA method, a NASBA Amplification kit (manufactured by Kainos) was used, and the NASBA method was carried out according to the operation procedure of the kit. However, the amplification time was shortened to 30 minutes instead of 90 minutes described in the instruction manual. For detection of the amplification product, a nucleic acid chromatographic test strip using the nucleotide sequence shown in SEQ ID NO: 3 as the first probe and the nucleotide sequence shown in SEQ ID NO: 4 as the second probe was used. In this case, the detection line was visually observed.

[ Result ]
When nucleic acid extracted from enterotoxin-producing Clostridium perfringens is used as a sample, the line is detected by nucleic acid chromatography. However, when nucleic acid extracted from non-enterotoxin-producing Clostridium perfringens is used as a sample, lines are detected on the nucleic acid chromatograph. Was not detected. That is, it was found that enterotoxin-producing Clostridium perfringens having the cpe gene can be detected, but Clostridium perfringens that does not have the cpe gene cannot be detected. Therefore, it was found that the primer pair and the probe pair can specifically detect enterotoxin-producing Clostridium perfringens (see FIG. 4).

[ Detection of Enterotoxin-producing Clostridium perfringens in food samples ]
Whether or not enterotoxin-producing Clostridium perfringens in food samples can be detected from the results obtained in Examples 1 to 4 above was actually examined using a curry material. Curry materials include vegetables, meats, fruits, seasonings, spices, oils and the like. As curry material, curry store curry (manufactured by House Foods Co., Ltd.) was used, and 10 ⁶ cfu of cultured enterotoxin-producing C. perfringens per 1 g of curry material was added to prepare a food sample containing C. perfringens.

  A food sample containing C. perfringens or a curry material to which C. perfringens was not added was diluted with physiological saline to a concentration of 10%, and homogenized for 1 minute with an exiser 400 (manufactured by Organo). Thereafter, in order to remove impurities, filtration was performed using glass fiber filter paper (manufactured by GE Healthcare), and an extracted nucleic acid was prepared from the filtrate using Extractage II (manufactured by Tosoh Corporation).

  In this case, as the forward primer, the nucleotide sequence shown in SEQ ID NO: 1 is used, and as the reverse primer, the nucleotide sequence shown in SEQ ID NO: 2 with the RNA polymerase promoter sequence added to the 5 ′ end is used. NASBA amplification reaction was carried out. As a reagent necessary for carrying out the NASBA method, a NASBA Amplification kit (manufactured by Kainos) was used, and the NASBA method was carried out according to the operation procedure of the kit.

  However, the amplification time was shortened to 20 minutes instead of 90 minutes described in the instruction manual. For detection of the amplification product, a nucleic acid chromatographic test strip using the nucleotide sequence shown in SEQ ID NO: 3 as the first probe and the nucleotide sequence shown in SEQ ID NO: 4 as the second probe was used. The detection line was visually observed.

[ Result ]
Lines are not detected when food samples that do not contain Clostridium perfringens are used as food samples, but lines are detected when food samples containing enough enterotoxin-producing Clostridium perfringens enough to cause food poisoning are used. Was confirmed (see FIG. 5).

[ Detection of enterotoxin-producing Clostridium perfringens in plasma ]
In order to investigate the optionality of the sample to be examined, an experiment was conducted to determine whether enterotoxin-producing Clostridium perfringens can be detected by using blood component plasma as a sample in addition to food samples. The cultured enterotoxin-producing Clostridium perfringens was added to plasma at 10 ⁶ cfu per mL to obtain a plasma sample containing Clostridium perfringens.

  An extracted nucleic acid was prepared by treating a plasma sample containing Clostridium perfringens or a plasma sample without addition of Clostridium perfringens using Extragen II (manufactured by Tosoh Corporation). In this case, the nucleotide sequence shown in SEQ ID NO: 1 is used as the forward primer, and the nucleotide sequence shown in SEQ ID NO: 2 in which an RNA polymerase promoter sequence is added to the 5 ′ end side as the reverse primer. Amplification reaction by NASBA method was performed.

  In this case, a NASBA Amplification kit (manufactured by Kainos) was used as a reagent necessary for carrying out the NASBA method, and the NASBA method was carried out according to the operation procedure of the kit. However, the amplification time was set to 20 minutes, which was greatly shortened, not 90 minutes described in the instruction manual. For detection of the amplification product, a nucleic acid chromatographic test strip using the nucleotide sequence shown in SEQ ID NO: 3 as the first probe and the nucleotide sequence shown in SEQ ID NO: 4 as the second probe was used. The detection line was visually observed.

[ Result ]
Lines are not detected when plasma samples without added C. perfringens are used, but detection of lines is confirmed when plasma samples added with enterotoxin-producing C. perfringens enough to cause food poisoning are used. (See FIG. 6).

Claims (4)

A method for detecting Clostridium perfringens having an enterotoxin (cpe) gene, the method of detecting Clostridium perfringens comprising :
A target nucleic acid arbitrarily extracted from a specimen sample is converted into a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1, the nucleotide sequence shown in SEQ ID NO: 2 and RNA added to the 5 ′ end side by NASBA method. A first step of amplifying as a single-stranded nucleic acid using a primer pair comprising a reverse primer comprising a polymerase promoter sequence ;
The amplification product of the first step is subjected to a probe pair consisting of a first probe consisting of the nucleotide sequence shown in SEQ ID NO: 3 and a second probe consisting of the nucleotide sequence shown in SEQ ID NO: 4 by nucleic acid chromatography. used method of detecting Clostridium perfringens that a second step, characterized in that Ru comprising a detecting. The method for detecting Clostridium perfringens according to claim 1, wherein the RNA polymerase promoter sequence is a promoter sequence of T7 RNA polymerase represented by SEQ ID NO: 14 . One of the first probe and the second probe is carried on a development support, and the other is bound to a labeled polymer carrier,
The spreading support is modified with a carboxyl group;
The labeled polymer carrier is modified with a carboxyl group,
The first probe has an amino group added to the 3 ′ or 5 ′ terminal side of the nucleotide sequence shown in SEQ ID NO: 3,
The method for detecting Clostridium perfringens according to claim 1 or 2, wherein the second probe has an amino group added to the 3 'or 5' end of the nucleotide sequence represented by SEQ ID NO: 4. . A kit for the detection of Clostridium perfringens having the enterotoxin (cpe) gene, and detecting Clostridium perfringens ,
A forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 and an RNA polymerase promoter sequence added to the 5 ′ end thereof, A primer pair that amplifies the extracted target nucleic acid as a single-stranded nucleic acid by the NASBA method;
A probe pair comprising a first probe consisting of the nucleotide sequence shown in SEQ ID NO: 3 and a second probe consisting of the nucleotide sequence shown in SEQ ID NO: 4, and detecting an amplification product by the primer pair by nucleic acid chromatography And a kit for detecting C. perfringens.