JP5134071B2 - Method for avoiding inhibition in nucleic acid amplification reaction - Google Patents

Description

  The present invention relates to a method for avoiding inhibition of a nucleic acid amplification reaction by components contained in a medium, particularly bile components, and a nucleic acid using the method, when genetic testing is performed on bacterial cells obtained by a culture method It relates to the amplification method.

  Microbiological testing is indispensable as an indicator of the safety of food, drinking water, pharmaceuticals, etc., and as an aid in investigating the cause of pathogenic infection routes in mammals such as humans and livestock. As a result, if infection of various microorganisms is suspected, subsequent treatments, countermeasures, and the like are attempted.

  Microbiological tests in the food field are based on culture methods, and various methods and procedures are adopted as official methods according to the purpose. Microbe count measurement method for measuring the number of microorganisms in a sample from the number of colonies grown after culturing in a flat plate medium, etc., enrichment culture method used when the target bacteria are very few in the test material, various types of samples present Examples include a separation culture method for extracting a target microorganism from the above microorganisms, an identification test for examining morphological properties, biological properties, etc. of the separated and cultured microorganisms. For example, the test procedure for food poisoning bacteria is to inoculate suspected colonies by increasing the number of causative bacteria that are likely to be present in the test material, and then separating and cultivating the grown bacteria, followed by biochemical tests and immunological tests. A method is adopted in which a bacterial species is identified by inspection or the like, and it is determined whether or not it is a causative agent of food poisoning.

  However, in the microorganism inspection based on the culture method as described above, the number of steps is large, and depending on the culture method, the time for one culture is long. Therefore, with the development of genetic technology in recent years, genetic testing excellent in rapidity and differential diagnosis has been introduced as a method for testing microorganisms. It has come to be actively carried out, such as when microorganisms that are difficult to cultivate, slow-growing bacteria, and when the microorganism is a virus, when differentiating strains with pathogenicity, etc. It is especially useful if you want to.

  In genetic testing, materials containing nucleic acids such as DNA and RNA derived from viruses, bacteria, yeasts, fungi, etc. are subject to testing, but as described above, the culture medium with the above-mentioned enrichment culture may be used directly. . The genetic test is a method for recognizing the target gene of interest, and thus has high specificity, and has attracted attention as an alternative to a culture method that requires more time for separation culture, identification test, and time from the viewpoint of rapidity. However, since the bacterial cells that have been enriched and cultured in this way are often subjected to genetic testing together with the medium, some medium components may affect the genetic testing, particularly the nucleic acid amplification reaction.

  As a basic component of the medium, a selection agent suitable for the properties of the target microorganism is used in addition to a nutrient and a discrimination agent for distinguishing from microorganisms other than the target. For example, enterohemorrhagic Escherichia coli 0-157 enrichment medium (for example, mEC medium), which is a food poisoning bacterium, usually contains peptone as a nutrient, inorganic salts, etc., saccharides as a discrimination agent, and bile salts as a selective agent Contains bile components.

  Here, a case where E. coli is actually grown by performing enrichment culture using a food material (for example, cut vegetables or ground meat infiltrated with microorganisms) as a sample will be described. The grown Escherichia coli decomposes sugar (lactose), which is a distinguishing agent, to acidify the medium, and as a result, bile salts are precipitated in the medium as insoluble bile acids. When a medium containing this Escherichia coli is used as a test sample for genetic testing, centrifugal concentration is performed as a means for increasing sensitivity. At the same time, bile acids that have been insolubilized with the bacterial cells are also concentrated. Thereafter, when a nucleic acid derived from E. coli is amplified, it is known that this bile acid inhibits the amplification reaction.

  On the other hand, in a medium for enrichment of Salmonella (for example, EEM medium), bovine bile powder is used in large quantities as a bile component, so when used as a test sample as it is without any special treatment, It is known to inhibit nucleic acid amplification reactions. In addition, when centrifugal concentration is performed to increase sensitivity, the insoluble bile acids are concentrated together with the cells as described above, and the subsequent nucleic acid amplification reaction is inhibited. Thus, the following method has been studied as a method for avoiding inhibition of the amplification reaction by bile acids.

  For example, a method of recovering nucleic acid of cells that have been enriched and cultured by nucleic acid extraction operation is known. In this method, first, the cells are centrifuged together with the medium to precipitate the cells. Further, the precipitate is lysed with an enzyme or the like, deproteinized by a phenol / chloroform method or the like, and then precipitated and recovered with alcohol.

  There has also been proposed a method of diluting an inhibitor contained in a medium to a concentration that does not affect nucleic acid amplification. In this method, the final concentration of the medium component during the amplification reaction is diluted about 100 times, and at the same time, the proliferated cells are also diluted about 100 times, but when the inhibitor contains a lot of inhibitory substances. Will require further dilution. Originally, enrichment culture is performed in order to ensure the target amount of bacteria that can withstand the test, but in order to perform nucleic acid amplification, the result is that the cells that proliferated have been diluted. End.

  Further, a method for performing PCR using a culture medium after enrichment by centrifuging the cells, further washing the precipitates with PBS three times, and then suspending the precipitates in purified water. Is disclosed (see Non-Patent Document 1). However, this method also has a problem that the bile component is not sufficiently removed, particularly when the concentration of the bile component in the medium is high or the amount of the grown cells is small. Thus, conventionally, when subjecting the nucleic acid of the bacterial cells obtained by the enrichment culture method to the amplification reaction, there were various problems.

Sadamoto Tsukamoto “Clinics and Microorganisms”, 1996, Volume 23, Special Issue, p.801-806

  The present invention has been made paying attention to such a conventional problem, and in the case of carrying out a genetic test on bacterial cells obtained by a culture method, the test sample is concentrated in order to increase detection sensitivity. Another object of the present invention is to provide a method for avoiding inhibition of the nucleic acid amplification reaction by components contained in the medium, particularly bile components, and a nucleic acid amplification method using the method.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have performed a method for avoiding inhibition of nucleic acid amplification reaction by a medium component by centrifugal concentration and alkaline treatment of the enriched and cultured cells, and the method described above. The nucleic acid amplification method used was found and the present invention was completed.

That is, the present invention has the following configuration.
(1) In a method for amplifying a nucleic acid derived from bacterial cells, an enrichment culture solution containing bacterial cells obtained by a culture method is used as a test sample, and bile components in the culture solution are removed by centrifugal concentration and alkali treatment. A method for avoiding inhibition of nucleic acid amplification reaction by bile components.
(2) The method according to (1), wherein the culture method is an enrichment culture method using a liquid medium containing a bile component.
(3) The method according to (1) or (2), wherein the bile component is a bile salt or bovine bile powder.
(4) The method according to (1), wherein the alkali treatment is performed at a pH of 7.5 or more.
(5) A nucleic acid amplification method comprising the following steps.
(A) a step of centrifugally concentrating an enrichment culture solution containing bacterial cells to obtain a precipitate;
(B) adding an alkalizing agent to the precipitate so as to have a pH of 7.5 or more;
(C) a step of further centrifuging to obtain microbial cells as a precipitate,
(D) A step of amplifying the nucleic acid derived from the cells.
(6) A nucleic acid amplification method comprising the following steps.
(A) a step of adding an alkalizing agent to the enrichment culture solution containing the bacterial cells so as to have a pH of 7.5 or more;
(B) a step of further centrifuging to obtain microbial cells as a precipitate;
(C) A step of amplifying the nucleic acid derived from the cells.
(7) (5) or (6), wherein the alkalizing agent comprises at least one selected from alkali metal hydroxides, alkali metal carbonates, alkali metal phosphates and Good buffering agents. The method described.
(8) The nucleic acid amplification method is any one selected from the group consisting of LAMP method, PCR method, ICAN method, NASBA method, LCR method, SDA method, TRC method and TMA method ( The method according to 5) or (6).

  The present invention provides a medium that inhibits a nucleic acid amplification reaction carried out after culturing by carrying out a centrifugal treatment and an alkali treatment of the grown microbial cells together with the medium when genetic testing is performed on the microbial cells obtained by the culture method. It is possible to provide a method for avoiding inhibition of the amplification reaction by removing components, particularly bile components. By using this method, a complicated nucleic acid extraction operation after culturing is unnecessary, and further, a nucleic acid derived from a target cell can be amplified with high sensitivity without requiring a dilution operation of a growth medium. it can. Hereinafter, the present invention will be described in more detail.

The medium used in the present invention is not particularly limited as long as it contains a bile component such as bile salt or bovine bile powder as a component, but any medium that precipitates as a bile acid as a result of enrichment culture. For example, mEC (abbreviation of modified Escherichia coli ) medium, novobiocin-added mEC medium (added as a growth inhibitor of general bacteria other than the target pathogen), EEM (abbreviation of Enterobacteriaceae Enrichment Mannitol) medium, etc. Liquid medium for enrichment of food poisoning pathogens.

  The alkalizing agent used in the present invention is not particularly limited as long as it can dissolve the precipitated bile acid as a salt, but is selected from alkali metal hydroxides, alkali metal carbonates, alkali metal phosphates or Good buffer agents. An aqueous solution containing at least one component, for example, an aqueous solution containing an alkaline component such as potassium hydroxide, sodium hydroxide, sodium carbonate, disodium hydrogen phosphate, or trisaminohydroxymethane.

  Here, the alkalinizing agent used in the present invention is added to the precipitate obtained after centrifugal enrichment of the enrichment culture solution or directly added to the enrichment culture solution. Is preferably carried out at 7.5 or more. When the pH is less than 7.5, it is insufficient to remove the precipitated bile acid as a salt and then remove it by centrifugation.

  Examples of the nucleic acid amplification method used in the present invention include the LAMP method, PCR method, ICAN method, NASBA method, LCR method, SDA method, TRC method and TMA method. Of these, the LAMP method has high amplification efficiency, high specificity, does not require temperature control, and can be detected visually or with a low-cost detection device, so genetic testing for microbial testing in a wider range of facilities is possible. This is a technique that can be performed and can be said to be a more suitable nucleic acid amplification method.

  The LAMP method anneals its 3 'end to the template nucleotide and uses it as a starting point for complementary strand synthesis. By combining primers that anneal to the loop formed at this time, isothermal complementary strand synthesis reaction is performed. In this amplification method, the 3 ′ end of the primer always anneals to the region derived from the sample, so that the check mechanism based on complementary binding of the base sequence functions repeatedly, resulting in high sensitivity and It enables highly specific nucleic acid amplification reactions. Reagent kits for detecting food poisoning bacteria based on the LAMP method, such as enterohemorrhagic Escherichia coli detection reagent kits and Salmonella detection reagent kits, are sold by Eiken Chemical Co., Ltd., and these kits can be used.

  An example of embodiment in this invention is shown. A specified weight of a food material that may be contaminated with bacteria is collected, the food material is added to a specified volume of novobiocin-added mEC medium, treated with a stomacher at room temperature for 1 minute, and cultured for a certain period of time (enrichment culture). By collecting a certain amount of medium (including enriched and cultured cells) in a centrifuge tube and centrifuging, a mixture of bile acids precipitated by culture and the grown cells can be obtained as a precipitate. . Thereafter, the supernatant is removed, and an alkalizing agent is added to the precipitate and stirred well. The precipitated bile acid becomes a bile salt, dissolves, and is centrifuged again to extract only the cells as a precipitate. The bacterial cells collected in this way are heat-treated to elute the nucleic acid. The eluted nucleic acid can be subjected to the amplification reaction without being affected by bile acid (which inhibits the subsequent amplification reaction).

  Furthermore, an example of another embodiment of the present invention is shown. As described above, a certain amount of medium that has been cultivated for a certain period of time is collected in a centrifuge tube, and an alkalizing agent is added and stirred well. By performing the centrifugal treatment, the bile acid precipitated by the culture becomes a salt and is solubilized, so that it moves to the supernatant, and the proliferated cells can be separated as a precipitate. The bacterial nucleic acid collected in this manner can be subjected to an amplification reaction as described above.

  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. LAMP reaction using cells containing medium components (1) Sample preparation Add 225 mL of novobiocin-added mEC medium to 25 g of bean sprouts, stoma treatment at room temperature for 1 minute, then culture at 42 ° C. for 18 hours and 10% bean sprouts Got. To this food solution, enterohemorrhagic E. coli O157: H7 ( Escherichia coli ATCC43889) (hereinafter abbreviated as “E. coli”) was added to a concentration of 1.5 × 10 ^{2 to 4} cfu / mL. A diluted solution was prepared. Collect 50 μL of the bacterial dilution in a 0.2 mL PCR tube (hereinafter abbreviated as “PCR tube”), then add 50 μL of 0.05 mol / L sodium hydroxide aqueous solution (hereinafter abbreviated as “NaOH aqueous solution”) at 95 ° C. After heat treatment for 5 minutes, it was cooled to 0 ° C. Further, using a centrifuge Peti-Hachi Model 2816 (manufactured by WAKEN), the sample was centrifuged at 25 ° C., 6,000 rpm for 1 minute, and cooled to 0 ° C. again. did.
(2) Nucleic acid amplification reaction 5 μL of a sample was mixed with 20 μL of LAMP reaction reagent (Reaction Mix.VT 20 μL and Bst DNA polymerase 1 μL) prepared from a Loopamp (registered trademark) enterohemorrhagic Escherichia coli detection reagent kit (manufactured by Eiken Chemical Co., Ltd.). 20 μL from the solution), and using a real-time turbidimeter LA-320C (manufactured by Telamex) (hereinafter abbreviated as “LA-320C”), the change in turbidity (absorbance) at a wavelength of 650 nm is used as an index. The LAMP reaction was performed at 65 ° C. for 1 hour.

The result is shown in FIG. An increase in turbidity was observed after about 35 minutes for E. coli at a concentration of 1.5 × 10 ⁴ cfu / mL and after about 40 minutes for 1.5 × 10 ³ cfu / mL. ^{At 2} cfu / mL, no increase in turbidity was observed even after 60 minutes of reaction. According to the specifications of this reagent kit, if the turbidity does not increase within 60 minutes, it is evaluated as negative. Therefore, in this result, at low concentration (1.5 × 10 ² cfu / mL), Detection was not possible and the result was judged negative.

Example 2 LAMP Reaction Utilizing Centrifugal Concentration Method (1) Sample Preparation As in Example 1 (1), a 10% bean sprouts food solution was prepared to prepare a 3 concentration bacterial dilution. Centrifugal treatment of 1 mL of this bacterial dilution using a small microcentrifuge PMC-060 (TOMY) (hereinafter abbreviated as “Centrifuge A”) under conditions of 25 ° C. and 2,000 × g for 10 minutes Then, after removing the supernatant, 10 μL of 1 mol / L Tris · HCl buffer solution (pH 8.0) (hereinafter abbreviated as “TE buffer solution”) containing 0.5 mol / L EDTA and 10 μL of NaOH aqueous solution were added to the obtained precipitate. Was added and stirred well. The entire amount was transferred to a PCR tube, and a supernatant obtained by performing the same operation as that after the heat treatment in Example 1 (1) was used as a sample.
(2) Nucleic acid amplification reaction A LAMP reaction was carried out according to Example 1 (2).

The result is shown in FIG. Although the amount of cells used in Example 2 (1 mL as the amount of medium) is 100 times the amount of cells used in Example 1 (50 μL), 1.5 × 10 ⁴ cfu / mL Even at the concentration, the time for increasing the turbidity was very slow, about 55 minutes. This suggests that medium components may inhibit the LAMP reaction.

Example 3 FIG. Effect of bile components on LAMP reaction (1) Preparation of sample Select bile salt or bovine bile powder as a bile component that is widely used as a medium component and is predicted to inhibit amplification reaction, and LAMP reaction of each bile component The final concentration was 0 to 20 mg / mL, and E. coli was added as a template nucleic acid to the aqueous solution of each concentration to a concentration of 7.5 × 10 ⁴ cfu / test to prepare a test sample.
(2) Nucleic acid amplification reaction 5 μL of a sample was mixed with 20 μL of LAMP reaction reagent (Reaction Mix.VT 20 μL and Bst DNA polymerase 1 μL) prepared from a Loopamp (registered trademark) enterohemorrhagic Escherichia coli detection reagent kit (manufactured by Eiken Chemical Co., Ltd.). 20 μL from the solution, added to the fluorescent dye Oxazole Yellow 0.625 μg / mL), and LAMP at 65 ° C. for 2 hours using a fluorescence detector ABI PRISM 7000 Sequence Detection System (manufactured by Applied Biosystems) Reaction was performed. In Examples 1 and 2, inhibition of the LAMP reaction was evaluated by detecting turbidity associated with the production of magnesium pyrophosphate, which is a byproduct of the amplification reaction. However, since the possibility that the inhibition has affected the generation rate of turbidity rather than the amplification reaction itself was considered, in Example 3, the fluorescence method using the nucleic acid amplification product as the main product as an index was adopted. .

  The results are shown in FIGS. When no bile component was added, an increase in fluorescence intensity was observed after 12 to 13 minutes. The concentration delayed from about 2 times (25 to 30 minutes) from the rise time without addition was 7.5 mg / mL for both bile salts and bovine bile powder. Furthermore, the concentration at which an increase in fluorescence intensity was confirmed within 60 minutes was 12.5 mg / mL. From the results of Examples 1 to 3, it was inferred that the bile component contained in the medium component inhibited the LAMP reaction.

Example 4.2 LAMP reaction by 2-step centrifugal concentration method (example using novobiocin-added mEC medium)
(1) Preparation of sample The method of performing centrifugal concentration twice was examined. First, a 10% bean sprouts food liquid was prepared in the same manner as in Example 1 (1). To this food solution, E. coli was added to a concentration of 3.5 × 10 ⁴ cfu / mL to prepare a bacterial dilution. 1 mL of the bacterial dilution was centrifuged using a centrifuge A at 25 ° C. and 2,000 × g for 10 minutes. After removing the supernatant, the resulting precipitate was added to TE buffer, NaOH aqueous solution, Distilled water or physiological saline (1 mL) was added, and the mixture was thoroughly washed with stirring. Further, centrifugation was performed using the centrifuge A under the same conditions. After removing the supernatant, the precipitate was collected. Add 10 μL of TE buffer and 10 μL of aqueous NaOH solution to the precipitate obtained from each washing solution, stir and suspend well, transfer the entire amount to a PCR tube, and perform the same operations as those after the heat treatment in Example 1 (1). The supernatant obtained by performing was used as a sample.
(2) Preparation of Comparative Control Sample According to Example 2, a sample obtained by omitting the precipitate washing step was used as a comparative control.
(3) Nucleic acid amplification reaction A LAMP reaction was performed according to Example 1 (2).

  The result is shown in FIG. Even when any washing solution was used, an increase in turbidity was observed within 60 minutes. However, when TE buffer solution or NaOH aqueous solution was used, the time for turbidity increase compared to distilled water or physiological saline was used. Remarkably recovered. The pH when distilled water, physiological saline, TE buffer solution or NaOH aqueous solution was added to the precipitate obtained from the bacterial dilution was 6.4, 6.4, 7.7, 12.6, respectively. Met. These suggest that the bile components that inhibit the LAMP reaction were effectively removed by alkaline washing.

Example 5.2 LAMP reaction by 2-step centrifugal concentration method (example using EEM medium)
(1) Preparation of sample As in Example 4, a method of performing centrifugal concentration twice was examined. 225 mL of EEM medium was added to 25 g of minced chicken, treated with stomacher at room temperature for 1 minute, and cultured at 35 ° C. for 18 hours to obtain a 10% minced chicken food material solution. To this food liquid, Salmonella enteritidis ATCC13076 (hereinafter abbreviated as “Sal.”) Was added to a concentration of 3.5 × 10 ⁴ cfu / mL to prepare a bacterial dilution. A supernatant obtained by performing the same operation as that after centrifugation of Example 4 (1) for 1 mL of the bacterial dilution was used as a sample. In addition, only NaOH aqueous solution was used for the washing | cleaning liquid.
(2) Preparation of Comparative Control Sample According to Example 2, a sample obtained by omitting the precipitate washing step was used as a comparative control. That is, a 10% ground chicken food liquid similar to Example 5 (1) was prepared and added so that the Sal. Concentration was 3.5 × 10 ⁴ cfu / mL to prepare a bacterial dilution. 1 mL of this bacterial dilution was centrifuged using Centrifuge A at 25 ° C. and 2,000 × g for 10 minutes. After removing the supernatant, 10 μL of TE buffer and NaOH were added to the resulting precipitate. 10 μL of an aqueous solution was added, well agitated and suspended, the entire amount was transferred to a PCR tube, and the supernatant obtained by performing the same operations as those after the heat treatment in Example 1 (1) was used as a comparative control sample.
(3) Nucleic acid amplification reaction 5 μL of a sample was obtained from 20 μL of LAMP reaction reagent 20 μL (Reaction Mix.Sal 20 μL and Bst DNA polymerase 1 μL prepared from Loopamp (registered trademark) Salmonella detection reagent kit (Eiken Chemical Co., Ltd.)). LAMP reaction was carried out at 65 ° C. for 1 hour using LA-320C and the change in absorbance (turbidity) at a wavelength of 650 nm as an index.

  The result is shown in FIG. The time for the turbidity to rise was remarkably recovered by alkaline washing of the precipitate. The pH when the aqueous NaOH solution was added to the precipitate obtained from the bacterial dilution was 12.6. These facts suggest that the bile components that inhibit the LAMP reaction were removed by alkaline washing.

Example 6.1 LAMP reaction by 1-step centrifugal concentration method (example in which aqueous NaOH solution was added)
(1) Preparation of sample The method of performing centrifugal concentration once was examined. A 10% bean sprouts food liquid was prepared in the same manner as in Example 1 (1). Bacterial cells were added to this food solution so that E. coli had a concentration of 3.5 × 10 ⁴ cfu / mL to prepare a bacterial dilution. 0.25, 0.5, 0.6, or 1 mL of NaOH aqueous solution was added to 1 mL of this bacterial dilution, respectively, and further, using Centrifuge A, conditions of 25 ° C., 2,000 × g, 10 minutes The precipitate was recovered after removing the supernatant. To the precipitate, 10 μL of TE buffer was added and suspended. The total amount of this suspension was transferred to a PCR tube, and 10 μL of NaOH aqueous solution was further added, and then the supernatant obtained by performing the same operations as those after the heat treatment in Example 1 (1) was used as a sample.
(2) Preparation of comparative control sample In Example 6 (1), a sample without addition of NaOH aqueous solution was used as a comparative control sample.
(3) Nucleic acid amplification reaction A LAMP reaction was performed according to Example 1 (2).

  The result is shown in FIG. As the amount of NaOH aqueous solution added increased, the time for the turbidity to rise was remarkably recovered. In addition, the pH when adding 0 (no addition), 0.25, 0.5, 0.6, or 1 mL of an aqueous NaOH solution to the bacterial dilution was 6.5, 6.7, 7.5, 8.2 and 10.3. These facts indicate that the bile components that effectively inhibit the LAMP reaction can be effectively removed by increasing the amount of aqueous NaOH solution added to the medium (diluted bacteria) (making the medium pH 7.5 or higher). Suggests.

Example 7.1 LAMP reaction by 1-step centrifugal concentration method (example in which TE buffer solution was added)
(1) Preparation of sample A bacterial dilution was prepared in the same manner as in Example 6 (1). Add TE buffer so that the pH of 1 mL of this bacterial dilution is ˜8.0, and further centrifuge using centrifuge A at 25 ° C., 2,000 × g for 10 minutes, After removing the supernatant, the precipitate was collected. To the precipitate, 10 μL of TE buffer was added and suspended. The total amount of this suspension was transferred to a PCR tube, 10 μL of NaOH aqueous solution was further added, and then the supernatant obtained by performing the same operations as those after the heat treatment in Example 1 (1) was used as a sample.
(2) Preparation of Comparative Control Sample In Example 7 (1), a sample without addition of TE buffer was used as a comparative control sample.
(3) Nucleic acid amplification reaction A LAMP reaction was performed according to Example 1 (2).

  The result is shown in FIG. As in Example 6, the time during which the turbidity increased was remarkably recovered by performing the centrifugal treatment after raising the pH of the medium. This suggests that bile components that effectively inhibit the LAMP reaction can be removed by raising the pH.

Example 8 FIG. Comparison of detection sensitivity by LAMP method and PCR method (1) Preparation of sample 225 mL of novobiocin-added mEC medium was added to 25 g of shellfish radish, treated with stomacher for 1 minute at room temperature, cultured at 42 ° C. for 18 hours, and 10% shellfish radish A food liquid was obtained. To this food solution, E. coli was added so as to have a concentration of 2.4 × 10 ^{2 to 5} cfu / mL, thereby preparing a bacterial dilution. According to Example 6, the sample obtained by the method (one-step centrifugal concentration method) in which the bile components in the medium were removed by adding an alkalizing agent to the medium and then performing centrifugation was used as a sample.
(2) Preparation of Comparative Control Sample For LAMP, a sample obtained by a method that does not remove the bile component in the medium was used as a comparative control according to Example 1. For PCR, 10 μL of the bacterial dilution was collected in a 0.5 mL PCR tube, then 90 μL of TE buffer was added, heat-treated at 95 ° C. for 5 minutes, and then cooled to 0 ° C. Furthermore, using a centrifuge 5414 R (rotor No. F45-36-8, manufactured by Eppendorf), centrifuge at 4 ° C., 12,000 rpm for 10 minutes, and cool to 0 ° C. again. The obtained supernatant was used as a comparative control sample.
(3) Nucleic acid amplification reaction A LAMP reaction was performed according to Example 1 (2). The reaction time was 2 hours. For PCR reaction, add 5 μL of sample to 45 μL of PCR reaction reagent, heat denaturation at 94 ° C. for 1 minute, annealing at 55 ° C. for 1 minute, polymerase extension reaction at 72 ° C. for 1 minute (final cycle 10 minutes). As a total of 35 cycles. As the reaction apparatus, T3 Thermocycler (manufactured by Biometra) was used. After completion of the reaction, 3.5% of the reaction solution was subjected to 1% agarose gel electrophoresis to confirm the reaction product.

  The results are shown in Table 1. In both the LAMP method and the PCR method, the one-step centrifugal concentration method showed an improvement in detection sensitivity of 10 to 100 times compared to the comparative control. From this, it is suggested that the present invention has an effect that reaction inhibition can be avoided in various amplification reactions when a nucleic acid of bacterial cells that have been enriched and cultured in a medium containing a bile component is amplified.

  As described above, the present invention, when carrying out a genetic test on the bacterial cells obtained by the culture method, simultaneously concentrates the test sample in order to increase the detection sensitivity, and at the same time, the components contained in the medium, particularly bile It is possible to provide a method for avoiding inhibition of nucleic acid amplification reaction by components and a nucleic acid amplification method using the method.

It is a graph which shows a LAMP reaction when using the microbial cell containing a culture medium component. It is a graph which shows the LAMP reaction using the centrifugal concentration method. It is a graph which shows the influence on the LAMP reaction by a bile salt. It is a graph which shows the influence on the LAMP reaction by bovine bile powder. It is a graph which shows the LAMP reaction (Novobiocin-added mEC culture medium use example) by a two-step centrifugal concentration method. It is a graph which shows the LAMP reaction (EEM culture medium usage example) by a two-step centrifugal concentration method. It is a graph which shows LAMP reaction (NaOH aqueous solution addition example) by 1 step centrifugal concentration method. It is a graph which shows the LAMP reaction (TE buffer solution addition example) by 1 step centrifugal concentration method.

Claims (4)

A nucleic acid amplification method comprising the following step of removing a bile component in a culture solution using a liquid medium containing a bile component as a selective inhibitor of Gram-positive bacteria .
(A) a step of obtaining a precipitate by centrifugal concentration of an enrichment culture solution containing bacterial cells,
(B) An alkalinizing agent is added to the precipitate, and pH 7. 5 or more steps,
(C) A step of further centrifuging to obtain cells as a precipitate,
(D) A step of amplifying the nucleic acid derived from the cells. A nucleic acid amplification method comprising the following step of removing a bile component in a culture solution using a liquid medium containing a bile component as a selective inhibitor of Gram-positive bacteria .
(A) An alkalizing agent is added to the enrichment culture solution containing the cells, and pH 7. 5 or more steps,
(B) A step of further centrifuging to obtain cells as a precipitate,
(C) A step of amplifying the nucleic acid derived from the cells.   The method according to claim 1, wherein the alkalizing agent comprises at least one selected from alkali metal hydroxides, alkali metal carbonates, alkali metal phosphates, and Good buffering agents.   The nucleic acid amplification method consists of L A MP method, P C R method, I C AN method, N A S B A method, L C R method, S D A method, T R C method and T M A method The method according to claim 1, wherein the method is selected from the group.

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