Method for detecting Lactobacillus helveticus

Abstract

To provide a technique capable of specifically detecting Lactobacillus helveticus in a sample and quantifying it with high accuracy.SOLUTION: A method for detecting and / or quantifying Lactobacillus helveticus in a sample, comprises amplifying a region containing positions 37 to 62 in a specific sequence of DNA or RNA of the bacterium in the sample. The method uses a labeled DNA probe comprising the base sequence of positions 48 to 53 of the specific sequence or a base sequence complementary thereto. Among the nucleic acids in the labeled DNA probe, at least sugars constituting the nucleic acid corresponding to the base at position 52 of the specific sequence are crosslinked. The method is also preferably applicable when the sample may contain Lactobacillus gallinarum.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for detecting Lactobacillus helveticus contained in a sample.

Background Art

[0002] Although the fermentation of lactic acid bacteria has been utilized by humans since ancient times, in recent years, it has been found that even dead bacterial cells exhibit useful physiological activities in the human body. In particular, Lactobacillus helveticus (hereinafter also referred to as "L. helveticus") has attracted attention for having many physiological functions such as an anti-anxiety and anti-stress effect, a blood pressure regulating effect, a fatigue recovery effect, an anti-tumor effect, an immune activation effect, and an effect of improving learning and memory ability, and there are examples of being incorporated into functional foods and the like.

[0003] When formulating bacteria as an active ingredient of a functional food or medicine, it is important and in demand to quantitatively measure the amount of the bacteria. Conventionally, various methods have been developed and put into practical use for detecting and quantifying bacteria, and one of the main methods is a nucleic acid amplification method such as the PCR method. So far, the present inventor has proposed a method for quantifying dead cells using the digital PCR method (Patent Document 1).

[0004] As a method for identifying and quantifying the bacterial species of L. helveticus by gene testing, a method of performing PCR by an intercalator method using the sequence specificity of the Intergenic Spacer (IS) region has been proposed (Non-Patent Documents 1 to 2). ーter method by PCR has been proposed (Non-Patent Documents 1 to 2).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] However, Lactobacillus helveticus has almost the same gene sequence in the IS region as its related species Lactobacirus gallinarum (hereinafter also referred to as "L. gallinarum"). ) Therefore, it is difficult to detect and quantify L. helveticus separately from L. gallinarum by the method described above. In view of such a situation, an object of the present invention is to provide a technique capable of specifically detecting and highly accurately quantifying Lactobacirus helveticus in a sample.

Means for Solving the Problems

[0008] As a result of intensive studies to solve the above problems, the present inventors found a base sequence that differs from each other in the IS region between L. helveticus and L. gallinarum, and obtained the idea that such a sequence would enable specific detection of L. helveticus. However, since such a base sequence has a very small difference of 1 to 2 bases, it was not possible to distinguish and detect both by the ordinary PCR method. Therefore, after further consideration, when nucleic acid amplification of a specific region is performed using a DNA probe having LNA (Locked Nucleic Acid) at the position of a specific base sequence, ​The present invention was completed by conceiving that L. helveticus can be specifically detected and quantified.

[0009] That is, the present invention is a method for detecting and / or quantifying Lactobacillus helveticus in a sample, comprising amplifying a region containing positions 37 to 62 of SEQ ID NO: 1 of the DNA or RNA of the bacterium in the sample, using a labeled DNA probe containing the base sequence from positions 48 to 53 of SEQ ID NO: 1 or a complementary base sequence thereto, and characterized in that the sugar constituting at least the nucleic acid corresponding to the position of the base at position 52 of SEQ ID NO: 1 among the nucleic acids in the labeled DNA probe is crosslinked. In a preferred embodiment of the present invention, the probe comprises at least the base sequence from positions 1 to 16 of SEQ ID NO: 2 or a complementary base sequence thereto. In a preferred embodiment of the present invention, PCR is performed using a forward primer containing the base sequence shown in SEQ ID NO: 3 and a reverse primer containing the base sequence shown in SEQ ID NO: 4 to amplify the region.

Advantages of the Invention

[0010] According to the present invention, L. helveticus in a sample can be specifically detected and quantified with high precision. The method of the present invention can achieve accurate quantification of L. helveticus even in the presence of L. gallinarum.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0012] Next, the present invention will be described in detail. However, the present invention is not limited to the following embodiments and can be freely modified within the scope of the present invention.

[0013] The method of the present invention includes amplifying a specific base sequence region of the IS region (SEQ ID NO: 1) of DNA or RNA of L. helveticus in a sample. The base sequence of the region to be amplified includes positions 37 to 62 of SEQ ID NO: 1, preferably positions 32 to 67, and more preferably positions 13 to 191. Since this region is a gene sequence specifically found in L. helveticus, the bacterium can be detected by amplifying such a region.

[0014] In the method of the present invention, a labeled DNA probe containing the base sequence from position 48 to position 53 of SEQ ID NO: 1 or a complementary base sequence thereto is used. The base sequence corresponds to positions 7 to 12 of SEQ ID NO: 2. The length of the DNA probe is not particularly limited as long as it contains the above base sequence, preferably 15 to 35 bases, more preferably 16 to 30 bases, and even more preferably 17 to 25 bases. Also, the probe may have an arbitrary base sequence on the 5' side and / or 3' side of the above base sequence, but usually has a continuous sequence from position 47 of SEQ ID NO: 1 to the 5' side on the 5' side and a continuous sequence from position 54 of SEQ ID NO: 1 to the 3' side on the 3' side.

[0015] The region of the base sequence from position 48 to position 53 of SEQ ID NO: 1 is a region containing bases different from those of L. gallinarum in the IS region of L. helveticus. L. gallinarum is genetically very closely related to L. helveticus and the IS regions are almost the same. Therefore, it is necessary to increase the specificity of detection by making the DNA probe contain the different bases. The present inventors have found that L. helveticus and L. gallinarum differ at least in the base at position 52 of the IS region.

[0016] ​Therefore, in order to enhance the sensitivity of the probe even with a single-base difference, the DNA probe used in the present invention is one in which the sugar constituting the nucleic acid corresponding to at least the position of the base at the 52nd position of SEQ ID NO: 1 among the nucleic acids therein is cross-linked. The DNA probe used in the present invention preferably uses one in which the sugar constituting the nucleic acid corresponding to the positions of the bases from the 50th to the 52nd positions of SEQ ID NO: 1, more preferably from the 49th to the 53rd positions, and even more preferably from the 48th to the 53rd positions is cross-linked.

[0017] In addition, the DNA probe preferably contains at least the base sequence from the 1st to the 16th positions of SEQ ID NO: 2 or a base sequence complementary thereto. The said base sequence corresponds to positions 42 to 57 of SEQ ID NO: 1.

[0018] Here, cross-linking refers to a mode in which the carbon atoms at the 2'-position and 4'-position of the sugar (ribose) constituting diffusion are cross-linked (also called Locked Nucleic Acid (LNA) or Blidged Nucleic Acid (BNA)). Specific cross-linking modes include 5-membered ring cross-links such as 2'4'-BNA (LNA) and 3'-Amino-2'4'-BNA, 6-membered ring cross-links such as 2'4'-BNA(Me), 7-membered ring cross-links such as 2'4'-BNA, etc. Nucleic Acid (BNA)). Specific cross-linking modes include 5-membered ring cross-links such as 2'4'-BNA (LNA) and 3'-Amino-2'4'-BNA, 6-membered ring cross-links such as 2'4'-BNA NC (Me), 7-membered ring cross-links such as 2'4'-BNA COC etc. In the case of an LNA-modified DNA probe, even if it differs from the target site by only one base, the interaction between complementary bases is weakened and it becomes difficult to bind. On the other hand, in the case of a sequence complementary to the target site, the stability of the double strand increases and the Tm value becomes larger. Therefore, the difference in nucleic acid amplification becomes large depending on the presence or absence of complementarity between the base sequence of the target site and the probe, and the detection sensitivity increases.

[0019] The labeling of the DNA probe is not particularly limited, and examples include fluorescence quenching labels, chemiluminescent substances, radioactive substances, biotin, alkaline phosphatase, digoxigenin, peroxidase, etc. Among them, fluorescence quenching labels are preferred because detection and quantification methods are established in the technical field. The fluorescence quenching dye is not particularly limited, and examples thereof include fluorescein or its derivatives (such as FAM, HEX, JOE, TET, etc.), rhodamine or its derivatives (such as tetramethylrhodamine, tetramethylrhodamine isothiocyanate, carboxyrhodamine, x-rhodamine, sulforhodamine 101 acid chloride), BODIPY or its derivatives (such as BODIPY-FL, etc.), IowaBlack (5IABkFQ, 3IABkFQ), and the like. Also, the label is usually modified at the 3'-end and / or 5'-end of the DNA probe. In addition, a linker, a spacer, or the like may be added for labeling.

[0020] Typical embodiments of the method of the present invention include: (1) a step of providing a sample; (2) a step of amplifying a region containing positions 37 to 62 of SEQ ID NO: 1 of the DNA or RNA of the bacterium in the sample; (3) a step of hybridizing a labeled DNA probe containing the base sequence from position 48 to position 53 of SEQ ID NO: 1 or a complementary base sequence thereto to the amplification product that can be obtained in (2), and detecting and quantifying the amount of the amplification product by detecting the label.

[0021] The sample in (1) is not limited, and examples thereof include foods, biological samples, vaccine preparations, drinking water, industrial water, environmental water, wastewater, soil, or swab samples. In particular, since it is expected to be applied to the quantification of L. helveticus contained in the active ingredients of functional foods and the like, it is preferable to use food and drink as the test sample. Examples of food and drink include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit juice drinks, fermented drinks (including concentrated stock solutions and preparation powders of these drinks); frozen desserts such as ice cream, ice sherbet, and shaved ice; confectioneries such as chocolate, caramel, candy, cake, biscuits, and cookies; dairy products such as sterilized milk, processed milk, milk drinks, fermented milk, and butter; highly nutritious liquid foods such as enteral nutrition foods, milk for infants, and sports drinks; and functional foods such as foods for specified health uses and dietary supplements.

[0022] ​The sample may be prepared by conventional methods. For example, prior to subjecting it to nucleic acid amplification reaction, a treatment for extracting DNA or RNA may be performed. There is no particular limitation on the method for extracting DNA or RNA, and it can be carried out by conventional methods, and a commercially available DNA extraction kit may also be used.

[0023] Lactobacillus helveticus detected and / or quantified by the present invention may be viable cells or dead cells.

[0024] In a preferred embodiment of the present invention, the sample may contain other Lactobacillus bacteria, such as Lactobacirus gallinarum, Lactobacillus · paracasei, Lactobacillus plantarum, Lactobacillus gasseri, Lactobacillus salivarius It may also contain.

[0025] In particular, the method of the present invention can detect L. helveticus separately from L. gallinarum. In other words, the method of the present invention is a method for detecting L. helveticus without detecting L. gallinarum in the sample. Therefore, the present invention is suitable when the sample may contain Lactobacillus gallinarum. For example, when there are 10 10 cells / g or more of L. gallinarum in the sample, or when L. gallinarum is present 10 3 times or more that of L. helveticus, L. helveticus can be specifically detected. Note that the sample "may possibly contain" other bacteria such as L. gallinarum only means that there is a suspicion of containing it before measurement, and even if it is found that the sample does not contain L. gallinarum as a result of measurement, it is included in the scope of the method of the present invention.

[0026] Examples of the nucleic acid amplification in (2) include the PCR method, digital PCR method, LAMP method, LCR method, TMA method, SDA method, RT-PCR method, RT-LAMP method, NASBA method, TRC method, TMA method, etc. These techniques have already been established in the relevant technical field, and the method can be selected according to the purpose. The nucleic acid amplification method used in the method of the present invention is preferably the PCR method, particularly the digital PCR method, but is not limited thereto.

[0027] Examples of the DNA polymerase used for nucleic acid amplification include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT and their mutants, and any of them can be used.

[0028] In the nucleic acid amplification in (2) and the hybridization of the DNA probe in (3), conditions such as temperature, time, number of cycles, pH, cation concentration, etc. may be appropriately adjusted according to the common knowledge in the relevant technical field.

[0029] In the nucleic acid amplification in (2), usually, in addition to the labeled DNA probe described above, a reagent containing components necessary for nucleic acid amplification is used. Such components vary depending on the nucleic acid amplification reaction to be performed, and known methods can be used respectively. For example, when using the PCR reaction, it is preferably to contain at least a labeled DNA probe, DNA polymerase, primer set, deoxyribonucleoside triphosphates (dNTPs), and magnesium salt. The concentration of each component may be appropriately adjusted according to the target experiment. A reagent containing these necessary components mixed or individually can also be used as a kit for detecting L. helveticus.

[0030] The primer set is not particularly limited as long as it can amplify the region containing positions 37 to 62 of SEQ ID NO: 1, and can be arbitrarily designed. For example, a DNA fragment containing the base sequence shown in SEQ ID NO: 3 can be used as the forward primer, and a DNA fragment containing the base sequence shown in SEQ ID NO: 4 can be used as the reverse primer.

[0031] (3) As the mode of detection and quantification, although not particularly limited, for example, when a fluorescent label is used for the labeling of the probe, the presence or absence of nucleic acid amplification can be determined by detecting the fluorescent signal derived from the probe using an apparatus such as a chemilumifluorometer. Then, by measuring the fluorescence intensity, the nucleic acid amplification product can be quantified and converted into the number of bacteria by a conventional method.

Examples

[0032] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0033] [1] Preparation of samples (1) Preparation of heat-killed suspension of L. helveticus The freeze-dried cells of L. helveticus MCC-1848 NITE BP-01671 strain were anaerobically cultured in MRS broth containing 0.5% raffinose. 1 mL of the culture solution was dispensed into a 15 mL test tube and autoclaved (87 °C, 1 min) to prepare heat-killed cells. Then, it was naturally cooled to 45 °C and further cooled to 25 °C by water cooling. Then, it was centrifuged (6000×g, 10 min, 4 °C), and after removing the supernatant, the heat-killed cell pellet was suspended in 0.8 mL of 0.1% Tween80-PBS. The heat-killed cells were counted by microscopy, and a heat-killed suspension of L. helveticus with 3.1×10 cells / mL was obtained. 9 9 cells / mL was obtained.

[0034] (2) Preparation of heat-killed suspension of L. gallinarum In the same manner as in (1), using the freeze-dried cells of L. gallinarum ATCC33199 strain, a heat-killed suspension of L. gallinarum with 1.3×10 9 cells / mL was obtained.

[0035] (3) Preparation of standard samples 3 g of sterilized non-fat dry milk free of L. helveticus and L. gallinarum was collected in a 50 mL sterilized tube, and 27.0 mL of 0.1% Tween80-PBS was added and diluted 10-fold. 1 mL of this was aliquoted into a 15 mL sterilized tube, and 9 mL of 0.1% Tween80-PBS was added for further 10-fold dilution, ultimately preparing a 100-fold dilution, which was used as the sample matrix for standards. Next, the L. helveticus dead cell suspension prepared in (1) was diluted with the above sample matrix for standards to obtain a diluted suspension of 1.08×10 9 cells / mL. This was further diluted 100-fold with the sample matrix for standards to prepare a standard sample (1 concentration) of 1.08×10 7 cells / mL. Similarly, dilutions were performed to prepare standard samples (5 concentration points; 0, 5.38×10 6 , 1.08×10 7 , 2.15×10 7 , 4.3×10 7 cells / mL) of L. helveticus dead cells.

[0036] (4) Preparation of samples containing L. gallinarum dead cells The L. gallinarum dead cell suspension prepared in (2) was diluted 15-fold with the sample matrix for standards prepared in (3) to prepare a 100-fold dilution of non-fat dry milk containing 8.6×10 7 cells / mL of L. gallinarum. 2. Experimental method

[0037] (5) DNA extraction of L. helveticus and L. gallinarum The standard sample of L. helveticus dead cells prepared in (3) and the sample containing L. gallinarum prepared in (4) were each collected in 0.2 mL microtubes, subjected to cold centrifugation (8000×g, 5 min, 4°C), and the supernatant was removed. Commercially available zirconia beads (450 mg of φ = 0.5 mm and 450 mg of φ = 3.0 mm) were added to the pellet, and the vortex was set to the maximum The cells were stirred at a high rotation speed for a total of 2 minutes to disrupt the cell walls of each cell. Subsequently, 0.2 mL of DNA purification solution for each cell was obtained manually using the genomic DNA extraction kit QuickGene SP kit DNA tissue Cat. No. S P-DT (manufactured by KURABO).

[0038] [2] Detection and quantification of L. helveticus (1) Example 1: Detection and quantification of L. helveticus Digital PCR was performed according to the following procedure to amplify the specific base sequence of the IS region of L. helveticus. [1] The DNA purification solution of L. helveticus obtained in (5) was diluted 6-fold with TE buffer. 2 μL of this was added to and mixed with 18 μL of the PCR master mix shown in Table 1 (10-fold dilution). 13.6 μL of the mixture was filled into a digital PCR chip for a digital PCR thermal cycler (manufactured by Applied Biosystems, Model: ProFlex Dual Flat).

[0039]

Table 1

[0040] Subsequently, each digital PCR chip was set in a digital PCR thermal cycler (manufactured by Applied Biosystems, Model: ProFlex Dual Flat), and the thermal cycle shown in Table 2 was performed.

[0041]

Table 2

[0042] After digital PCR, the number of wells emitting green fluorescence and their fluorescence intensities were measured using a dedicated chip reader, and the amplification products were measured for each chip. Based on the measurement results of the amplification products, the copy number of the specific nucleotide sequence of the IS region of L. helveticus contained in each sample subjected to digital PCR was calculated using dedicated cloud-based analysis software according to the Poisson distribution.

[0043] (2) Example 2: Confirmation of Specificity Digital PCR was performed in the same manner as in (1), except that the DNA purification solution of L. gallinarum obtained in [1](5) was used instead of the DNA purification solution of L. helveticus.

[0044] (3) Results The standard curves of Example 1 when digital PCR was performed using the DNA extracted from the standard sample of L. helveticus dead cells in (1) as a template are shown in FIG. 1 respectively. Since the PCR amplification products were detected in direct proportion to the concentration of L. helveticus dead cells in the standard sample, it can be seen that the method of the present invention can quantitatively detect the bacterium. Furthermore, Table 3 shows the measured values of the bacterial cell amounts calculated by comparing the amount of amplification products when digital PCR was performed using the DNA extracted from the L. gallinarum dead cell-containing sample in (2) as a template with the standard curve obtained in (1). L. gallinarum was not detected in Example 2. On the other hand, although PCR amplification products were detected in direct proportion to the dead cell concentration even by the method using the TaqMan probe of Comparative Example 1 (FIG. 2), since L. gallinarum was detected and quantified in Comparative Example 2, it can be seen that the probe cannot specifically detect L. helveticus. From these results, it can be seen that the method of the present invention can quantitatively detect L. helveticus with high precision, distinguishing it from L. gallinarum. From these facts, it can be understood that L. helveticus can be quantitatively detected with high precision, distinguishing it from L. gallinarum, by the method of the present invention.

[0045]

Table 3

Claims

Claim 1 A method for detecting and / or quantifying Lactobacillus helveticus in a sample, comprising: amplifying a region comprising positions 37 to 62 of SEQ ID NO: 1 of the DNA or RNA of the bacterium in the sample; using a labeled DNA probe comprising the nucleotide sequence from positions 48 to 53 of SEQ ID NO: 1 or a nucleotide sequence complementary thereto; and characterized in that the sugar constituting at least the nucleic acid corresponding to the positions of the bases from positions 48 to 53 of SEQ ID NO: 1 among the nucleic acids in the labeled DNA probe is cross-linked. A method. Claim 2 The method according to claim 1, wherein the probe comprises at least the nucleotide sequence from positions 1 to 16 of SEQ ID NO: 2 or a nucleotide sequence complementary thereto. Claim 3 The method according to claim 1 or 2, wherein amplification of the region is carried out by performing PCR using a forward primer comprising the nucleotide sequence shown in SEQ ID NO: 3 and a reverse primer comprising the nucleotide sequence shown in SEQ ID NO:

4. ​

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