Selection and quantitative method for Bifidobacterium longum subsp. longum RAPO in a sample

Abstract

The present invention relates to a method for detecting and quantifying Bifidobacterium longum subsp. longum RAPO (KCTC13773BP) in a sample. By using the present invention, Bifidobacterium longum RAPO in a sample can be detected and accurately quantified with high sensitivity.

Description

Technology Field

[0001] The present invention relates to Bifidobacterium longum in a sample ( Bifidobacterium longum subsp. longum This relates to a method for detecting and quantitatively measuring RAPO (KCTC13773BP) bacteria. Background Technology

[0003] One method for measuring the amount of specific bacteria in a sample is the Colony-forming unit (CFU) method. However, when using the Colony-forming unit (CFU) method, there is a problem of low accuracy because the specificity is low, even when using selective media.

[0004] As an alternative to this, there is a method to measure the total amount of a specific bacterium within a microorganism by calculating the amount of DNA in the sample using the real-time PCR method. However, this method also has the problem that it is difficult to accurately measure the amount of DNA in small sample sizes. Prior art literature

[0006] Korean Registered Patent No. 10-2428043 (July 28, 2022) describes a method for quantifying the number of bacteria in a sample using a PCR method. Korean Registered Patent No. 10-2363462 (February 10, 2022) describes a method for quantifying the number of bacteria in a sample using a PCR method. The problem to be solved

[0007] The present invention aims to develop and provide a method capable of detecting Bifidobacterium longum RAPO bacteria in a sample and accurately quantifying the total number of bacteria with high sensitivity. means of solving the problem

[0009] The present invention provides a primer set consisting of a forward primer, a reverse primer, and a probe that can detect the nucleic acid sequence of SEQ ID NO. 4 present in the genome of Bifidobacterium longum RAPO (KCTC13773BP) through PCR.

[0010] In the primer set of the present invention, the primer set is preferably composed of a forward primer of SEQ ID NO. 1, a reverse primer of SEQ ID NO. 2, and a probe of SEQ ID NO. 3.

[0011] In addition, the present invention comprises the steps of: (a) extracting DNA from a sample; and (b) performing digital PCR by adding a forward primer of SEQ ID NO. 1, a reverse primer of SEQ ID NO. 2, and a probe of SEQ ID NO. 3 to the extracted DNA to measure the DNA concentration of the Bifidobacterium longum RAPO (KCTC13773BP) strain in the sample. The present invention provides a method for quantifying the number of Bifidobacterium longum RAPO (KCTC13773BP) strains in a sample, characterized by including: step (c) of calculating the total number of Bifidobacterium longum RAPO (KCTC13773BP) strains by substituting the DNA concentration of Bifidobacterium longum RAPO (KCTC13773BP) strains measured in step (b) into a standard curve showing the correlation between the DNA concentration of Bifidobacterium longum RAPO (KCTC13773BP) strains and the number of Bifidobacterium longum RAPO (KCTC13773BP) strains after step (b);

[0012] In the method for quantifying the number of Bifidobacterium longum RAPO (KCTC13773BP) bacteria in a sample of the present invention, the sample may be, for example, feces. Effects of the invention

[0014] When using the present invention, Bifidobacterium longum RAPO bacteria in a sample can be detected, and the total number of bacteria can be accurately quantified with high sensitivity. Brief explanation of the drawing

[0016] Figure 1 is a 'standard curve showing the correlation between DNA concentration of RAPO strains and the number of RAPO strains,' where the X-axis represents the total number of RAPO strains in the sample and the Y-axis represents the DNA concentration (copies / µl) calculated using the digital PCR method on the sample. Specific details for implementing the invention

[0017] The present invention provides a primer set consisting of a forward primer, a reverse primer, and a probe that can detect the nucleic acid sequence of SEQ ID NO. 4 present in the genome of Bifidobacterium longum RAPO (KCTC13773BP) through PCR.

[0018] The present invention involves PCR amplifying a specific nucleic acid sequence (Sequence No. 4) within the genome of Bifidobacterium longum RAPO, and through the detection of this specific nucleic acid sequence, it was confirmed that only Bifidobacterium longum RAPO can be specifically amplified among strains of the same genus.

[0019] A primer set for amplifying a specific nucleic acid sequence of Bifidobacterium longum RAPO (SEQ No. 4) is preferably a forward primer of SEQ No. 1, a reverse primer of SEQ No. 2, and a probe of SEQ No. 3. When such primers are used, the accuracy of PCR amplification is excellent, and PCR amplification proceeds smoothly. In addition, the probe of SEQ No. 3 is a probe that increases amplification specificity during the PCR step.

[0020] Meanwhile, the present invention comprises the steps of: (a) extracting DNA from a sample; and (b) performing digital PCR by adding a forward primer of SEQ ID NO. 1, a reverse primer of SEQ ID NO. 2, and a probe of SEQ ID NO. 3 to the extracted DNA to measure the DNA concentration of the Bifidobacterium longum RAPO (KCTC13773BP) strain in the sample. The present invention provides a method for quantifying the number of Bifidobacterium longum RAPO (KCTC13773BP) bacteria in a sample, characterized by including: step (c) of calculating the total number of Bifidobacterium longum RAPO (KCTC13773BP) bacteria by substituting the DNA concentration of Bifidobacterium longum RAPO (KCTC13773BP) bacteria measured in step (b) into a standard curve showing the correlation between the DNA concentration of Bifidobacterium longum RAPO (KCTC13773BP) bacteria and the number of Bifidobacterium longum RAPO (KCTC13773BP) bacteria after step (b). In this case, the sample may be, for example, feces.

[0021] There are various types of strains in the gut microbiome, and even within the same genus, there are diverse microorganisms, making it difficult to select and quantify only specific strains.

[0022] Generally available methods for measuring the amount of specific bacteria in a sample include plating the sample onto a selective medium of the strain to be measured, culturing it, and then measuring the number. However, when using the Colony-forming Unit (CFU) method as described above, there is a problem of low accuracy due to low specificity, even when using a selective medium. Additionally, there is a method to measure the total amount of specific bacteria within microorganisms by calculating the amount of DNA in the sample using the real-time PCR method, but this method also has the problem of being difficult to accurately measure the amount of DNA in small sample volumes.

[0023] However, in the present invention, a DNA sequence (Sequence No. 4) unique to Bifidobacterium longum RAPO was discovered, and a primer set capable of specifically detecting it was developed. Then, using a digital PCR method, the total number of Bifidobacterium longum RAPO bacteria in a sample could be accurately quantified with high sensitivity.

[0024] Bifidobacterium longum targeted in the present invention ( Bifidobacterium longum subsp. longum The RAPO (KCTC13773BP) strain is a strain registered in Korean Patent No. 10-2074445 (2020.01.31) and Korean Patent No. 10-2307603 (2021.09.27).

[0025] Meanwhile, in the method for quantifying the number of Bifidobacterium longum RAPO (KCTC13773BP) bacteria in a sample according to the present invention, step (a) involves extracting DNA from the bacteria contained in the sample and measuring the DNA concentration. If the DNA concentration is less than 10 ng / µl, it is preferable to re-extract DNA from the sample. However, if this is not possible, it is preferable to concentrate the DNA so that the DNA concentration becomes 10 ng / µl or higher. This is because if the DNA concentration is too low, the PCR reaction may not proceed properly. As an example, a centrifugal vacuum concentrator may be used for concentration. At this time, there is no upper limit to the DNA concentration. As an example, the DNA concentration may be measured using a spectrophotometer or a fluorescent substance.

[0026] Meanwhile, in step (a) above, after extracting DNA from the cells contained in the sample, it is recommended to measure the absorbance at 260 nm and 280 nm and re-extract the DNA if the purity (260 / 280 nm) is less than 1.65. However, if this is not possible, it is recommended to purify the DNA to achieve a purity of 1.65 or higher. This is because various impurities, such as RNA or proteins, may be extracted along with DNA during the DNA extraction process, which can interfere with the PCR reaction and lower the accuracy. For the purification mentioned above, a DNA purification kit may be used as an example.

[0027] Meanwhile, in the present invention, a sample containing or expected to contain a RAPO strain may be used. For example, feces obtained after having experiment participants consume a probiotic product containing a RAPO strain may be used as a sample.

[0028] In the present invention, the primer of SEQ ID NO. 1 is a forward primer that exhibits excellent PCR amplification specificity for RAPO strains, and the primer of SEQ ID NO. 2 is a reverse primer that exhibits excellent PCR amplification specificity for RAPO strains. Additionally, SEQ ID NO. 3 is a probe that enhances amplification specificity during the PCR step.

[0029] Meanwhile, in the present invention, digital PCR refers to a PCR technology that quantifies DNA or RNA in a sample by dividing DNA in a sample into 8,500 microreaction vessels and replicating it. That is, digital PCR is a PCR technology that quantifies DNA or RNA in a sample by checking for amplification in microreaction vessels containing the divided sample.

[0030] In this case, the number of microreaction vessels where amplification occurs and a PCR positive reaction takes place is called the partition positive value. It is recommended to dilute or concentrate the sample so that the partition positive value displayed for each sample is 50, or the total partition value containing DNA is 95% or less. The reason for the 95% figure is that DNA does not fit into all 8,500 microreaction vessels, so it is calculated as the ratio of the number of positive reactions to the total partition value. By adjusting the partition positive value as described above, the accuracy of DNA or RNA quantified in the sample using digital PCR can be improved.

[0031] Meanwhile, in the present invention, the 'standard curve showing the correlation between the DNA concentration of RAPO strains and the number of RAPO strains' in step (c) may be derived by measuring the number of RAPO bacteria in a sample and quantifying the DNA concentration of RAPO in the sample using various methods; preferably, the DNA concentration of RAPO in the sample is quantified using a digital PCR method. In the following example, the standard curve graph of FIG. 1 could be derived.

[0033] The contents of the present invention will be explained in more detail below through the following examples. However, the scope of the present invention is not limited to the following examples but includes variations of equivalent technical concepts.

[0035] [Example 1: Method for Quantifying RAPO Strains Using RAPO Strain-Specific PCR Primers and dPCR Technology]

[0036] Digital PCR is a type of PCR technology that digitizes the PCR amplification process to accurately measure the amount of DNA or RNA in a sample. In this embodiment, Bifidobacterium longum ( Bifidobacterium longum subsp. longumUsing RAPO (hereinafter referred to as 'RAPO') specific PCR primers, probes, and digital PCR technology, the DNA value of RAPO in the sample was measured, and the number of RAPO strains in the sample was quantified using this.

[0038] 1) Construction of a standard curve representing the correlation between DNA concentration and bacterial count of RAPO strains in the sample

[0039] Prepare a total of 1 ml of saline with 10 mg of RAPO strain powder, and serially dilute to the same volume to 10 -8 After diluting to a ratio of [amount], 100 µl was removed from the final diluted product to prepare a RAPO strain suspension. A SYBR dilution was prepared as described above by adding 10 µl of SYBR to 990 µl of TDW. The prepared RAPO strain suspension and SYBR dilution were mixed at a ratio of 100:1 and reacted for 15 minutes. After adding 200 µl of the reaction mixture to each 96-well plate, the total number of bacteria in the RAPO strain suspension was measured using a FACS instrument.

[0040] Meanwhile, DNA was extracted from 10 mg of RAPO strain powder and diluted at various ratios to prepare RAPO DNA diluents (Table 2). A dPCR mixture was prepared by mixing 6.5 µl of RNase-Free Water, 3 µl of QIAcuity Probe Master Mix (4x), and 0.5 µl of a 1:1:1 mixture of 10 pmol forward primer (Table 1, SEQ No. 1), 10 pmol reverse primer (Table 1, SEQ No. 2), and 10 pmol probe (Table 1, SEQ No. 3). 2 µl of the prepared RAPO DNA diluent was mixed into the dPCR mixture.

[0041] Using a digital PCR instrument with Channel FAM, Exposure duration 700, and Gain 8, DNA concentration (copies / µl) was measured after 1 cycle of 2 minutes at 95°C and 50 cycles of 15 seconds at 95°C and 30 seconds at 60°C. The size of the product amplified using RAPO-specific PCR primers is 61 bp, and it has the sequence of SEQ ID NO. 4 (Table 1).

[0042] Sequence number division nucleotide sequence Amplification product size (bp) 1 RAPO-specific PCR primer set Forward 5'-GCCGACCCAGCCAAAC-3' 61 2 Reverse 5'-CGAGCGCGGTGTCCT-3' 3 Probe 5'-FAM-ACCCGGCCAACCAA-BHQ-1-3' 4 Product amplified using RAPO-specific PCR primers 5'-GCCGACCCAGCCAAACAGGCTTCCACAGTCGACCCGGCCAACCAACAGGACACCGCGCTCG-3'

[0044] Subsequently, a 'standard curve showing the correlation between RAPO strain DNA and the number of bacteria in the sample' was constructed with the total number of bacteria in the measured RAPO strain suspension on the x-axis and the DNA concentration (copies / µl) of the measured RAPO DNA dilution on the y-axis (Table 2, Fig. 1).

[0045] Dilution drainage dPCR concentration (copies / µl) Total bacterial count / g 1 - 4.45.E+10 0.0002 4533 8.89.E+06 0.0001 2547 4.45.E+06 0.00005 942 2.22.E+06 0.00001 161 4.45.E+05 0.000005 59 2.22.E+05

[0047] Looking at Figure 1, it can be seen that as the total number of bacteria in the RAPO strain suspension increases, the DNA concentration (copies / µl) increases proportionally. The above results mean that the graph in Figure 1 can be used as a standard curve representing the correlation between the DNA concentration of the RAPO strain and the number of RAPO strains.

[0049] 2) Implementation of the method for measuring RAPO strain concentration according to the present invention

[0050] Fecal samples were collected from participants after they had consumed a probiotic product containing the RAPO strain for three months. DNA was extracted directly from the fecal samples without dilution to prepare fecal RAPO DNA extracts. The DNA concentration of the fecal RAPO DNA extracts was prepared to be at least 10 ng / µl, and the purity (260 / 280 nm) was at least 1.65 (Table 3).

[0051] Sample number DNA concentration (ng / µl) Purity (260 / 280nm) 1 646.535 1.99 2 724.658 1.92 3 654.251 1.92 4 819.874 1.83 5 1228.400 1.85

[0053] Subsequently, the dPCR concentration was measured using a digital PCR instrument and introduced into the standard curve graph prepared in 1) above to calculate the total number of bacteria in the sample (Table 4).

[0054] Sample number dPCR concentration (copies / µl) Total bacterial count / g 1 32595 1.31.E+09 2 4115 1.67.E+08 3 3810 1.55.E+08 4 6395 2.58.E+08 5 28855 1.16.E+09

[0056] [Example 2: Identification of RAPO strains and other Bifidobacterium strains using RAPO strain-specific PCR primers and dPCR technology]

[0057] In this embodiment, the Bifidobacterium longum identified in Example 1 ( Bifidobacterium longum subsp. longum We attempted to differentiate the DNA of RAPO strains from other Bifidobacterium strains using a RAPO-specific PCR primer set and digital PCR technology.

[0059] 1) DNA extraction and concentration measurement of RAPO strains and other strains

[0060] RAPO strains and 10 other Bifidobacterium strains ( Bifidobacterium longum subsp. longum KCTC 3128, Bifidobacterium longum subsp. infantis ATCC 15697, Bifidobacterium longum subsp. infantis ATCC 15702, Bifidobacterium longum subsp. infantis ATCC 25962, Bifidobacterium longum subsp. suis ATCC 27533, Bifidobacterium animalis subsp. animalis ATCC 25527, Bifidobacterium breve Strain S1 ATCC 15700, Bifidobacterium bifidum ATCC 29521, Bifidobacterium adolescentis ATCC 15703, Bifidobacterium pseudocatenulatum ATCC 27919) was inoculated into a culture medium and cultured at 37°C under anaerobic conditions (Table 5). DNA was extracted from the culture medium of each bacterium, and its concentration and purity were measured (Table 6).

[0061] Sample number Sample Name culture medium 1 Bifidobacterium longum subsp. longum RAPO RAPO 최적배양배지 2 Bifidobacterium longum subsp. long KCTC 3128 BD MRS broth 3 Bifidobacterium longum subsp. child ATCC 15697 ATCC 260 → MRS 4 Bifidobacterium longum subsp. child ATCC 15702 ATCC 2107 5 Bifidobacterium longum subsp. child ATCC 25962 ATCC 2107 6 Bifidobacterium longum subsp. their own ATCC 27533 ATCC 2107 7 Bifidobacterium animalum subsp. animal ATCC 25527 ATCC 2107 8 Bifidobacterium breve Strain S1 ATCC 15700 ATCC 2107 9 Bifidobacterium bifidum ATCC 29521 BD MRS broth 10 Bifidobacterium adolescentis ATCC 15703 ATCC 2107 11 Bifidobacterium pseudocatenulatum ATCC 27919 ATCC 2107

[0062] sample number sample name DNA concentration (ng / ㎕) 수도 (260 / 280nm) 1 Bifidobacterium longum subsp. long RAPO 30.3 1.86 2 Bifidobacterium longum subsp. long KCTC 3128 9.57 1.78 3 Bifidobacterium longum subsp. child ATCC 15697 29.9 1.83 4 Bifidobacterium longum subsp. child ATCC 15702 218 1.89 5 Bifidobacterium longum subsp. child ATCC 25962 824 1.91 6 Bifidobacterium longum subsp. their own ATCC 27533 124 1.88 7 Bifidobacterium animalum subsp. animal ATCC 25527 992 1.63 8 Bifidobacterium breve Strain S1 ATCC 15700 846 1.94 9 Bifidobacterium bifidum ATCC 29521 1,060 1.80 10 Bifidobacterium adolescentis ATCC 15703 388 1.99 11 Bifidobacterium pseudocatenulatum ATCC 27919 386 1.98

[0064] 2) 복 균주의 RAPO 균주와 피 균주기 그리스 마이리말이 하다

[0065] Based on the measured concentration values, dilution was performed to equalize the DNA concentration values ​​of all samples. The DNA concentration of all samples was diluted to 10 ng / µl using RNase-Free Water.

[0066] Meanwhile, a dPCR mixture was prepared by mixing 6.5 µl of RNase-Free Water, 3 µl of QIAcuity Probe Master Mix (4x), and 0.5 µl of a 1:1:1 mixture of 10 pmol forward primer (Table 1, SEQ No. 1), 10 pmol reverse primer (Table 1, SEQ No. 2), and 10 pmol probe (Table 1, SEQ No. 3). 2 µl of the prepared sample DNA dilution was mixed into the dPCR mixture.

[0067] Using a digital PCR instrument with Channel FAM, Exposure duration 700, and Gain 8, DNA concentration (copies / µl) was measured after 1 cycle of 2 minutes at 95°C and 50 cycles of 15 seconds at 95°C and 30 seconds at 60°C. The size of the product amplified using RAPO-specific PCR primers is 61 bp, and it has the sequence of SEQ ID NO. 4 (Table 1).

[0068] Subsequently, the dPCR concentration (copies / µl) derived from the dPCR program was verified. A concentration value of 24,992 copies / µl was detected in the RAPO strain sample, and no valid value (partition positive value > 50) was obtained in all other samples (Table 7). The above results indicate that specific detection and quantitative measurement of the RAPO strain among Bifidobacterium strains are possible using the primer set of the present invention.

[0069] sample number sample name dPCR concentration (copies / ㎕) partition 양성 1 Bifidobacterium longum subsp. long RAPO 24,992.2 8260 2 Bifidobacterium longum subsp. long KCTC 3128 0.8 2 3 Bifidobacterium longum subsp. child ATCC 15697 1.3 4 4 Bifidobacterium longum subsp. child ATCC 15702 1.2 3 5 Bifidobacterium longum subsp. child ATCC 25962 0.6 2 6 Bifidobacterium longum subsp. their own ATCC 27533 2.7 7 7 Bifidobacterium animalum subsp. animal ATCC 25527 0.6 2 8 Bifidobacterium breve Strain S1 ATCC 15700 1.2 3 9 Bifidobacterium bifidum ATCC 29521 0.2 1 10 Bifidobacterium adolescentis ATCC 15703 18.2 47 11 Bifidobacterium pseudocatenulatum ATCC 27919 7.9 20

Claims

Claim 1 A primer set consisting of a forward primer of SEQ ID NO. 1, a reverse primer of SEQ ID NO. 2, and a probe of SEQ ID NO. 3, capable of detecting the nucleic acid sequence of SEQ ID NO. 4 present in the genome of Bifidobacterium longum RAPO of accession number KCTC13773BP via PCR. Claim 2 delete Claim 3 A step of extracting DNA from a sample (a); a step of performing digital PCR by adding a forward primer of SEQ ID NO. 1, a reverse primer of SEQ ID NO. 2, and a probe of SEQ ID NO. 3 to the extracted DNA to measure the DNA concentration of the Bifidobacterium longum RAPO strain of accession ID NO. KCTC13773BP in the sample (b); and a step of calculating the total number of Bifidobacterium longum RAPO strains of accession ID NO. KCTC13773BP after step (b), by substituting the DNA concentration of the Bifidobacterium longum RAPO strain of accession ID NO. KCTC13773BP measured in step (b) into a standard curve showing the correlation between the DNA concentration of the Bifidobacterium longum RAPO strain of accession ID NO. KCTC13773BP and the number of Bifidobacterium longum RAPO strains of accession ID NO. KCTC13773BP (c); wherein the sample Method for quantifying the number of Bifidobacterium longum RAPO bacteria of accession number KCTC13773BP. Claim 4 A method for quantifying the number of Bifidobacterium longum RAPO bacteria of accession number KCTC13773BP in a sample, characterized in that, in paragraph 3, the sample is feces.

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