KR20240018800A - One step sample pretreatment method for rapid quantitative test of oral bacteria - Google Patents

Abstract

The present invention relates to a one-step sample preparation method for rapid quantitative testing of oral bacteria. In detail, in order to perform simple and fast bacterial analysis in the field, qPCR is performed immediately after one-step sample preparation without a separate bacterial DNA extraction process. A pretreatment method that can be implemented was developed. In order to dissolve bacteria well, the composition of the lysis buffer is important. If you want to perform PCR analysis directly without extracting DNA, it must not contain various components that inhibit the reaction, and adding enzymes increases costs and requires maintenance and storage. Problems may arise, and a method was developed that adds thermal lysis and some physical stimulation to maximize bacterial lysis while optimizing the composition of the lysis buffer. By using the one-step sample preprocessing method developed based on the present invention, the final bacterial rapid test capable of qPCR analysis within 1 hour can be used to detect not only oral diseases such as periodontitis and gingivitis, but also various systemic diseases related to oral health at an early stage. It is expected that it can be effectively used for diagnosis and preventive management.

Description

One step sample pretreatment method for rapid quantitative test of oral bacteria}

The present invention relates to a one-step sample preparation method for rapid quantitative testing of oral bacteria.

The oral microbiome refers to the microorganisms present in the oral cavity and their entire genetic information. It is the most common oral disease and infectious disease when the ecological balance of the oral microbiota is shifted to an imbalanced state or when the complexity of the oral microbiota increases. Dental caries and periodontal disease, which are diseases, occur. In addition, periodontal disease is known to increase the risk of developing systemic diseases such as diabetes, cardiovascular disease, and dementia, and in the case of rheumatoid arthritis, the risk of developing rheumatoid arthritis has been reported to increase by up to 1.93 times. This is Porphyromonas , a representative gram-negative anaerobic bacterium that causes periodontitis. It is known that gingivalis secretes peptidylarginine deiminase (PADs), which contributes to the production of anti-CCP antibodies (anti-cyclic citrullinated peptide Ab) in rheumatoid arthritis. Therefore, accurate analysis of oral bacteria can be of great help not only in the diagnosis of oral diseases but also in the prevention and management of systemic diseases.

Oral bacterial testing can be largely divided into sample preparation and real-time PCR analysis. While real-time PCR analysis is performed on a device, sample preparation must be performed by professional personnel and involves several steps using devices and instruments. Because a complex process must be implemented, a labor-intensive process is required.

Korean Patent No. 10-1467371 (registered on November 25, 2014)

The object of the present invention is to provide a sample pretreatment composition containing a bacterial lysis buffer containing Tris-HCl, NaCl, SDS, Nonidet P-40, and Chelex-100S as an active ingredient, and a sample pretreatment method using the composition. It is provided.

In order to achieve the above object, the present invention includes the steps of adding a sample to a bacterial lysis buffer containing Tris-HCl, NaCl, SDS, Nonidet P-40, and Chelex-100S; and reacting the bacterial lysate to which the sample was added at 70 to 95° C. for 10 to 30 minutes while vortexing or tapping the sample.

In addition, the present invention is a bacterial lysate (lysis) containing a final concentration of 50mM Tris-HCl (pH 7.4), 150mM NaCl, 0.1 to 0.2% SDS, 1% Nonidet P-40 and 2.5 to 20% Chelex-100S. Provides a composition for sample pretreatment containing buffer) as an active ingredient.

The present invention relates to a one-step sample preparation method for rapid quantitative testing of oral bacteria. In detail, in order to perform simple and fast bacterial analysis in the field, qPCR is performed immediately after one-step sample preparation without a separate bacterial DNA extraction process. A pretreatment method that can be implemented was developed. In order to dissolve bacteria well, the composition of the lysis buffer is important. If you want to perform PCR analysis directly without extracting DNA, it must not contain various components that inhibit the reaction, and adding enzymes increases costs and requires maintenance and storage. Problems may arise, and a method was developed that adds thermal lysis and some physical stimulation to maximize bacterial lysis while optimizing the composition of the lysis buffer. By using the one-step sample preprocessing method developed based on the present invention, the final bacterial rapid test capable of qPCR analysis within 1 hour can be used to detect not only oral diseases such as periodontitis and gingivitis, but also various systemic diseases related to oral health at an early stage. It is expected that it can be effectively used for diagnosis and preventive management.

Figure 1 shows qPCR analysis results (3 types of bacteria) by temperature change.
Figure 2 shows qPCR analysis results ( S. mutans ) by temperature change.
Figure 3 shows qPCR analysis results according to change in reaction time ( S. mutans , 70°C).
Figure 4 shows qPCR analysis results (3 types of bacteria) with or without physical stimulation.
Figure 5 shows qPCR results (three types of Gram-positive bacteria) for optimizing bacterial lysate for 1-step pretreatment.

The present invention includes the steps of adding a sample to a bacterial lysis buffer containing Tris-HCl, NaCl, SDS, Nonidet P-40, and Chelex-100S; and reacting the bacterial lysate to which the sample was added at 70 to 95° C. for 10 to 30 minutes while vortexing or tapping the sample.

Preferably, the bacterial lysis buffer has a final concentration of 50mM Tris-HCl (pH 7.4), 150mM NaCl, 0.1 to 0.2% SDS, 1% Nonidet P-40 and 2.5 to 20% Chelex-100S. It may include, but is not limited to this.

Preferably, the sample includes feces, urine, tears, saliva, external secretions of the skin, external secretions of the respiratory tract, external secretions of the intestinal tract, external secretions of the digestive tract, plasma, serum, blood of an object suspected of containing bacteria. , spinal fluid, lymphatic fluid, body fluid, and tissue, but is not limited thereto. More preferably, the sample may be saliva.

More preferably, the sample may be a sample containing Streptococcus sobrinus, Streptococcus mutans , or Eikenella corrodens , but is not limited thereto.

In addition, the present invention is a bacterial lysate (lysis) containing a final concentration of 50mM Tris-HCl (pH 7.4), 150mM NaCl, 0.1 to 0.2% SDS, 1% Nonidet P-40 and 2.5 to 20% Chelex-100S. Provides a composition for sample pretreatment containing buffer) as an active ingredient.

Preferably, the sample may be saliva, and more preferably, the sample contains Streptococcus sobrinus , Streptococcus mutans , or Eikenella corrodens . It may be a sample, but is not limited thereto.

The composition may additionally include a buffer solution for pH adjustment necessary for effective sample pretreatment.

Below, the present invention will be described in detail according to examples that do not limit the present invention. Of course, the following examples of the present invention are only intended to embody the present invention and do not limit or limit the scope of the present invention. Accordingly, what can be easily inferred by an expert in the technical field to which the present invention belongs from the detailed description and examples of the present invention is interpreted to fall within the scope of the rights of the present invention.

< Example 1> Bacterial lysate for 1-step pretreatment ( lysis buffer) production

A bacterial lysate was prepared using ingredients such as various surfactants, chaotropic reagents, and salts, which are chemical substances with bacterial lytic power. Since the lysate itself is used as a template for qPCR, a bacterial lysate that does not destroy the bacterial wall and interfere with qPCR amplification must be selected.

The types of candidate bacterial lysates for 1-step pretreatment are shown in Table 1.

bacterial lysate Furtherance One Lysis buffer 1-1 (LB1-1) 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% Nonidet P-40 2 Lysis buffer 1-2 (LB1-2) 50mM Tris-HCl (pH 7.4), 150mM NaCl, 5% Nonidet P-40 3 Lysis buffer 2
(LB2) 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% Nonidet P-40, 0.1% SDS, 2mM EDTA 4 Lysis buffer 3
(LB3) 50mM Tris-HCl (pH 7.4), 6M guanidinium HCl , 300mM NaCl , 1% Nonidet P-40 , 2mM EDTA 5 Lysis buffer 4-1
(LB4-1) 50mM Tris-HCl (pH 7.4), 150mM NaCl, 0.1% SDS , 1% Nonidet P-40 6 Lysis buffer 4-2
(LB4-2) 50mM Tris-HCl (pH 7.4), 150mM NaCl, 0.2% SDS , 1% Nonidet P-40 7 Lysis buffer 5
(LB5) 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5 M NaOH , 0.1% SDS 8 Lysis buffer 6
(LB6) 10 mg/ml SB3-14 *, 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% Nonidet P-40 9 Lysis buffer 7
(LB7) EMIM Ac **: 10mM Tris-HCl (9:1)

* SB3-14: 3-(N,N-Dimethylmyristylammonio)propanesulfonate

**EMIM Ac: 1-ethyl-3-methylimidazolium acetate

< Example 2> Selection of bacterial lysate and optimization of lysis conditions

Regarding bacterial lysates prepared by fixing a reaction temperature of 95°C and a reaction time of 10 minutes for a total of 9 types of lysate and performing 4 tappings (at 2 minutes and 30 second intervals), Gram-positive bacteria have a hard cell wall. qPCR was performed using a primer (F-primer) for Firmicutes and a 16s rRNA primer (16s-primer) for Gram-negative bacteria, and the lysate that amplified PCR well was selected as the first.

1. First selection of bacterial lysate

(1) Produce 3 types of Gram-positive bacteria ( S. sobrinus , S. mutans , L. casei , 1×10 ⁷ colony forming unit (CFU)) and 1 type of Gram-negative bacteria ( E. corrodens , 1×10 ⁷ CFU). 200 μL of the 9 bacterial lysates were added and incubated at 95°C for 10 minutes, followed by tapping four times. At this time, the commercially available reagent PCRopsis ^TM Reagent SRVD (Entopsis) was used as a positive control of the bacterial lysate.

(2) To confirm the bacterial lytic ability of the candidate lysates, centrifugation (12000 rpm, 10 minutes) was performed and 1 μL of the supernatant was used as a template for qPCR. For the three types of Gram-positive bacteria, make a final 20 μL of 10 μL 2 denaturation 98°C; 1 second, binding 56°C; 1 second, height 72°C; 30 seconds, 30 cycles were performed. For one type of Gram-negative bacteria, make a final 20 μL of 10 μL 2 denaturation 98°C; 1 second, binding 68°C; 30 seconds, height 72°C; 45 seconds, 30 cycles were performed. DNA of each bacterium was used as a positive control for qPCR, and DW was used as a negative control.

(3) As a result, PCR amplification was confirmed only in the lysates of LB1-1, LB4-1, and LB4-2 for 1 × 10 ⁷ CFU of bacteria.

The cycle threshold value (Ct value) results of qPCR using 9 types of bacterial lysates are shown in Table 2.

Bacteria Cycle threshold (Ct) value positivity LB1-1 LB1-2 LB2 LB3 LB4-1 F-primer S. sobrinus 17.41 24.95 0 0 24.72 24.72 S. mutans 15.97 20.48 0 0 20.26 20.26 L. casei 17.80 23.58 0 0 24.12 24.12 16s-primer E. corrodens 18.60 24.72 0 0 23.07 23.07 Bacteria Cycle threshold (Ct) value LB4-2 LB5 LB6 LB7 thanks S.R.V.D. voice F-primer S. sobrinus 24.09 0 0 24.09 22.09 0 S. mutans 20.58 0 0 20.58 19.82 0 L. casei 25.31 0 0 25.31 25.42 0 16s-primer E. corrodens 22.38 0 0 22.38 23.58 0

2. Optimization of bacterial lysis conditions

The Ct values of the first selected bacterial lysates were similar overall, and the bacterial lysis conditions were optimized using the LB4-1 solution. However, Chelex-100-small (Chelex-100S) was added to the LB4-1 solution.

(1) Reaction temperature

Bacterial lysis was performed while the reaction temperature was kept at room temperature (RT) and 95°C, respectively. At this time, the reaction time was fixed at 10 minutes. qPCR was performed using F-primer for Gram-positive bacteria and 16s primer for Gram-negative bacteria, and conditions with low Ct values were selected (Ct value is a relative measure of sample DNA concentration; the better the bacteria are lysed under the same reaction conditions) The DNA concentration in the sample becomes high, resulting in a low Ct value).

① Treat Gram-positive bacteria S. sobrinus , S. mutans , and Gram-negative bacteria E. corrodens (1× ¹⁰⁷ CFU/mL) with 1 mL of LB4-1 solution containing 10% Chelex-100S, and incubate for 10 minutes at RT and 95°C. It was left for a minute to dissolve the bacteria.

② Centrifugation (12000 rpm, 10 minutes) was performed, 1 μL of the supernatant was used as a template for qPCR, and qPCR was performed using F-primer & 16s primer. DNA of each bacterium was used as a positive control, and DW was used as a negative control.

③ As a result, it was confirmed that for 1×10 ⁷ CFU/mL of bacteria, more bacteria were destroyed at 95°C than at RT, resulting in a lower Ct value.

The qPCR analysis results (3 types of bacteria) by temperature change are shown in Table 3 and Figure 1.

Bacteria CT value Positive control RT 95℃ F-primer S. sobrinus 19.47 28.73 27.41 S. mutans 18.19 27.83 26.78 16s primer E. corrodens 18.6 27.75 24.72

Next, bacterial lysis was performed by vortexing four times at reaction temperatures of room temperature (RT), 70, and 95°C, respectively. At this time, the reaction time was fixed at 10 minutes.

① Gram-positive bacteria S. mutans (1×10 ⁷ CFU/mL) was treated with 1 mL of LB4-1 solution containing 10% Chelex-100S, reacted at RT, 70, 95°C for 10 minutes, and vortexed 4 times (2 minutes and 30 seconds apart). This was performed to dissolve the bacteria.

② Centrifugation (12000 rpm, 10 minutes) was performed, 5 μL of the supernatant was used as a template for qPCR, and qPCR was performed using S. mutans primer-probe ( Sm PP). Mix the final 20 μL with 10 μL 2X master mix, 2 μL bacterial PP ( Sm PP), and 3 μL DW, and perform initial denaturation (95°C; 10 minutes - 1 cycle) in a qPCR equipment. 2-step PCR conditions, denaturation 95 ℃; 15 seconds, binding & elongation 60°C; It was carried out for 1 minute - 45 cycles.

③ Similar to the above results, it was confirmed that the greatest amount of bacterial destruction occurred at 95°C for bacteria of 1×10 ⁷ CFU/mL. It was judged that an increase in reaction time would be necessary to achieve bacterial lysis at lower temperatures.

The qPCR analysis results by temperature change ( S. mutans ) are shown in Table 4 and Figure 2.

Bacteria CT value Positive control RT 70℃ 95℃ Sm -primer S. mutans 18.94 27.98 26.52 25.64

(2) reaction time

Samples were prepared by fixing the reaction temperature at 95°C, LB4-1 solution (Chelex-100S added), and tapping 4 times, changing the reaction time to 5 to 15 minutes each, and proceeding with bacterial lysis. qPCR was performed using F-primer for Gram-positive bacteria and 16s primer for Gram-negative bacteria.

① Gram-positive bacteria S. sobrinus , S. mutans and Gram-negative bacteria E. corrodens (1×10 ⁷ CFU/mL) was treated with 1 mL of L4 bacterial lysate containing 10% Chelex-100S and incubated at 95°C for 5, 10, and 15 minutes, respectively, while tapping four times to dissolve the bacteria.

② Centrifugation (12000 rpm, 10 minutes) was performed, 1 μL of the supernatant was used as a template for qPCR, and qPCR was performed using F-primer & 16s primer. DNA of each bacterium was used as a positive control, and DW was used as a negative control.

③ As a result, for 1×10 ⁷ CFU/mL bacteria, 10 and 15 minutes at 95°C showed similar Ct values, so the reaction time was selected as 10 minutes. However, when heat treatment is performed at 95℃, the tube used in the reaction is hot, so caution is required when using it, and it is difficult to immediately recover the sample.

The qPCR results (3 types of bacteria, 95°C) according to change in reaction time are shown in Table 5.

Bacteria CT value Positive control Reaction temperature 95℃ 5 minutes 10 minutes 15 minutes F-primer S. sobrinus 18.13 25.98 24.36 24.14 S. mutans 18.1 26.60 25.84 25.82 16s primer E. corrodens 18.92 23.80 22.98 22.15

Next, samples were prepared by fixing the reaction temperature at 70°C, LB4-1 solution (Chelex-100S added), and vortexing 4 times, changing the reaction time to 10 to 30 minutes each, and proceeding with bacterial lysis. For S. mutans, qPCR was performed using Sm PP.

① Gram-positive bacteria S. mutans (1×10 ⁷ CFU/mL) was treated with 1 mL of LB4-1 solution containing 10% Chelex-100S, and the bacteria were dissolved by vortexing four times at 70°C for 10, 20, and 30 minutes, respectively. .

② Centrifugation (12000 rpm, 10 minutes) was performed, 5 μL of the supernatant was used as a template for qPCR, and qPCR was performed using S. mutans primer-probe ( Sm PP). Mix the final 20 μL with 10 μL 2X master mix, 2 μL bacterial PP ( Sm PP), and 3 μL DW, and perform initial denaturation (95°C; 10 minutes - 1 cycle) in a qPCR equipment. 2-step PCR conditions, denaturation 95 ℃; 15 seconds, binding & elongation 60°C; It was carried out for 1 minute - 45 cycles.

③ As a result , for ¹ Therefore, it was determined that a reaction time of more than 20 minutes would be necessary for reaction at 70°C. In the 70℃ reaction, the tube is not hot, so sample recovery is possible right away.

The qPCR results (S. mutans, 70°C) according to change in reaction time are shown in Table 6 and Figure 3.

Bacteria CT value Positive control Reaction temperature 70℃ 95℃ 10 minutes 20 minutes 30 minutes 10 minutes Sm -primer S. mutans 18.94 26.52 25.48 22.64 25.64

(3) Chelex-100 type and concentration

Chelex-100 by type: Small, medium, and large products depending on dry mesh size. Chelex-100 small (S, 75-150 ㎛), a molecular biology grade cation exchange resin, and Chelex-100, a general analytical grade cation exchange resin. Samples were prepared using Chelex-100L, which has the largest wet bead size among 100 medium (M, 150-300 ㎛) and Chelex-100 large (L, 300-1180 ㎛).

① Gram-positive bacteria S. sobrinus , S. mutans (1×10 ⁷ CFU/mL), reaction temperature was fixed at 95°C, reaction time was 10 minutes, and 10% Chelex-100S and 10% Chelex-100L were treated with 1 mL of LB4-1 bacterial lysate, followed by physical stimulation (Vortex & Samples were prepared according to Tapping.

② S. sobrinus , S. mutans Bacterial lysate was used as a template, qPCR was performed using F-primer, and conditions with a low Ct value were selected.

③ As a result, Chelex-100S was selected.

The qPCR analysis results according to Chelex-100 type are shown in Table 7.

Bacteria CT value Chelex -100S Chelex -100L positivity 95℃ positivity 95℃ Vor Tap Vor Tap F-primer S. sobrinus 13.9 19.8 20.1 13.9 21.4 21.6 S. mutans 21 26.8 26.6 21 28.2 28.4

By concentration of Chelex-100S: qPCR was performed on samples prepared by changing the concentration (5% to 20%) of Chelex-100S, which was selected among Chelex-100 types.

① Gram-positive bacteria S. sobrinus , S. mutans (1×10 ⁷ CFU/mL) with fixed reaction temperature of 95°C, reaction time of 10 minutes, and physical stimulation by tapping, and 2.5%, 5%, 10%, 15%, and 20% Chelex-100S added to LB4-1. Bacteria were lysed by treating with 1 mL of bacterial lysate.

② S. sobrinus , S. mutans , and E. corrodens bacterial lysates were used as templates, and qPCR was performed using F-primer & 16s primers to select the concentration of Chelex-100S.

③ As a result, 10% Chelex-100S was selected.

The qPCR analysis results according to Chelex-100S concentration are shown in Table 8.

Bacteria Cycle threshold value ( Ct value ) positivity Chelex -100S 2.5% 5% 10% 15% 20% F-primer S. sobrinus 19.86 27.32 27.95 26.76 26.97 26.56 S. mutans 18.47 27.06 26.63 26.02 25.93 26.92 16s primer E. corrodens 18.45 24.71 23.82 22.48 22.30 22.14

(4) Optimization of physical stimulation

Samples were prepared by fixing the LB4-1 solution (Chelex-100S added) at a reaction temperature of 95°C for 10 minutes and proceeding with bacterial lysis while changing the physical stimulation between leaving as is, vortexing, and tapping. qPCR was performed using F-primer for Gram-positive bacteria and 16s primer for Gram-negative bacteria.

① Gram-positive bacteria S. sobrinus , S. mutans and Gram-negative bacteria E. corrodens (1×10 ⁷ CFU/mL) was treated with 1 mL of L4 bacterial lysate with 10% Chelex-100S added, reacted at 95°C for 10 minutes, left as is, then 4 times tapping and 4 times vortexing to remove bacteria. dissolved.

② Centrifugation (12000 rpm, 10 minutes) was performed, 1 μL of the supernatant was used as a template for qPCR, and qPCR was performed using F-primer & 16s primer. DNA of each bacterium was used as a positive control, and DW was used as a negative control.

③ As a result, it was confirmed that vortexing or tapping is better for ¹

The qPCR analysis results (3 types of bacteria) with and without physical stimulation are shown in Table 9 and Figure 4.

Bacteria CT value Positive control Static Vortexing Tapping F-primer S. sobrinus 19.47 27.41 26.01 26.49 S. mutans 18.19 26.78 25.77 25.59 16s primer E. corrodens 18.6 24.72 23.07 22.38

3. Final selection of bacterial lysate

Using optimized bacterial lysis conditions, specific probe-primer for each of the three Gram-positive bacteria ( S. sobrinus , S. mutans, L. casei ) - S. sobrinus probe-primer ( Ss PP) , Sm PP, and L. casei probe-primer ( Lc PP) - were produced and the final bacterial lysate was selected by applying the first three bacterial lysates at low bacterial counts.

At this time, two commercially available reagents - PCRopsis ^TM Reagent SRVD (Entopsis) and One-Step DNA/RNA extraction buffer (CHAIBio) were added to the positive control group, which was used as the standard for selecting the final bacterial lysate.

① Bacteria S. sobrinus , S. mutans , L. casei (2×10 ³ & 4×10 ³ CFU/mL), 1 mL of 3 types of bacterial lysate (lysis buffer 990 μL + bacteria 10 μL) was added, reaction temperature 95°C, reaction time 10 minutes, physical stimulation tapping, 10% Bacterial lysate was prepared by treatment with Chelex-100S. As a positive control group of bacterial lysate, bacterial lysate was prepared by treating with commercially available reagents Goma SRVD and Linin One-step.

② To check the lytic power of the bacterial lysate, centrifugation (12000 rpm, 10 minutes) was performed, 5 μL of the supernatant was used as a template for qPCR, and qPCR was performed using each bacterial PP. Mix the final 20 μL with 10 μL 2X master mix, 2 μL bacterial PP (S.s PP, S.m PP, L.c PP), and 3 μL DW, and perform initial denaturation (95°C; 10 minutes - 1 cycle) in a qPCR device. 2- step PCR conditions, denaturation 95°C; 15 seconds, binding & elongation 60°C; It was carried out for 1 minute - 45 cycles.

③ The positive control group of qPCR used DNA of each bacteria, and the negative control group used DW.

④ As a result, PCR amplification was confirmed in the bacterial lysate of L4 for all bacteria performed (Ct value 33 ~ 40, based on 10 bacteria), and it was finally selected as the bacterial lysate for 1-step pretreatment. Two commercially available positive controls also achieved PCR amplification, but for small numbers of bacteria, there were bacterial species that were not amplified, and they showed higher Ct values (38 to 40, based on 10 bacteria) than the PCR of the selected L4 bacterial lysate.

The qPCR results for optimizing the bacterial lysate for 1-step pretreatment are shown in Table 10 and Figure 5.

Bacteria Number of bacteria
(CFU/mL) CT value Positive control LB1-1 LB4-1 LB4-2 S.R.V.D. One step S. sobrinus 4 x 10 ³ 15.37 37.30 36.31 38.03 36.98 37.46 2 x 10 ³ 15.37 36.37 36.43 - 38.98 37.95 S. mutans 4 x 10 ³ 17.70 33.59 33.14 34.20 36.67 38.18 2 x 10 ³ 17.70 34.83 34.84 35.82 36.88 36.81 L. casei 4 x 10 ³ 19.32 - 38.56 38.26 39.32 38.83 2 x 10 ³ 19.32 - 39.67 - - -

In conclusion, the following optimized 1-step pretreatment protocol was established (based on Lab. scale).

- Bacterial lysate: LB4-1 (based on SDS/nonidetP-40) + Chelex-100S (10% w/v)

- Heat treatment: temperature 95℃, 10 minutes (or 70℃, more than 20 minutes)

- Physical stimulation: Vortexing 4 times or more (performed at 2 minutes and 30 second intervals for 10 minutes)

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

Adding the sample to a bacterial lysis buffer containing Tris-HCl, NaCl, SDS, Nonidet P-40, and Chelex-100S; and
A sample pretreatment method comprising reacting the bacterial lysate to which the sample was added at 70 to 95°C for 10 to 30 minutes while vortexing or tapping. The method of claim 1, wherein the bacterial lysis buffer (lysis buffer) contains 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1 to 0.2 % SDS, 1 % Nonidet P-40, and 2.5 to 20 % Chelex-100S. A sample pretreatment method characterized by including the final concentration. The sample preparation method according to claim 1, wherein the sample is saliva. The sample pretreatment method of claim 3, wherein the sample is a sample containing Streptococcus sobrinus, Streptococcus mutans , or Eikenella corrodens . A bacterial lysis buffer containing a final concentration of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1 to 0.2 % SDS, 1 % Nonidet P-40, and 2.5 to 20 % Chelex-100S was used as the active ingredient. A composition for sample pretreatment comprising: The composition for sample pretreatment according to claim 5, wherein the sample is saliva. The composition for sample pretreatment of claim 6, wherein the sample is a sample containing Streptococcus sobrinus, Streptococcus mutans , or Eikenella corrodens . .