KR20160093994A - RAPD primer for discrimination of lactic acid bacteria and pathogenic bacteria and uses thereof - Google Patents

Abstract

The present invention relates to a novel randomly amplified polymorphic DNA (RAPD) primer, and to a method for distinguishing between lactic acid bacteria and pathogenic bacteria using the same. The novel RAPD primer of the present invention is confirmed to stably amplify specific genes of each virus, and to express high repeatability. Accordingly, the novel RAPD primer and the method for distinguishing between lactic acid bacteria and pathogenic bacteria using the same can be effectively used for rapidly and accurately distinguishing various strains.

Description

RAPD primers for distinguishing lactic acid bacteria and pathogens and their uses {RAPD primers for discrimination of lactic acid bacteria and pathogenic bacteria and uses thereof]

The present invention relates to a novel RAPD (randomly amplified polymorphic DNA) primer and a method for distinguishing pathogenic bacteria from lactic acid bacteria.

Lactic acid bacteria are involved in natural fermentation or are widely used as industrial starters such as yogurt and cheese. In particular, Lactobacillus casei ( L. casei ) is widely used in useful fermentation products, and thus, a rapid separation into new strains excellent in fermentation efficiency and excellent in fermentation has become a main theme of research . L. casei is known to form stable microflora in the intestine, inhibiting the growth of other harmful microorganisms, thereby maintaining the intestinal environment in a healthy state and releasing anticancer substances at the same time. Along with these characteristics, it is another advantage of L. casei that it is safe to reach to the intestine without being killed from above due to its strong survival in acidity. However, lactobacillus strains present in nature are so diverse that their physiological characteristics are also diverse. Therefore, despite the long desire to select only excellent strains rapidly, the physiological and biochemical information of the target strains has not been sufficiently accumulated and systematic classification is still difficult.

Recently, the development of polymerase chain reaction (PCR) has enabled the study of randomly amplified polymorphic DNA (RAPD) using random primers. The RAPD method has been used as a means to compare the genetic relationships of several plants and microorganisms because it has the advantage of being accurate and comparing many strains at a time. Therefore, it is expected that it will be possible to select useful lactic acid bacteria at the genetic level by using RAPD. However, because of the difficulty in matching the experimental conditions up to now, it has not been applied and a primer having a specific base sequence is required for the RAPD experiment. It is difficult to independently develop and apply it.

Korean Patent Publication No. 10-2011-0007543

The present invention provides a novel RAPD primer that can be used for distinguishing lactic acid bacteria and pathogens by a randomly amplified polymorphic DNA (RAPD) method, and a method for rapidly and accurately distinguishing lactic acid bacteria and pathogens using the same.

The present invention provides Random Amplified Polymorphic DNA (RAPD) primers for discriminating pathogenic bacteria and lactic acid bacteria comprising one or more of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 3.

The present invention provides a PCR detection kit for the detection of lactic acid bacteria and pathogens, which comprises one or more RAPD (Random Amplified Polymorphic DNA) primers consisting of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 3.

The present invention also relates to a method for isolating chromosomal DNA from a strain (step 1); Performing PCR by mixing one or more RAPD (Random Amplified Polymorphic DNA) primers consisting of the nucleotide sequences of SEQ ID NOS: 1 to 3 with the chromosomal DNA isolated in the first step; And a step of electrophoresis of the sample amplified by the PCR of the second step. The present invention also provides a method of distinguishing lactic acid bacteria and pathogens.

According to the present invention, it has been confirmed that the novel RAPD primer of the present invention can stably amplify a specific gene of each bacterium and exhibits high repeatability. Therefore, the novel RAPD primer of the present invention and the distinction of lactic acid bacteria and pathogens The method can be effectively used to quickly and accurately distinguish various strains.

Fig. 1 shows the result of electrophoresis of the separated chromosomal DNA bands. Lane 1 is the chromosomal DNA isolated from E. coli JM109, lane 2 is the chromosomal DNA isolated from E. coli BL21, and lane 3 is the lactic acid bacteria host Lane casei YA-90 , lane 4 is chromosomal DNA isolated from commercially available L. casei YA-90 , and lane 5 is chromosomal DNA isolated from L. buchneri JCM 1115 . Lane 6 is a chromosomal DNA isolated from the pathogen Listeria monocytogenes , isolated from Staphylococcus aureus , a pathogen of lane 7, and lane 8 is a chromosomal DNA isolated from the pathogen Streptococcus mutans .
Figure 2 shows the result of randomly amplified polymorphic DNA (RAPD) confirmed by PCR with rp1 (random primer 1).
Figure 3 shows the result of randomly amplified polymorphic DNA (RAPD) confirmed by PCR with rp2 (random primer 2).
FIG. 4 shows the results of randomly amplified polymorphic DNA (RAPD) confirmed by PCR using rp3 (random primer 3).

The present invention can provide a Random Amplified Polymorphic DNA (RAPD) primer for discriminating pathogenic bacteria and lactic acid bacteria comprising one or more of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 3.

The lactic acid bacteria of the present invention may be selected from the group consisting of lactobacillus casei , lactobacillus buchneri, lactobacillus acidophilus and lactobacillus lactis , , And more preferably lactobacillus casei (L. casei ), but the present invention is not limited thereto.

In addition, the pathogen of the present invention may be Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Streptococcus mutans, but is not limited thereto.

The present invention can provide a PCR detection kit for distinguishing lactic acid bacteria and pathogens, which comprises one or more RAPD (Random Amplified Polymorphic DNA) primers consisting of the nucleotide sequences of SEQ ID NOS: 1 to 3.

The present invention also relates to a method for isolating chromosomal DNA from a strain (step 1); Performing PCR by mixing one or more RAPD (Random Amplified Polymorphic DNA) primers consisting of the nucleotide sequences of SEQ ID NOS: 1 to 3 with the chromosomal DNA isolated in the first step; And a step of electrophoresis of the sample amplified by the PCR of the second step.

More specifically, the step of isolating the chromosomal DNA can be carried out by a modified alkaline-soluble miniprep method. The miniprep method can further treat lysozyme, and the phenol treatment can be rapidly performed for 20 seconds to 30 seconds in order to prevent the DNA from being decomposed by physical force, but the present invention is not limited thereto.

According to one embodiment of the present invention, it was confirmed that 1 to 2 DNA bands are displayed for every primer in all the bacteria used in the present invention. In the case of rp1, it was difficult to distinguish between Escherichia coli (BL21) of the second lane and L. casei YA-90 of the third and fourth lanes by the RAPD results as shown in Table 3 and FIG. 2, And DNA bands were confirmed. From Table 4 and Fig. 3, which are the results of rp2, and Table 5 and Fig. 4, which are the results of rp3, all of the strains to be tested were sufficiently distinguished from each other by only one primer, And the band at the same position in the lane No. 4 were confirmed, amplification of the specific gene of the primers according to the present invention and high repeatability were confirmed. In addition, when the primer according to the present invention was used, L. casei and L. buchneri could be clearly distinguished even with the same lactic acid bacteria.

Therefore, when rp2 and rp3 of the present invention are used alone or in combination with rp1 in appropriate combination, lactic acid bacteria and / Pathogens can be clearly distinguished.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

≪ Referential Example 1 >

Taq DNA polymerase and dNTP were purchased from Promega (USA), and lysozyme, SDS, NaOH, and isopropyl alcohol were purchased from Sigma Co. (USA) were used. The primer was made by Macrogen (Korea). The media reagents were purchased from Difco Laboratories (USA) for sale in the pre-mixture state. Electrophoretic reagents, general reagents and glassware used the best available products available on the market.

Gradient PCR was performed using a Takara PCR thermal Cycler Dick Gradient machine (Japan). Primus MWG-AG Biotec thermal cycler (USA) was used for general PCR.

≪ Example 1 > Culture of a strain

1. Strain used

Two strains of L. casei YA-90 were used in the experiment. A total of 8 strains were used for the experiment. Two L. casei YA-90 strains were purchased and purchased separately from the one originally identified and preserved in this laboratory. Respectively. Theoretically, these two should be genetically identical, and we would like to reaffirm this through RAPD. If these two patterns always show the same pattern, this is evidence that the experiment is stable and repeatable. In addition, JM109 and BL21 were identified and stored in the laboratory. L. buchneri JCM 1115 was isolated and isolated from the marsh area of Jeju, Listeria monocytogenes , Staphylococcus aureus, and Streptococcus mutans In the laboratory, the previously identified and stored samples were used.

2. Culture and storage of strain

As shown in Table 1, 0.1% of each strain was inoculated into a suitable medium for each strain, and cultured for 15 hours. Then, glycerol was added to a final concentration of 50% and stored at -70 ° C for long-term storage. For short-term preservation for 2 weeks or less, the bacteria were cultured in a colony state on an agar plate and stored at 4 ° C.

In the case of lactic acid bacteria, the culture broth cultured in 10% skim milk was kept frozen at -20 ° C and 0.1% cultured in MRS (Difco Laboratories, USA) MRS medium was subcultured 2-3 times continuously to maintain the activated state of the cells in a high state.

Strain name badge JM109 LB BL21 LB L. casei YA-90 (laboratory) MRS L. casei YA-90 (commercial product) MRS L. buchneri JCM 1115 MRS Listeria monocytogenes BHI Staphylococcus aureus BHI Streptococcus mutans TS

≪ Example 2 > Preparation of PCR primer

Randomly amplified polymorphic DNA (RAPD) was used to design three random primers for rapid identification of E. coli, lactic acid bacteria, and pathogenic bacteria. The primer was designed according to the following criteria and was manufactured by Macrogen (Korea).

1) The nucleotide sequence of the primer should appear at least 10 times in the genome of the bacterium. 2) The distribution of GC and AT should be adjusted to make the Tm value close to the growth condition (40 ℃), 3) Between 30 ° C and 38 ° C, slightly lower than the Tm value.

In addition, no degeneracy was given to prevent excessive amplification of DNA bands.

As a result, three random primers as shown in Table 2 were prepared.

primer
name order Length Tm (占 폚) Annealing temperature
(Annealing temperature) rp1 GCC CTC AGG CG (SEQ ID NO: 1) 11 40 30 ℃ rp2 GCG CCA GGT GG (SEQ ID NO: 2) 11 40 30 ℃ rp3 CAG GCT TCG ACC (SEQ ID NO: 3) 12 40 30 ℃

Theoretically, the nucleotide sequence of rp1 and rp2 appears once every 4 ¹¹ = 4,194,303 bp, and the nucleotide sequence of rp3 appears every 1.7 x 10 ^7. Therefore, if the bacterial genome is considered to be about 3 × 10 ⁹ bp, The band was expected to be amplified, and several DNA bands were identified because they did not actually provide degeneracy. The prepared primer stock solution was stored and stored at -20 ° C and diluted with TE buffer immediately before the experiment.

Example 3: Chromosomal DNA (Chromosomal DNA) pure separation

Chromosomal DNA suitable for PCR reaction (Chromosomal DNA was minified in the alkaline lysis miniprep method. The lysozyme was further used to remove the cell wall of L. casei and the phenol treatment was rapidly performed in as mild conditions as possible to minimize the degradation of the DNA by physical forces during processing. Enzyme reaction buffer was 0.2 M sucrose and 0.5 M Tris-Cl (pH 8.0) solution. The overall procedure for Chromosomal DNA isolation is as follows.

First, 0.5 ml of glycine was added to 3 ml of the appropriate medium, 0.1% of the yeast was inoculated, and the cells were incubated at 37 ° C for at least 15 hours. The bacterial cells were transferred to a microfuge tube and centrifuged at 12,000 g for 3 minutes Respectively.

The cell pellet was washed with ST buffer [0.2 M sucrose, 0.5 M Tris-Cl (pH 8.0)], and the washed cell suspension was resuspended in 400 μl of enzyme reaction buffer, followed by addition of lOμM lysozyme The reaction was carried out at room temperature for 10 minutes.

Thereafter, 25 μl of 10% sodium dodecyl sulfate (SDS), 5 μl of proteinase K (10 mg / ml) and 5 μl of RNAase A (10 mg / ml) (25: 24: 1), and the mixture was stirred for 20 seconds and then centrifuged at 12,000 g for 5 seconds.

After centrifugation, the supernatant was transferred to a new microfuge tube, 450 μl of isopropyl alcohol was added, and the DNA was recovered by centrifugation at 12,000 g for 5 seconds.

The recovered DNA was washed with 70% alcohol, and the DNA was dissolved in 100 μl of sterilized water and stored at 4 ° C. At this time, the final concentration of DNA was 10 ng / μl.

As described above, chromosomal DNA isolated from eight strains was confirmed by electrophoresis.

As a result, as shown in FIG. 1, all of them formed a band of about 20 kb. The DNA of Staphylococcus aureus and Streptococcus mutans in 7 and 8 lanes was confirmed as a very weak band. These results suggest that the cell wall of the two strains is relatively thick and the recovery rate of chromosomal DNA is low. However, since the amplification of the RAPD band was well performed in the PCR result, the modified chromosomal DNA separation method in this study was E. coli, , And pathogenic bacteria, and the whole process took less than 1 hour.

Example 4 PCR reaction and randomly amplified polymorphic DNA (RAPD) analysis

1. PCR reaction

The PCR reaction solution was prepared as follows.

2.5 μl of separate chromosomal DNA, 5 μl of dNTP, 2.5 μl of dNTP, 2.5 μl of reaction buffer (200 mM Tris-Cl (pH 8.0), 15 mM MgCl ₂ , 1 mg / ml gelatin) and 100 pmoles of each primer Mu l) were mixed in a microfuge tube. 0.2-0.5 units of Taq DNA polymerase and H ₂ O were added to the mixed solution, and 25 μl of the whole reaction solution was prepared.

PCR was performed by post-denaturation at 95 ° C for 30 sec, denaturation at 95 ° C for 15 sec, annealing at 30 ° C to 38 ° C for 30 sec, extension at 72 ° C for 30 sec and post-extension at 72 ° C for 15 sec And the total number of reactions was 40 cycles.

The annealing temperature was set to be lower than Tm, and rp1 and rp2 were performed at 30 ° C and rp3 at 38 ° C.

Thereafter, the PCR reaction results were confirmed by electrophoresis.

2.5 μl of loading buffer [0.25% bromophenol blue, 0.25% xylene cyanol FF, 15% Ficoll (type 400)] was added to 12.5 μl of the whole reaction solution and the mixture was diluted with 1.2% agarose gel at 8 V / cm After electrophoresis, the cells were stained with 0.5 μg / ml ethidium bromide solution, and the size and intensity of the band were visually observed on a UV transilluminator. When photographing was required, the gel was photographed and stored for 45 minutes in H ₂ O.

2. Checking RAPD results

2-1. Confirmation of RAPD result by rp1 primer

As a result of RAPD using the rp1 primer, DNA bands were amplified in all strains other than Staphylococcus aureus corresponding to lane 7 as shown in Table 2 and Fig. The number of amplified bands was one to two, which was generally simple. 3 and 4 were the same L. casei YA-70, the RAPD results were expected to be the same, and the confirmation results were the same as expected.

From the above results, it was confirmed that rp1 repeatedly acts stably on the same bacteria.

However, lanes 1 and 2 were Escherichia coli (JM109, BL21), which was difficult to distinguish from L. casei YA-70 only by the band size. Thus, we have identified the need to use additional primers for the purpose of RAPD, which distinguishes by the size of the DNA band.

lane Strain name Number of amplified bands Band size (bp) One JM109 2 980, 250 2 BL21 One 650 3 L. casei YA-90 (laboratory) One 650 4 L. casei YA-90 (commercial product) One 650 5 L. buchneri JCM 1115 One 200 6 Listeria monocytogenes One 660 7 Staphylococcus aureus none - 8 Streptococcus mutans One 480

2-2. Confirmation of RAPD results by rp2 primer

As a result of RAPD using rp2 primer, DNA bands were amplified in all 8 strains as shown in Table 3 and Fig. 3, and the number of DNA bands varied from 1 to 2. The band of L. casei YA-90 (commercial product) of lane 4 was rather hazy, but the same band as L. casei YA-90 (laboratory) of lane 3 was visually confirmed.

As a result, the DNA bands 3 and 4 were confirmed at the same positions, and it was confirmed that the results of the rp2 primer test for L. casei YA 90 were specific and stable repeatedly. In addition, since the patterns of bands in different strains were different from each other, it was confirmed that even if rp2 primer alone is used, it can be effectively used for distinguishing Escherichia coli, lactic acid bacteria and other pathogens. A very small band of over 50 bp found in Streptococcus mutans , lane 8, was identified as a primer dimer.

lane Strain name Number of amplified bands Band size (bp) One JM109 One 520 2 BL21 2 520, 480 3 L. casei YA-90 (laboratory) One 240 4 L. casei YA-90 (commercial product) One 240 5 L. buchneri JCM 1115 2 600, 250 6 Listeria monocytogenes 2 180, 90 7 Staphylococcus aureus One 200 8 Streptococcus mutans One 140

2-3. Confirmation of RAPD results by rp3 primer

As a result of RAPD using rp2 primer, L. casei YA-90 of lanes 3 and 4 as shown in Table 5 and Fig. 4 were the same as those of rp1 and rp2.

From the above results, it was confirmed that rp3 exhibits stable repeatability for the same bacteria.

In addition, all other strains showed different bands, and the number of bands varied from 1 to 2.

From the above results, it was confirmed that rp3 was also sufficient for distinguishing E. coli, lactic acid bacteria, and other pathogens alone.

A small band (about 50 bp) found at the bottom of all lanes was identified as a primer dimer, with an additional cloudy band of about 460 bp found in L. buchneri JCM 1115 , the fifth lane, but more cloudy than the 200 bp band below And a non-specific amplified DNA band.

lane Strain name Number of amplified bands Band size (bp) One JM109 2 480, 140 2 BL21 2 220, 140 3 L. casei YA-90 (laboratory) One 490 4 L. casei YA-90 (commercial product) One 490 5 L. buchneri JCM 1115 2 200, 170 6 Listeria monocytogenes One 210 7 Staphylococcus aureus One 230 8 Streptococcus mutans One 250

From the above results, it was confirmed that one or two DNA bands were observed for every primer used in the present invention. In addition, in the case of rp1, it was difficult to distinguish between the E. coli (BL21) of the second lane and the L. casei YA-90 of the third and fourth lanes only by the RAPD result, but the specific DNA band was confirmed .

In the case of the rp2 and rp3 primers, all of the strains to be tested were sufficiently distinguished from each other by only one primer, and bands at the same positions were confirmed in the lanes 3 and 4 of the same lactic acid bacteria, the specific amplification of the primers according to the present invention And high repeatability. In addition, when the primer according to the present invention was used, L. casei and L. buchneri could be clearly distinguished even with the same lactic acid bacteria.

Therefore, when rp2 and rp3 of the present invention are used alone or in combination with rp1 in appropriate combination, L. casei and The pathogens can be distinguished.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> INDUSTRY ACADEMIC COOPERATION FOUNDATION KEIMYUNG UNIVERSITY <120> RAPD primer for discrimination of lactic acid bacteria and          pathogenic bacteria and uses thereof <130> ADP-2015-0024 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 11 <212> DNA <213> random primer 1 <400> 1 gccctcaggc g 11 <210> 2 <211> 11 <212> DNA <213> random primer 2 <400> 2 gcgccaggtg g 11 <210> 3 <211> 12 <212> DNA <213> random primer 3 <400> 3 caggcttcga cc 12

Claims (5)

A Random Amplified Polymorphic DNA (RAPD) primer for distinguishing lactic acid bacteria and pathogens comprising one or more of the nucleotide sequences represented by SEQ ID NO: 1 to SEQ ID NO: 3. The RAPD primer according to claim 1, wherein the lactic acid bacterium is selected from the group consisting of Lactobacillus casei (L. casei ) and Lactobacillus buchneri . The method of claim 1, wherein the pathogen is selected from the group consisting of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Wherein the RAPD primer is selected from the group consisting of Streptococcus mutans . A PCR detection kit for discriminating lactic acid bacteria and pathogens, which comprises one or more RAPD (Random Amplified Polymorphic DNA) primers consisting of the nucleotide sequences of SEQ ID NOS: 1 to 3. Isolating the chromosomal DNA from the strain (step 1);
Performing PCR by mixing one or more RAPD (Random Amplified Polymorphic DNA) primers consisting of the nucleotide sequences of SEQ ID NOS: 1 to 3 with the chromosomal DNA isolated in the first step; And
And electrophoresing the sample amplified by the PCR of the second step.

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