KR101767744B1

KR101767744B1 - A primer and probe set for specific and rapid quantitative detection of Lactobacillus kefiri and a real-time PCR method using the same

Info

Publication number: KR101767744B1
Application number: KR1020150143686A
Authority: KR
Inventors: 서건호; 김동현; 김홍석; 강일병; 정다나
Original assignee: 건국대학교 산학협력단
Priority date: 2015-10-14
Filing date: 2015-10-14
Publication date: 2017-08-14
Also published as: KR20170043957A

Abstract

The present invention relates to a primer, a probe set and a real-time PCR method using the primer, the probe set and the real-time PCR method for detecting a specific rapid quantification of Lactobacillus kefiri lactic acid bacteria. Can be actively used in the fermentation process research.

Description

Technical Field [0001] The present invention relates to a primer, a probe set, and a real-time PCR method using the Lactobacillus kefiri lactic acid bacteria-specific rapid quantitative detection method and a real-time PCR method using the primer, a probe set, and a rapid-quantitative detection method using Lactobacillus kefiri and a real-

The present invention relates to a primer, a probe set, and a real-time PCR method using Lactobacillus kefiri lactic acid bacteria-specific rapid quantitative detection.

Lactobacillus kefiri is the first lactic acid bacterium isolated from Kepir fermented milk, a longevity food in the Caucasus region of the former Soviet Union. As a result of recent studies on this bacterium, various beneficial effects such as antibacterial effect, toxin neutralization ability, inhibition of intestinal epithelial cell adhesion to food poisoning bacteria, blood cholesterol lowering effect, immunoregulatory ability and extracellular polysaccharide production ability are revealed It is recognized as a new probiotic lactic acid bacteria species.

Kefir fermented milk derived from this bacterium is a complex fermented food in which more than 50 kinds of lactic acid bacteria, acetic acid bacteria and yeast form a poultry. Regular intake of kefir fermented milk is known to provide a variety of health benefits such as regular action, anti-obesity action, anti-inflammatory action, and anti-microbial action. In order to maximize the health benefit of such Kefir fermented milk, Lb. it is necessary to provide an appropriate fermentation environment for kefiri .

In order to understand the quantitative relationship between various microbial groups at the time of fermentation of kefir and to study the fermentation conditions for maximizing the activity of this bacterium, selective quantification technology for this bacterium is required, And a method for quantifying it has not been developed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above needs, and an object of the present invention is to provide Lb. and to provide a primer and a probe capable of selectively detecting quantitatively kefiri .

A further object of the present invention is to provide a method and apparatus for detecting and / and a real-time PCR method capable of selectively quantitatively detecting kefiri .

In order to accomplish the above object, the present invention provides a method for producing Lactobacillus kefiri Specific A primer set consisting of the nucleotide sequence shown in SEQ ID NO: 1 to 2 and a probe comprising the nucleotide sequence shown in SEQ ID NO: 3.

The present invention also provides a primer set and a probe of the present invention as an active ingredient, wherein Lactobacillus kefiri A composition for detection is provided.

The present invention also provides a primer set and a probe of the present invention as an active ingredient, wherein Lactobacillus kefiri A kit for detection is provided.

The present invention also relates to a method for preparing a nucleic acid sample, which comprises hybridizing a nucleic acid sample obtained from a sample with a primer set consisting of a nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: 2; And detecting the amplified target nucleic acid sequence, wherein Lactobacillus kefiri A method for detecting a strain is provided.

In one embodiment of the present invention, the amplified nucleic acid sequence may be further hybridized with a probe comprising the nucleotide sequence of SEQ ID NO: 3, but the present invention is not limited thereto.

In another embodiment of the present invention, the sample is preferably a kefir, but is not limited thereto.

In one embodiment of the present invention, the amplification of the target nucleic acid sequence is preferably performed by a polymerase chain reaction, but is not limited thereto.

In one embodiment of the present invention, the detection is preferably a real time detection, but is not limited thereto.

In addition, the present invention relates to a method for detecting the presence or absence of Lb. lactic acid in a Kefir fermented milk by extracting genomic DNA from Kefir fermented milk, and then performing a PCR using the primer set and the probe of claim 1. kefiri 's Thereby providing a method for quantifying the number.

In the present invention, the nucleotides can be synthesized and produced by an appropriate method (for example, chemical synthesis) according to methods known in the art. Nucleotides are also commercially available.

The term "primer" refers to a single primer that can act as a starting point for template-directed DNA synthesis in a suitable buffer at a suitable temperature (for example, four different nucleoside triphosphates and polymerization enzymes) Strand < / RTI > oligonucleotide.

Suitable lengths of the primers are typically 15-35 nucleotides, depending on various factors such as temperature and use of the primer. Short primers may generally require lower temperatures to form a sufficiently stable hybridization complex with the template. The terms "forward primer" and "reverse primer" refer to primers that bind to the 3 'and 5' ends of a constant region of a template amplified by a polymerase chain reaction, respectively.

The primer may be hybridized or annealed at one site of the template to form a double-stranded structure. Conditions for nucleic acid hybridization suitable for forming such a double-stranded structure are well known to those skilled in the art.

The term "probe" means a linear oligomer having a natural or modified monomer or linkage comprising deoxyribonucleotides and / or ribonucleotides that can be hybridized to a particular polynucleotide sequence . For example, the probe may be single stranded to increase the efficiency of hybridization.

The probe may be a sequence that is completely complementary to a polynucleotide sequence to be a template, but may be a substantially complementary sequence to the extent that it does not interfere with specific hybridization.

The composition may be a composition for amplifying a target nucleic acid. Amplification may be a known method for nucleic acid amplification. The amplification may be, for example, DNA amplification or RNA amplification. The amplification method may be one that requires thermal cycling or is performed at isothermal. The amplification method may be selected from the group consisting of polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), ligase chain reaction (LCR), strand displacement amplification (SDA), rolling circle amplification (RCA), and the like. The amplification method may also include an RNA amplification method. For example, reverse transcription (RT) or reverse transcription-PCR. Amplification may be to increase the number of copies of the target nucleic acid sequence or its complementary sequence. "PCR" may be a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase.

In addition, the composition may further comprise known materials required for amplification of the target nucleic acid. For example, the composition may further comprise a nucleic acid polymerase, a buffer necessary for its activity, cofactors, and / or a substrate. The nucleic acid polymerase may be a DNA polymerase, an RNA polymerase, a reverse transcriptase, or a combination thereof. Reverse transcription may be the synthesis of DNA strands complementary to the RNA sequence using RNA as a template. The nucleic acid polymerase may have a strand displacement activity. For example, it may be one or more reverse transcriptase derived from retroviruses such as HIV, MMLV, and AMV. The nucleic acid polymerase may not have 3 '-> 5' exonuclease activity. The composition may comprise a material necessary for reverse transcription or PCR amplification.

In addition, the present invention relates to a primer set of the present invention and a Lactobacillus kefiri And may be a kit for detecting a strain.

Also, for example, the probe may be labeled with a fluorescence resonance energy transfer (FRET) pair. In addition, for example, the 5'end of the probe may be labeled with one or more fluorescent markers selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, and NED, May be labeled with one or more fluorescent quenchers selected from the group consisting of 6-TAMRA, BHQ-1,2,3, and molecular groove binding non-fluorescence quencher (MGBNFQ).

The kit may be a kit for amplifying a target nucleic acid. Amplification may be a known method for nucleic acid amplification. The amplification may be, for example, DNA amplification or RNA amplification. The amplification method may be one that requires thermal cycling or is performed at isothermal. The amplification method may be selected from the group consisting of polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), ligase chain reaction (LCR), strand displacement amplification (SDA), rolling circle amplification (RCA), and the like. The amplification method may also include an RNA amplification method. For example, reverse transcription (RT) or reverse transcription-PCR. Amplification may be to increase the number of copies of the target nucleic acid sequence or its complementary sequence. "PCR" may be a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase.

In addition, the kit may further comprise known materials required for amplification of the target nucleic acid. For example, the kit may further comprise a nucleic acid polymerase, a buffer required for its activity, a cofactor, and / or a substrate. The nucleic acid polymerase

A DNA polymerase, an RNA polymerase, a reverse transcriptase, and combinations thereof. Reverse transcription may be the synthesis of DNA strands complementary to RNA sequences using RNA as a template. The nucleic acid polymerase may have a strand displacement activity. For example, it may be a reverse transcriptase derived from retroviruses such as HIV, MMLV, and AMV. The nucleic acid polymerase may not have 3 '-> 5' exonuclease activity. The kit may comprise a material necessary for reverse transcription or PCR amplification. The kit may further include instructions for use to amplify the target nucleic acid.

The present invention will be described.

In order to understand the quality control and fermentation process of Kefir , Lb. We have developed a real-time PCR method to quantitatively detect kefiri .

As can be seen from the present invention, the Primer / Probe set developed in the present invention has Lb. kefiri Positive reaction was observed only in bacteria. In addition, Lb. < RTI ID = 0.0 > L. < / RTI > present in kefir fermented milk produced under various fermentation conditions. kefiri was quickly quantified. In conclusion, the newly developed real-time PCR method of the present invention is expected to be actively utilized in quality control of kefir fermented milk and fermentation process of kefir.

Figure 1 shows the partial alignment of the recA gene sequence of 15 Lactobacillus species . The locations of the LK_508F / R primers and LK_508P probes developed in the present invention are shown in boxes.
Figure 2 shows the sensitivity and specificity of a newly developed real-time PCR using a standard strain,
Fig. 3 Lb. kefiri Standard curve for quantification.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example 1: Development of primer / probe set

The primer / probe set developed in the present invention is Lb. The rec A gene of kefiri was targeted, the sequence of which is shown in Table 1 below.

Primer / Probe Name order density LK_508F
(forward primer) 5'-GGGAGATGCCCATGTTGGT -3 '(SEQ ID NO: 1) 300 nM LK_508R
(reverse primer) 5'-AAGCTTTCGAAGTGCCTGTGA -3 '(SEQ ID NO: 2) 900 nM LK_508P (probe) 5'-FAM-TGCAAGCACGACTGAT-3'-TAMRA (SEQ ID NO: 3) 250 nM

Table 1 shows Lb. Kefiri Represents primer / probe set for specific quantitative detection.

With respect to the primer and probe design of the present invention, as shown in Fig. 1, when the target species of other genes (16S rRNA gene, etc.) and other regions of the gene are targeted, the species of Lb. We could not design a specific primer / probe set because kefiri and sequence were close to each other. However, in the present invention, since the primer / probe set for the region is designed with the recA gene as the target, among Lb. Only kefiri could be selectively detected and quantified.

Example 2: Standard strain and Food separator Verification of Sensitivity / Specificity Used

In order to evaluate the sensitivity / specificity of the primer / probe set developed in the present invention, 23 standard strains and 40 food isolates were used for verification experiments. The list of used strains is shown in Table 2 below.

Test strain Result Acetobacter aceti ATCC23746 - Bifidobacterium adolescents ATCC15703 - Bifidobacterium longum BL-720 - Enterococcus faecalis ATCC19433 - Escherichia coli ATCC25922 - Lactobacillus acidophilus ATCC4357 - Lactobacillus acidophilus (isolated from kefir grain, n = 1) - Lactobacillus brevis ATCC8287 - Lactobacillus crispatus KCTC3174 - Lactobacillus delbrueckii subsp. bulgaricus ATCC11842 - Lactobacillus gasseri KCTC3163 - Lactobacillus helveticus KCTC3545 - Lactobacillus jensenii KCTC5194 - Lactobacillus johnsonii JCM1022 - Lactobacillus kefiranofaciens subsp. kefiranofaciens ATCC43761 - Lactobacillus kefiranofaciens subsp. kefirgranum DSM10550 - Lactobacillus kefiranofaciens (isolated from kefir grain, n = 24) - Lactobacillus kefiri ATCC35411 + Lactobacillus kefiri (isolated from kefir grain, n = 13) + Lactobacillus casei ATCC393 - Lactobacillus paracasei KCTC13169 - Lactobacillus parakefiri KCTC5087 - Lactobacillus parakefiri KCTC5044 - Lactobacillus fermentum ATCC11739 - Lactococcus lactis subsp. lactis (isolated from kefir grain, n = 1) - Lactococcus lactis subsp. cremoris (isolated from kefir grain, n = 1) - Leuconostoc mesenteroides KCTC13302 - Streptococcus salivarius subsp. thermophilus KCTC5091 -

Genomic DNA was extracted from the above strains and a PCR reaction solution as shown in Table 3 below was prepared. Thereafter, the reaction mixture was reacted at 50 ° C for 2 minutes and at 95 ° C for 10 minutes using a real-time PCR system (CFX96, BioRad), and the reaction cycle consisting of 15 seconds at 95 ° C and 60 seconds at 57 ° C was repeated 40 times Repeat and get results.

material Volume TaqMan? Universal PCR Master Mix (Applied Biosystems) 12.5 μL LK_508F 2.5 μL LK_508R 2.5 μL LK_508P. 2.5 μL Extracted genomic DNA 5 μL total 25 μL

Table 3 is a table for preparation of PCR reaction solution.

Example 3: Kefir Existing within Lb. kefiri Quantification of bacteria

Lactobacillus kefiri ATCC 35411 was inoculated in 10% skim milk by anaerobic incubation in MRS medium at 27 ° C for 72 hours. After 10-fold dilution with 10% skim milk, the cells were counted on MRS medium. At the same time, genomic DNA was extracted from the inoculum and diluted stepwise, and real-time PCR reaction was performed in the same manner as above. A standard calibration curve was prepared by correlating the Ct value obtained from the real-time PCR with the number of the sample solution obtained from the MRS medium. Genomic DNA was extracted from 1 ml of Kefir fermented milk fermented at three temperature conditions (20 ° C, 25 ° C, 30 ° C) and three Kefir grain-milk ratios (2%, 5%, 10% The Ct value obtained by performing the real-time PCR in the same manner as above was substituted into the standard calibration curve to determine the Lb. kefiri 's The water was quantified.

The results of the above example of the present invention are presented as mean + standard deviation using SPSS (version 18.0, SPSS Inc.). Statistical significance between the quantitative values was analyzed by analysis of variance (ANOVA; Tukey's method). P value <0.05 was considered statistically significant.

The results of the above embodiment are as follows.

(1) Sensitivity and specificity

In a total of 63 standard strains, Lb. kefiri separated from kefir grains and ATCC35411 (n = 13) Lb. Only kefiri showed positive reaction, and the remaining 49 strains showed negative reaction (Fig. 2).

(2) Lb. present in kefir and kefir grains. Quantification of kefiri

Lb. present in kefir and kefir grains. In order to quantify kefiri , a standard calibration curve as shown in Fig. 3 was obtained.

Using the standard calibration curve above, the concentration of Lb. The results of quantifying kefiri bacteria are shown in Table 4 below.

Fermentation temperature Grain-milk ratio Kefir Number of bacteria per 1 ml of fermented milk (Log CFU, mean ± standard deviation) 20 ℃ 2% 2.31 ± 0.27 5% 2.37 ± 0.17 10% 4.5 ± 0.02 D 25 ℃ 2% 2.67 ± 0.13 A 5% 3.14 ± 0.11 B 10% 4.58 ± 0.16 D 30 ℃ 2% 3.21 ± 0.24 B 5% 3.92 ± 0.22 C 10% 4.85 ± 0.14 D

Table 4 summarizes Lb. content in the kefir fermented milk fermented under various conditions. Number of kefiri fungi

* Other alphabets in the same column mean that there is a statistically significant difference between the two values (P <0.05)

As can be seen from the above table, the higher the fermentation temperature of the kefir fermented milk, the higher the grain-milk ratio, the higher the Lb. The number of kefiri is high.

<110> Konkuk University Industrial Cooperation Corp <120> A primer and probe set for specific and rapid quantitative detection of Lactobacillus kefiri and a real-time PCR method using the same <130> HY151241 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gggagatgcc catgttggt 19 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 aagctttcga agtgcctgtg a 21 <210> 3 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 3 tgcaagcacg actgat 16

Claims

delete

SEQ ID NO: 1 and SEQ ID NO primer set consisting of the nucleotide sequence of SEQ ID NO: 2 and Lactobacillus kefiri specific detection composition comprising a probe consisting of a nucleotide sequence according to 3 as an active ingredient.

SEQ ID NO: 1 and SEQ ID NO primer set consisting of the nucleotide sequence of SEQ ID NO: 2 and Lactobacillus kefiri specific detection kit comprising a probe consisting of a nucleotide sequence according to 3 as an active ingredient.

A nucleic acid sample, obtained from the sample,
A primer set consisting of a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2;
Amplifying the target nucleic acid sequence;
Hybridizing the amplified nucleic acid sequence with a probe comprising the nucleotide sequence of SEQ ID NO: 3; And
And detecting the amplified target nucleic acid sequence,
Characterized in that the sample is a kefir. &Lt; RTI ID = 0.0 > 21. < / RTI >

delete

5. The method of claim 4, wherein the amplification of the target nucleic acid sequence is by polymerase chain reaction.

5. The method of claim 4, wherein the detection is real time detection.

After extracting genomic DNA from the Kefir fermented milk, a PCR reaction was carried out using a primer set consisting of the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 and a nucleotide sequence of SEQ ID NO: 3, The calibration curve was used to calculate Lb. kefiri 's A method for quantitatively quantifying a number.