KR102237098B1 - Method for detection and quantification of probiotic yeast Saccharomyces unisporus using real-time polymerase chain reaction - Google Patents

Abstract

The present invention relates to a Saccharomyces unisporus detection primer set composed of base sequences of SEQ ID NOS: 1 and 2; a composition for detection and quantification of Saccharomyces unisporus, wherein the composition comprises the primer set; a kit; and a method for detection and quantification of Saccharomyces unisporus. A real-time polymerase chain reaction is performed, and thus only Saccharomyces unisporus can be rapidly and accurately detected and quantified. Thus, the present invention can be useful in the food field.

Description

{Method for detection and quantification of probiotic yeast Saccharomyces unisporus using real-time polymerase chain reaction}

The present invention relates to a method for detecting and quantifying Saccharomyces unisporus using real-time polymerase chain reaction.

Saccharomyces unisporus is a probiotic yeast found in fermented foods such as kefir, cumis, and cheese. These fermented foods are made in a mixed fermentation state of various microorganisms such as lactic acid bacteria, acetic acid bacteria, and yeast, and this yeast prevents excessive accumulation of lactic acid in the food based on its metabolic ability to use lactic acid as an energy source. It plays a role in helping to increase the number of lactic acid bacteria among fermented foods. In addition, in recent years, a number of studies to develop Saccharomyces unisporus as probiotics based on the non-pathogenic and probiotic properties of Saccharomyces unisporus are ongoing.

As such, as the potential usefulness of Saccharomyces unisporus in the fermented food industry and its functionality as a new probiotic is gradually increasing, Saccharomyces unisporus is rapidly searched and identified in various food materials, and The demand for quantifiable technology is expected to increase gradually. However, there is not yet a culture medium capable of discriminating Saccharomyces unisporus at the species level, and it is difficult to selectively use Saccharomyces unisporus even in other molecular biological methods or immunological confirmation methods. .

Korean Patent Publication No. 10-2015-0116319

An object of the present invention is to provide a primer set for detection of Saccharomyces unisporus consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2.

Another object of the present invention is to provide a composition for detection or quantification of Saccharomyces unisporus comprising the primer set.

Another object of the present invention is to provide a kit for detection or quantification of Saccharomyces unisporus comprising the composition.

Another object of the present invention is a primer set consisting of nucleotide sequences of SEQ ID NOs: 1 and 2 for DNA isolated from a sample; And it provides a method for detecting Saccharomyces unisporus comprising the step of performing and analyzing a real-time polymerase chain reaction using a probe consisting of the nucleotide sequence of SEQ ID NO: 3 It is to do.

Another object of the present invention is a primer set consisting of nucleotide sequences of SEQ ID NOs: 1 and 2 for DNA isolated from a sample; And a saccharide to the Ct value obtained by using a probe consisting of a nucleotide sequence of SEQ ID NO: 3 performs real-time polymerase chain reaction (real-time polymerase chain reaction) comprising the step of substituting a standard curve My process Younis porous (Saccharomyces unisporus ) to provide a method of quantification.

In order to achieve the above object, the present invention provides a primer set for detection of Saccharomyces unisporus consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2.

In addition, the present invention provides a composition for detection or quantification of Saccharomyces unisporus comprising the primer set.

In an embodiment of the present invention, the composition may further include a probe consisting of the nucleotide sequence of SEQ ID NO: 3.

In one embodiment of the present invention, the probe may have a fluorescent labeling factor (reporter dye) bound to the 5'-end, and a fluorescence inhibitor (quencher) bound to the 3'-end, and the fluorescence Coloring agents are FAM (6-carboxyfluorescein), HEX (hexachloro-6-carboxyfluorescein), TET (tetrachlorofluorescein), Texas red, rhodamine green, rhodamine red, and tetramethylrhodamine. (tetramethylrhodamine), NED (N-(1-naphthyl) ethylenediamine), Cy5 (cyanine-5) and Cy3 (cyanine-3) may be selected from the group consisting of, the quenching agent TAMRA (6-carboxytetramethylrhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), DDQ (Deep Dark Quencher), Blackberry Quencher, Iowa black and NFQ-MGB ( Nonfluorescent quencher-minor groove binder) may be selected from the group consisting of, but is not limited thereto.

In addition, the present invention provides a kit for detection or quantification of Saccharomyces unisporus comprising the composition.

In an embodiment of the present invention, the kit may further include a DNA polymerase, dNTP, and a buffer.

In an embodiment of the present invention, the kit may be performed by a real-time polymerase chain reaction method.

In addition, the present invention is a primer set consisting of the nucleotide sequence of SEQ ID NO: 1 and 2 for the DNA isolated from the sample; And it provides a method for detecting Saccharomyces unisporus comprising the step of performing and analyzing a real-time polymerase chain reaction using a probe consisting of the nucleotide sequence of SEQ ID NO: 3 do.

In one embodiment of the present invention, the sample may be a microorganism present in food.

In one embodiment of the present invention, when the Ct value is confirmed in the analysis, it may be determined that Saccharomyces unisporus is present in the sample.

In addition, the present invention is a primer set consisting of the nucleotide sequence of SEQ ID NO: 1 and 2 for the DNA isolated from the sample; And a saccharide to the Ct value obtained by using a probe consisting of a nucleotide sequence of SEQ ID NO: 3 performs real-time polymerase chain reaction (real-time polymerase chain reaction) comprising the step of substituting a standard curve My process Younis porous (Saccharomyces unisporus ) provides a method for quantification.

In one embodiment of the present invention, the sample may be a microorganism present in food.

A primer set consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2 according to the present invention; And by performing a real-time polymerase chain reaction using a probe consisting of the nucleotide sequence of SEQ ID NO: 3 , only Saccharomyces unisporus can be detected and quantified quickly and accurately, so it can be usefully used in the food field. .

1 is a result of verifying sensitivity and specificity by performing real-time PCR using a primer set and a probe for detection of Saccharomyces unisporus.
FIG. 2 shows a standard calibration curve prepared by corresponding to the Ct value obtained in real-time PCR and the number of yeast in the sample solution obtained by plate-plating on Potato dextrose agar.

The present invention is a primer set for detection or quantification of Saccharomyces unisporus in probiotic yeast; A composition for detection or quantification of Saccharomyces unisporus comprising the primer set; Kit; A method of detecting Saccharomyces unisporus using this; And a method of quantification.

The term "primer" in the present invention is a base sequence having a short free 3'-terminal hydroxyl group, which can form a base pair with a complementary template, and the starting point for template strand copying It refers to a short nucleotide sequence that acts as. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (DNA polymerase or reverse transcriptase) and four different deoxynucleoside triphosphates (dNTPs) at an appropriate buffer and temperature.

The primer set of the present invention is composed of forward and reverse primers for amplifying genes specifically present in Saccharomyces unisporus. The primer set of the present invention sufficiently considers conditions such as the length of the primer, the Tm value, the GC content of the primer, and the prevention of the formation of a dimer by the self-complementary sequence in the primer, and Saccharomyces unisporus. It is carefully designed to maximize the sensitivity and specificity for detection of (Saccharomyces unisporus ).

The primer set of the present invention can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known method. These nucleotide sequences can also be modified through various methods known in the art. Examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides and modifications between nucleotides, such as uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.). ) Or to a charged linker (e.g. phosphorothioate, phosphorodithioate, etc.). In addition, the nucleotide sequence of the primer set of the present invention can be modified using a label that can directly or indirectly provide a detectable signal.

The probe of the present invention may be labeled with a fluorescent material at the end of the probe. A fluorescent labeler (reporter dye) may be bound to the 5'-end of the probe, and a fluorescence suppressor (quencher) may be bound to the 3'-end.

Any material developed to date may be used as the fluorescent labeling factor, for example, 6-carboxyfluorescein (FAM), hexachloro-6-carboxyfluorescein (HEX), tetrachlorofluorescein (TET), and Texas red. , Rhodamine green, rhodamine red, tetramethylrhodamine, NED (N-(1-naphthyl) ethylenediamine), Cy5 (cyanine-5) or Cy3 (cyanine-3) May be used, preferably FAM (6-carboxyfluorescein) may be used.

The fluorescence inhibitors include TAMRA (6-carboxytetramethylrhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), DDQ (Deep Dark Quencher), and Blackberry Quencher. ), Iowa black or and NFQ-MGB (nonfluorescent quencher-minor groove binder) may be used, preferably NFQ-MGB (nonfluorescent quencher-minor groove binder) may be used.

In the present invention, the polymerase chain reaction comprises: (i) an initial denaturation step; (ii) repeating one cycle consisting of denaturation, annealing, and extension several to tens of times; And (iii) a final heat treatment step; Alternatively, it may be carried out through a thermal cycle program that suitably transforms these steps.

Specifically, PCR conditions in the present invention include initial denaturation by heat treatment at 90 to 95°C for 5 to 20 minutes, and then denaturation at 90 to 95°C for 10 seconds to 2 minutes; Annealing step (annealing) for 10 seconds to 2 minutes at 54 ~ 60 ℃ (Tm); And PCR amplification may be performed by repeating the synthesis step (extension) a total of 10 to 50 times at 70 to 75°C for 10 seconds to 2 minutes, and the reaction temperature and reaction time conditions may be appropriately modified by a person skilled in the art. . More preferably, the denaturation was performed at 55° C. for 2 minutes and 95° C. for 10 minutes and then repeated 40 to 50 times at 95° C. for 15 seconds and 60 seconds at 60° C.

In the present invention, any method known in the art may be used to analyze the size of the PCR amplification product. For example, the size of the amplification product can be known through comparison with a target size marker by agarose or polyacrylamide gel electrophoresis.

In the present invention, the real-time polymerase chain reaction is an experimental method that enables absolute or relative quantification of the target DNA by fluorescence emission at the same time as the amplification of the target DNA, and the step of analyzing by electrophoresis is omitted, and the degree of amplification of the amplified product is automated. By digitizing, detection and quantification are possible quickly and accurately. In the case of performing the above method, it can be performed using a probe that specifically binds to the amplified sequence in the target DNA and tagged with a fluorescent labeling factor and a fluorescent inhibitor, and a fluorescent substance such as SYBR-Green without the use of a probe. It can also be done using. The results of the real-time polymerase chain reaction can be analyzed using a Ct (cycle threshold) value.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

Example 1. Development of a primer set

The present inventors developed a primer set and a probe that specifically binds to the probiotic yeast Saccharomyces unisporus , and the sequence thereof is shown in Table 1 below.

Primer/Probe name order density SUDH_F
(forward primer) 5'-GACGTCCTGCTGGACATCTTC-3' (SEQ ID NO: 1) 250 nM SUDH_R
(reverse primer) 5'-GGCCGCCTCGCTAACC-3' (SEQ ID NO: 2) 250 nM SUDH_P
(probe) 5'- FAM-TACTAGGTTTTACCAATTCG-3'-MGB-NFQ
(SEQ ID NO: 3) 250 nM

Example 2. Verification of sensitivity and specificity using standard and food-separated yeast strains

In order to evaluate the sensitivity and specificity of the developed primer set and probe, a verification experiment was conducted using 15 standard yeast strains and yeast strains isolated from food. The list of yeast strains used is shown in Table 2 below.

Test strain Result Saccharomyces unisporus KU26S1 + Saccharomyces unisporus KU2603 + Saccharomyces unisporus DHIB7 + Candida inconspicua ATCC 16783 - Candida sake ATCC 22021 - Kluyveromyces aestuarii ATCC 18862 - Kluyveromyces dobzhanskii ATCC 24175 - Kluyveromyces lactis var. lactis KCTC 17616 - Kluyveromyces marxianus ATCC 46537 - Kluyveromyces marxianus KCTC 17212 - Kluyveromyces marxianus 16241A1 - Kluyveromyces marxianus 16245M1 - Kluyveromyces nonfermentans KCTC 17682 - Saccharomyces cerevisiae ATCC 6037 - Saccharomyces cerevisiae ATCC 7752 -

After extracting genomic DNA from the 15 strains, a PCR reaction solution as shown in Table 3 was prepared. Thereafter, the reaction solution was reacted at 50℃ for 2 minutes and at 95℃ for 10 minutes using a real-time PCR system (CFX96, BioRad), followed by 40 reaction cycles consisting of 15 seconds at 95℃ and 60 seconds at 60℃. Repeatedly obtained the result. After the reaction was completed, the data was analyzed using CFX manager software v3.1 (BioRad).

material Volume TaqMan® Universal PCR Master Mix
(Applied Biosystems) 12.5 μL SUDH_F 2.5 μL SUDH_R 2.5 μL SUDH_P 2.5 μL Extracted genomic DNA 5 μL total 25 μL

As a result of performing TaqMan probe real-time PCR using the primer set and probe, Saccharomyces unisporus among a total of 15 strains Only IBDH7 , KU26S1 and KU2603 produced a specific PCR product with a 63 bp amplification product, and showed a positive reaction (FIG. 1), and all of the remaining 12 strains showed a negative reaction.

Therefore, when real-time PCR was performed using the prepared primer set and probe, it was confirmed that it showed 100% sensitivity and 100% specificity in detecting Saccharomyces unisporus. I did.

Example 3. Saccharomyces unisporus present in food ( Saccharomyces unisporus ) Of quantification

The present inventors performed an experiment to quantify Saccharomyces unisporus contained in food. First, Saccharomyces unisporus After stepwise dilution of the DHIB7 strain, a real-time PCR reaction was performed, and a standard calibration curve was prepared by matching the Ct value obtained from the real-time PCR with the number of yeast in the sample solution obtained by plate-plating on Potato dextrose agar. Then, after extracting a genomic DNA from Kefir fermented milk 1ml of the food, the same way as real-time the Ct values obtained by performing PCR with saccharide present in the fermented milk peer Ke By substituting the standard curve My process Younis porous (Saccharomyces unisporus ) of The number was quantified.

[Equation 1]

Y = -3.583 X + 35.426 (R ² = 0.992)

As a result of quantifying the number of Saccharomyces unisporus present in kefir fermented milk using the standard calibration curve, ^{3.24 x 10 5} CFU/ml, at 36 hours fermentation in kefir fermented milk 5.36 x 10 ⁵ CFU / ml, when fermented for 48 hours, 8.36 x 10 ⁴ CFU / ml of Saccharomyces unisporus was found to be contained.

Accordingly, the primer set of the present invention and the probe saccharide as MY process Younis porous (Saccharomyces unisporus) only specific can be detected by, my process Younis porous (Saccharomyces unisporus) the rapid quantitative within 1.5 hours saccharide present in the food It was confirmed that it can be done.

<110> Konkuk University Industrial Cooperation Corp <120> Method for detection and quantification of yeast using real-time polymerase chain reaction <130> 2019-I293 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SUDH_F (forward primer) <400> 1 gacgtcctgc tggacatctt c 21 <210> 2 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> SUDH_R (reverse primer) <400> 2 ggccgcctcg ctaacc 16 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SUDH_P (probe) <400> 3 tactaggttt taccaattcg 20

Claims (14)

Including a primer set consisting of the nucleotide sequence of SEQ ID NO: 1 and 2 and a probe consisting of the nucleotide sequence of SEQ ID NO: 3
A composition for specific detection or quantification of Saccharomyces unisporus. delete delete The method of claim 1,
The probe is a composition characterized in that a fluorescent labeling factor (reporter dye) is bound to the 5'-end, and a fluorescence suppressor (quencher) is bound to the 3'-end. The method of claim 4,
The fluorescent labeling factor is FAM (6-carboxyfluorescein), HEX (hexachloro-6-carboxyfluorescein), TET (tetrachlorofluorescein), Texas red (texas red), rhodamine green (rhodamine green), rhodamine red (rhodamine red), tetra A composition, characterized in that it is selected from the group consisting of methylrhodamine, NED (N-(1-naphthyl) ethylenediamine), Cy5 (cyanine-5), and Cy3 (cyanine-3). The method of claim 4,
The fluorescence inhibitors are TAMRA (6-carboxytetramethylrhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), DDQ (Deep Dark Quencher), and Blackberry Quencher. , Iowa black (Iowa black) and NFQ-MGB (nonfluorescent quencher-minor groove binder) composition, characterized in that selected from the group consisting of. A kit for specific detection or quantification of Saccharomyces unisporus comprising the composition of claim 1. The method of claim 7,
The kit further comprises a DNA polymerase, dNTP, and a buffer. The method of claim 7,
The kit is a kit, characterized in that carried out by a real-time polymerase chain reaction (real-time polymerase chain reaction) method. A primer set consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2 for the DNA isolated from the sample; And specificity of Saccharomyces unisporus in the sample comprising the step of performing and analyzing a real-time polymerase chain reaction using a probe consisting of the nucleotide sequence of SEQ ID NO: 3 Detection method. The method of claim 10,
The method, characterized in that the sample is a microorganism present in food. The method of claim 10,
When the Ct value is confirmed in the analysis, it is determined that Saccharomyces unisporus is present in the sample. A primer set consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2 for the DNA isolated from the sample; And substituting the Ct value obtained by performing a real-time polymerase chain reaction using a probe consisting of the nucleotide sequence of SEQ ID NO: 3 to a standard calibration curve. ( Saccharomyces unisporus ) specific quantification method. The method of claim 13,
The method, characterized in that the sample is a microorganism present in food.

Images