KR-20260065688-A - PLASMID-SPECIFIC MULTIPLEX PRIMER SETS FOR DETECTING STARTER CULTURE FOR KIMCHI

Abstract

The primer set composition of the present invention can be utilized in a multiplex PCR method, and since it allows for testing many samples at once, it enables differentiation between similar microbial species at low cost and high efficiency. In addition, it can accurately detect only the starter culture Leuconostoc mesenteroides WiKim0121 strain in kimchi, and by rapidly identifying the starter culture in kimchi, it can be utilized for quality control of kimchi, which is undergoing fermentation at any given moment.

Inventors

• 이모은
• 송정희
• 김다운
• 김호명
• 장지윤

Assignees

• 한국식품연구원

Dates

Publication Date

20260511

Application Date

20241101

Claims (5)

1. A primer set composition for detecting the Leuconostoc mesenteroides WiKim0121 strain, comprising one or more primer sets selected from the group consisting of: a primer set consisting of the nucleotide sequences of SEQ ID NO. 1 and SEQ ID NO. 2; a primer set consisting of the nucleotide sequences of SEQ ID NO. 3 and SEQ ID NO. 4; a primer set consisting of the nucleotide sequences of SEQ ID NO. 5 and SEQ ID NO. 6; and a primer set consisting of the nucleotide sequences of SEQ ID NO . 7 and SEQ ID NO. 8.
2. A kit for detecting the Leuconostoc mesenteroides WiKim0121 strain comprising a primer set composition according to claim 1.
3. a) a step of amplifying a target sequence by performing an amplification reaction using DNA isolated from a sample as a template and a primer set composition according to claim 1; and b) a step of detecting the amplification product obtained from the amplification step to determine the presence or absence of the Leuconostoc mesenteroides WiKim0121 strain; comprising a method for detecting the Leuconostoc mesenteroides WiKim0121 strain.
4. In paragraph 3, A method for detecting a strain, wherein the amplification reaction is one or more selected from the group consisting of polymerase chain reaction (PCR), multiplex-PCR, real-time PCR, ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, strand displacement amplification, and Qβ replicase.
5. In paragraph 4, A method for detecting a strain in which the detection of the amplified product is one or more selected from the group consisting of gel electrophoresis, capillary electrophoresis, DNA chip, radiometric measurement, fluorescence measurement, and phosphorescence measurement.

Description

Plasmid-Specific Multiplex Primer Set for Detecting Starter Culture for Kimchi Production The present invention relates to a plasmid-specific multi-complex primer set for detecting starter cultures for kimchi production and a detection method using the same. Kimchi is a non-sterilized fermented food that ferments naturally through the interaction of various microorganisms present in raw materials; as such, the succession patterns and community development of microbial communities are important factors determining kimchi quality. Starter cultures are being developed and applied as a method to improve the quality of industrially mass-produced kimchi (extending shelf life, standardization, enhancing hygiene, imparting functionality, etc.), and measuring the dominance rate of starter cultures within kimchi—that is, the management and traceability of starter cultures—is a critical issue in terms of quality control and maintenance. While plate culture is the most commonly used method for identifying and monitoring specific microorganisms, or fermentation starter cultures, from food, it has disadvantages such as long incubation times, the need to use selective media, and the requirement to define specific culture conditions. In particular, it is very difficult to distinguish between strains of lactic acid bacteria using plate culture because their nutritional requirements and culture conditions are nearly identical. As alternatives to plate culture methods, polymerase chain reaction (PCR)-based RADP, PCR-PFLP, and 16S rDNA-based PCR are known, and denaturing gradient gel electrophoresis (DGGE) is a method for measuring microorganisms during the mixing process. Compared to plate culture methods, PCR methods are sensitive, selective, fast, economical, and convenient, and multiplex PCR is characterized by the ability to measure a large number of microorganisms at once and to distinguish between similar microorganisms. PCR detection methods utilizing primer sets developed to date include sets designed to detect specific microbial species from food (e.g., Weissella koreensis in kimchi) and to detect only specific strains within the same species (e.g., Bifidobacterium bifidum BGN4). While these primers were developed to specifically bind only to the chromosomal DNA or 16S rRNA sequences of target microorganisms, in the case of lactic acid bacteria, the chromosomal DNA nucleotide sequences and 16S rRNA sequences among species exhibit very high homology, making it difficult to identify specific nucleotide sequences capable of distinguishing particular strains. Plasmids are DNA molecules that exist independently of the chromosomes within bacterial cells and can replicate autonomously; consequently, even within the same species of lactic acid bacteria, the number and size of intracellular plasmids vary depending on the strain. In other words, there are differences in their nucleotide sequences. Meanwhile, the Leuconostoc mesenteroides WiKim0121 strain, deposited under accession number KFCC11890P, is a probiotic that possesses the general intestinal health and immune-enhancing effects of lactic acid bacteria and is known to ferment kimchi to an excellent flavor by producing metabolites such as lactic acid and mannitol, which are generally known as the main factors in producing the cool and clean taste of kimchi. Accordingly, there is a need to develop a new technology that can rapidly identify Leuconostoc mesenteroides WiKim0121 used as a starter culture in kimchi by using a set of multiple complex primers that can specifically bind to the plasmid nucleotide sequence of the Leuconostoc mesenteroides WiKim0121 strain developed as a starter culture for kimchi production. Figure 1 is a figure showing the genome map of the Leuconostoc mesenteroides WiKim0121 strain according to one embodiment of the present invention. FIG. 2 is a figure showing the PCR results using a primer set specific to the Leuconostoc mesenteroides WiKim0121 strain according to one embodiment of the present invention. Figure 3 is a figure showing the results of evaluating the detection specificity for the Leuconostoc mesenteroides WiKim0121 strain according to one embodiment of the present invention. Figure 4 is a figure showing the results of evaluating the detection sensitivity of WiKim0121 in kimchi prepared using the Leuconostoc mesenteroides WiKim0121 strain according to one embodiment of the present invention as a starter culture. Figure 5 is a figure showing the results of a comparative evaluation of WiKim0121 detection according to the fermentation period of kimchi prepared using Leuconostoc mesenteroides WiKim0121 as a starter culture according to one embodiment of the present invention. The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below but may be implemen