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US-12618117-B2 - Composition for discriminating Lactobacillus acidophilus strains and discrimination method using same

US12618117B2US 12618117 B2US12618117 B2US 12618117B2US-12618117-B2

Abstract

The present disclosure relates to a composition for discriminating the species Lactobacillus acidophilus , which enables microorganisms belonging to the species Lactobacillus acidophilus to be discriminated, detected and identified simply, quickly and accurately from a target sample. Therefore, the present disclosure can be effectively used in the development of fermented milk, baby food, dairy products, livestock feed, cosmetics, health supplements, drugs for intestinal disorders, raw materials, etc. using the Lactobacillus acidophilus sp. strain.

Inventors

  • Young-Do NAM
  • SeungPyo Hong
  • Won-Hyong CHUNG
  • Mi Young Lim
  • So-Young Lee
  • Yong-Soo Park
  • Jisu Kang
  • Yun-Tai KIM
  • Hee Soon SHIN
  • Ji-hee SHIN

Assignees

  • KOREA FOOD RESEARCH INSTITUTE

Dates

Publication Date
20260505
Application Date
20190529
Priority Date
20180921

Claims (10)

  1. 1 . A method for identifying presence of Lactobacillus acidophilus strains Lactobacillus acidophilus LA1 , Lactobacillus acidophilus YT1, Lactobacillus acidophilus ATCC 4356, or Lactobacillus acidophilus NCFM in a target sample comprising a mixture of Lactobacillus acidophilus strains, comprising: a) using a primer composition to obtain a PCR product through polymerase chain reaction (PCR) by using a template comprising DNA isolated from the target sample to be discriminated; wherein the primer set comprises: i) a primer set consisting of a forward primer having the sequence of SEQ ID NO: 7 and a reverse primer having the sequence of SEQ ID NO: 16; and/or ii) a primer set consisting of a forward primer having the sequence of SEQ ID NO: 7 and a reverse primer having the sequence of SEQ ID NO: 15; wherein the forward primer further comprises one or more labels selected from the group consisting of a fluorophore, a chromophore, a chemiluminophore, a magnetic particle and a radioisotope, wherein the one or more labels is linked to the 5′-end of the forward primer; and b) identifying the presence of Lactobacillus acidophilus LA1, Lactobacillus acidophilus YT 1 , Lactobacillus acidophilus ATCC 4356, or Lactobacillus acidophilus NCFM from the PCR product; wherein identifying the presence of Lactobacillus acidophilus LA1 , Lactobacillus acidophilus YT1, Lactobacillus acidophilus ATCC 4356, or Lactobacillus acidophilus NCFM comprises determining a size of the PCR product; wherein the size of the PCR product obtained in step a) corresponds to the presence of Lactobacillus acidophilus LA1 , Lactobacillus acidophilus YT1, Lactobacillus acidophilus ATCC 4356, or Lactobacillus acidophilus NCFM; wherein if the size of the PCR product is 150-200 bp, then the Lactobacillus acidophilus strain that is present is Lactobacillus acidophilus LA1; wherein if the size of the PCR product is 300-350 bp, then the Lactobacillus acidophilus strain that is present is Lactobacillus acidophilus YT1; wherein if the size of the PCR product is 800-1000 bp, then the Lactobacillus acidophilus strain that is present is Lactobacillus acidophilus ATCC 4356 or Lactobacillus acidophilus NCFM.
  2. 2 . The method according to claim 1 , wherein the primer set consists of the forward primer having the sequence of SEQ ID NO: 7 and the reverse primer having the sequence of SEQ ID NO: 15.
  3. 3 . The method of claim 1 , wherein the forward primer further comprises the chromophore linked to the 5′-end of the forward primer.
  4. 4 . The method of claim 1 , wherein the forward primer further comprises the chemiluminophore linked to the 5′-end of the forward primer.
  5. 5 . The method of claim 1 , wherein the forward primer further comprises the magnetic particle linked to the 5′-end of the forward primer.
  6. 6 . The method of claim 1 , wherein the forward primer further comprises the radioactive isotope linked to the 5′-end of the forward primer.
  7. 7 . The method of claim 1 , wherein the PCR product has a size of 165 bp and the Lactobacillus acidophilus strain is Lactobacillus acidophilus LA1.
  8. 8 . The method of claim 1 , wherein the PCR product has a size of 320 bp and the Lactobacillus acidophilus strain is Lactobacillus acidophilus YT1.
  9. 9 . The method of claim 1 , wherein the PCR product has a size of 837 bp and the Lactobacillus acidophilus strain is Lactobacillus acidophilus ATCC 4356.
  10. 10 . The method of claim 1 , wherein the PCR product has a size of 898 bp and the Lactobacillus acidophilus strain is Lactobacillus acidophilus NCFM.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a § 371 national stage entry of International Application No. PCT/KR2019/006460, filed May 29, 2019, which claims priority to Korean Patent Application No. 10-2018-0113928, filed on Sep. 21, 2018, Korean Patent Application No. 10-2019-0040985, filed on Apr. 8, 2019, and Korean Patent Application No. 10-2019-0040986, filed on Apr. 8, 2019, the entire contents of which are incorporated herein by reference. SEQUENCE LISTING The instant application contains a Sequence Listing, which has been submitted electronically in ASCII format, and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 6, 2025, is named G1035-19001_SequenceListing.txt and is 11,449 bytes in size. TECHNICAL FIELD The present disclosure relates to a composition for discriminating the species Lactobacillus acidophilus, more particularly to a universal primer composition capable of specifically discriminating and identifying the species acidophilus from the genus Lactobacillus quickly and accurately, a primer composition for discriminating and identifying individual Lactobacillus acidophilus strains, and a primer composition for discriminating a Lactobacillus acidophilus YT1 strain. The present disclosure also relates to a kit including the primer composition and a discrimination method using the same. BACKGROUND ART Lactic acid bacteria are bacteria which degrade carbohydrates into lactic acid through metabolism. They are used to ferment foods such as yogurt, lactic acid bacteria drinks, kimchi, etc. Lactic acid bacteria exhibit various effects depending on strains. They can be classified into the five genera of Streptococcus, Lactobacillus, Leuconostoc, Bifidobacteria and Pediococcus. The microorganisms belonging to the genus Lactobacillus among them are homofermentative or heterofermentative lactobacillus bacteria and are usually found during fermentation of dairy products or vegetables. The microorganisms in the genus Leuconostoc are heterofermentative and are mainly involved in the fermentation of vegetables. Among the microorganisms belonging to the genus Lactobacillus, the species Lactobacillus acidophilus is used in various products because it has strong adherence ability to human small intestine, can endure gastric acid or bile acid due to excellent acid resistance and has many probiotic characteristics of lactic acid bacteria, such as lowering of cholesterol, improvement of diarrhea and constipation, enhancement of immunity, etc. In addition, with proven usability through various researches on applicability as probiotics, production of useful products, inhibition of the growth of harmful microorganisms, co-culture with other lactic acid bacteria or yeasts, change in gut flora, antibiotic adaptability, assimilation of cholesterol, etc., development and utilization of more functional food additives, sanitary goods, cosmetics, etc. are expected. Therefore, the importance of detection, discrimination and identification methods capable of specifically discriminating the species acidophilus from the genus Lactobacillus is increasing gradually. The most representative method of discriminating the species of microorganisms developed thus far is the analysis of the gene base sequence information of 16S rRNA. This method is disadvantageous in that the criteria for determining the 16S rRNA gene base sequence are not applicable to some Lactobacillus acidophilus strains and the method is time-consuming and labor-intensive. Recently, Genesig (UK) developed a kit for discriminating the species Lactobacillus acidophilus based on the base sequence of the recombinase A (recA) gene of Lactobacillus acidophilus. However, it is limited in that only 95% of the Lactobacillus acidophilus strains in the NCBI database can be detected. In addition, since many mutations can be made to the specific genes of the microorganisms, the methods targeting specific genes are limited in detecting the various microorganisms of the species Lactobacillus acidophilus. In addition, although the NGS analysis technology allows discrimination of the species of a specific microorganism based on the whole genome analysis result of the microorganism, it is disadvantageous in that the complicated process of comparing genome data and identifying the species and drawing up the genealogy of each strain is necessary and the method is time-consuming and labor-intensive. Also, the method is limited in detecting a number of species at the same time. The inventors of the present disclosure have made efforts to develop a species-specific universal primer composition capable of specifically detecting Lactobacillus acidophilus by identifying the CRISPR region of Lactobacillus acidophilus having useful effects for human body, which is distinguished from the microorganisms of other species, and a specific primer composition capable of specifically detecting individual Lactobacillus acidophilus strains by a