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Special primer for aided-identifying strains of lactobacillus and application thereof

A Lactobacillus genus, milk identification technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of high labor intensity and long time consumption

Inactive Publication Date: 2010-09-01
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these technical means are time-consuming, labor-intensive, the identification results are affected by factors such as culture conditions, inevitable preferences and defects, etc.

Method used

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  • Special primer for aided-identifying strains of lactobacillus and application thereof
  • Special primer for aided-identifying strains of lactobacillus and application thereof
  • Special primer for aided-identifying strains of lactobacillus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, the acquisition of specific primer pair

[0025] The RNA polymerase β subunit is encoded by the rpoB gene, which has RNA polymerase activity and catalyzes the synthesis of RNA. The rpoB gene of most bacteria is highly conserved and is a single copy (Kapur, V., Li, L., Iordanescu, S., Hamrick, M., Wanger, A., Kreiswirth, B., Musser, J., 1994 .Characterizationby automated DNA sequencing of mutations in the gene(rpoB)encoding the RNApolymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas.Journal of Clinical Microbiology.32,1095-1098.).

[0026]According to L. brevis CGMCC 1.2028 (NC_008497.1), L. casei CGMCC 1.2435 (NC_008526.1), L. casei BL23 (NC_010999.1), L. acidophilus NCFM (NC_006814.1), L. acidophilus ATCC 4796 (000) , ACHN L.plantarum subsp.plantarum JCM1149(NZ_ACGZ00000000),L.rhamnosus GG(NC_013198),L.helveticus DSM 20075(NZ_ACLM00000000),Lactobacillus johnsonii ATCC 33200(NZ_ACGR00000000),L.crisp...

Embodiment 2

[0029] Embodiment 2, the application of special primers in the identification of Lactobacillus strains

[0030] After starvation for 1 week, zebrafish were fed with common pellets for 3 days, and fish species with the same size (body weight 0.19±0.02g) were selected for the experiment.

[0031] 1. Sample preparation

[0032] 17 experimental strains:

[0033] Lactobacillus brevis CGMCC 1.2028; (1.2028);

[0034] Lactobacillus casei subspecies (Lactobacillus casei) CGMCC 1.2435; (1.2435);

[0035] Lactobacillus casei subspecies (Lactobacillus casei) ATCC 393, namely Lactobacillus casei BL23; (BL23);

[0036] Lactobacillus delbrueckii subsp. bulgaricus CICC 6103; (6103);

[0037] Lactobacillus delbrueckii subsp. bulgaricus CICC 6097; (6097);

[0038] Lactobacillus reuteri (Lactobacillus reuteri) CICC 6132; (6132);

[0039] Lactobacillus reuteri (Lactobacillus reuteri) CICC 6119; (6119);

[0040] Lactobacillus acidophilus JCM1132; (1132)

[0041] Lactobacillus plantarum su...

Embodiment 3

[0092] Example 3. Application of special primers to identify the colonization of Lactobacillus strains in the zebrafish intestine

[0093] 1. Preparation of PBS and bacterial agent feed

[0094] PBS buffer (pH7.4) formula:

[0095] NaCl 8g

[0096] KCl0.2g

[0097] Na 2 HPO 4 1.42g

[0098] K H 2 PO 4 0.27g

[0099] Add water to dissolve, dilute to 1L, sterilize, store at room temperature

[0100] Preparation of "PBS pellets": Spray 100ml of PBS buffer evenly on 100g of ordinary pellets, and air-dry at 20°C.

[0101] Preparation of "LAB granules": Centrifuge 15 kinds of lactic acid bacteria, collect bacteria, resuspend in PBS, centrifuge, wash twice, evenly spray on 100g of ordinary granules, dry at 20°C, each concentration of lactic acid bacteria About 6×10 7 cells / gram of feed.

[0102] 2. Group feeding

[0103] After starvation for 1 week, zebrafish were fed with common pellets for 3 days, and fish species with the same size (body weight 0.19±0.02g) were...

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Abstract

The invention claims a special primer for aided-identifying strains of lactobacillus and the application thereof. The special primer is composed of a primer pair A and a primer pair B; the primer pair A is composed of the DNA of the sequences 13 and 14 in the sequence table; and the primer pair B is composed of the DNA of the sequences 15 and 16 in the sequence table. The PCR amplification is carried out on the total DNA of the strain to be detected by using the special primer; if the PCR product can be amplified by the primer pair A or the primer pair B, the strain to be detected is the candidate strain of lactobacillus; if no PCR product is amplified by the primer pair A or the primer pair B, the strain to be detected is the candidate strain of non-lactobacillus. In combination with the denaturing gradient gel electrophoresis (DGGE) or the sequencing technology, the invention further determines that the strain to be detected is the specific strain of lactobacillus. By adopting the special primer of the invention and combining with the PCR and the DGGE, the planting condition of the probiotics on the intestinal wall of aquatic animal can be directly identified. The method is the fastest, simplest, most convenient and effective method at present.

Description

technical field [0001] The invention relates to a special primer for assisting identification of Lactobacillus strains and application thereof. Background technique [0002] Whether it is in clinical medicine or in the prevention and control of livestock and poultry aquatic diseases, the infection of the intestinal tract by pathogenic bacteria is considered to be one of the ways to cause diseases. With the improvement of people's health awareness and the enhancement of drug resistance of pathogenic bacteria, biological therapy has gradually replaced traditional chemotherapy (such as applying antibiotics). Taking probiotics, such as Lactobacillus and Bifidobacterium, can effectively inhibit the growth of pathogenic bacteria in the intestine and benefit the intestinal microecological environment. However, to play a probiotic role, it is a necessary prerequisite to determine that probiotics can adhere tightly to intestinal mucosal epithelial cells. In some experiments, the re...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/04
Inventor 周志刚王雯雯姚斌曹雅男刘玉春何夙旭石鹏君孟昆
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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