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Molecular markers, detection primers and detection methods for identifying Lactobacillus plantarum and Lactobacillus pentosus

A technology of Lactobacillus pentosus and Lactobacillus plantarum, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., to achieve the effects of reduced detection cost, high specificity, and short detection cycle

Active Publication Date: 2022-05-06
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some scholars design interspecies-specific primers based on the recA gene sequence shared by Lactobacillus plantarum, combined with nested PCR technology, can distinguish L. plantarum from L. pentosus in Lactobacillus plantarum, but a second PCR amplification is required

Method used

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  • Molecular markers, detection primers and detection methods for identifying Lactobacillus plantarum and Lactobacillus pentosus
  • Molecular markers, detection primers and detection methods for identifying Lactobacillus plantarum and Lactobacillus pentosus
  • Molecular markers, detection primers and detection methods for identifying Lactobacillus plantarum and Lactobacillus pentosus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1: Identification of Lactobacillus plantarum

[0060] 1. Bacterial DNA extraction

[0061] Take 1 mL of the bacterial culture and centrifuge at 12000r / min for 5min, discard the supernatant, collect the bacteria, add 50μL of the lysate of the bacterial DNA extraction kit, suspend and mix well, place in a water bath at 100°C for 15min, ice bath for 3min, and centrifuge at 12000r / min After 10 minutes, the supernatant was taken for later use, and the supernatant contained bacterial DNA as template DNA.

[0062] 2. PCR amplification

[0063] PCR uses a 25 μL reaction system, in which 1.0 μL of the upstream and downstream specific primers of the target DNA at a concentration of 10 μmol / L, 0.5 μL of 1.5U Taq DNA polymerase, and 25 mmol / L MgCl 2 2 μL, 2 μL of 10 mmol / L dNTP, 2.5 μL of 10×PCR buffer, 2.0 μL of DNA template, and 25 μL of sterile deionized water. PCR amplification conditions were: 95°C pre-denaturation for 5 min; 95°C denaturation for 1 min, 58°C an...

Embodiment 2

[0066] Example 2: Identification of Pentobacter

[0067] 1. Bacterial DNA extraction

[0068] Take 1 mL of the bacterial culture and centrifuge at 12000r / min for 5min, discard the supernatant, collect the bacteria, add 50μL of the lysate of the bacterial DNA extraction kit, suspend and mix well, place in a water bath at 100°C for 15min, ice bath for 3min, and centrifuge at 12000r / min After 10 minutes, the supernatant was taken for later use, and the supernatant contained bacterial DNA as template DNA.

[0069] 2. PCR amplification

[0070] PCR uses a 25 μL reaction system, in which 1.0 μL of the upstream and downstream specific primers of the target DNA at a concentration of 10 μmol / L, 0.5 μL of 1.5U Taq DNA polymerase, and 25 mmol / L MgCl 2 2 μL, 2 μL of 10 mmol / L dNTP, 2.5 μL of 10×PCR buffer, 2.0 μL of DNA template, and 25 μL of sterile deionized water. PCR amplification conditions were: 95°C pre-denaturation for 5 min; 95°C denaturation for 1 min, 58°C annealing for 45 ...

Embodiment 3

[0073] Example 3: Identification of Lactobacillus plantarum and Lactobacillus pentosus in actual samples

[0074] 1. Sample processing

[0075] For cheese and yogurt samples sold in the market, take 1-2g and add them to 10mL MRS broth, incubate at 37°C for 48h, take 1mL of bacterial liquid, collect the bacterial cells by centrifugation, add 50μL of DNA lysate, suspend and mix well, and place in a water bath at 100°C for 15min , ice-bathed for 3 minutes, centrifuged at 12,000 r / min for 10 minutes, and the supernatant was taken as template DNA. In addition, an inoculation loop was used to streak and inoculate the MRS plate, cultured at 37°C for 48 hours, and a single colony was taken for DNA extraction, PCR identification and mass spectrometry identification.

[0076] 2. PCR amplification

[0077] PCR uses a 25 μL reaction system, in which 1.0 μL of the upstream and downstream specific primers of the target DNA at a concentration of 10 μmol / L, 0.5 μL of 1.5U Taq DNA polymerase, ...

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Abstract

The invention discloses molecular markers, detection primers and detection methods for identifying plantar lactobacillus and pentosus lactobacillus. Specific molecular markers for identifying Lactobacillus plantarum and Lactobacillus pentosus, the nucleotide sequences of which are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or 4. The present invention screens new targets for specific molecular detection of Lactobacillus plantarum and Lactobacillus pentosus by using the whole genome comparison analysis technology, and designs primers for the new molecular targets for PCR amplification, which can specifically identify the two target bacteria. Currently, these molecular markers are not available. See report. Compared with 16S rDNA PCR method and physiological and biochemical method, the method established by the present invention has higher specificity, simple operation, short detection cycle, no need for further sequencing, and low detection cost, especially suitable for rapid initial detection of L.plantarum and L.pentosus Sieve provides an important auxiliary identification method for food production enterprises and testing institutions.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a molecular marker, a detection primer and a detection method for identifying plant lactobacillus and pentosus lactobacillus. Background technique: [0002] In terms of the taxonomic status of the genus Lactobacillus, Lactobacillus plantarum and Lactobacillus pentosus belong to the same group of Lactobacillus plantarum, which have a good inhibitory effect on Gram-positive and negative pathogenic bacteria, and inhibit the growth of pathogenic bacteria by competing with pathogenic bacteria for nutrition . In addition, Lactobacillus plantarum can pass through the stomach and colonize the intestinal tract to play a beneficial role, and can regulate the composition of intestinal microecology. In terms of food applications, Lactobacillus plantarum is the main species of Lactobacillus in industrial fermented foods, and is widely used in the preparation of fermented foods, such...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/686C12Q2565/125
Inventor 张淑红黄远斌吴清平张菊梅杨广珠丁郁薛亮陈谋通孙奇凡叶青华吴诗古其会韦献虎张友雄
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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