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Molecular marker, detection primer and detection method for identifying lactobacillus helveticus, lactobacillus fermentum and lactobacillus acidophilus

A technology for Lactobacillus fermentum and Lactobacillus acidophilus, which is applied to the molecular markers of Lactobacillus fermentum and Lactobacillus acidophilus, the detection of primers, and the identification of Lactobacillus helveticus. It is difficult to popularize and apply problems in grassroots laboratories, and achieve the effects of short detection cycle, low detection cost and high reliability

Active Publication Date: 2020-12-04
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

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) has a good effect in the identification of lactic acid bacteria, but the instrument is expensive and difficult to popularize and apply in grassroots laboratories
16S rDNA sequence homology analysis has been widely used for the identification of lactic acid bacteria, but it needs to be further sequenced for the relative species of Lactobacillus with high homology cannot be accurately distinguished
Grazia and Yu carried out the identification and evolution analysis of Lactobacillus helveticus and Lactobacillus acidophilus based on the PCR amplification and sequencing of pepC, pepN and htrA and hsp60, pheS and tuf genes respectively. These methods are helpful for the distinction of Lactobacillus, but established Based on a limited number of housekeeping genes

Method used

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  • Molecular marker, detection primer and detection method for identifying lactobacillus helveticus, lactobacillus fermentum and lactobacillus acidophilus
  • Molecular marker, detection primer and detection method for identifying lactobacillus helveticus, lactobacillus fermentum and lactobacillus acidophilus
  • Molecular marker, detection primer and detection method for identifying lactobacillus helveticus, lactobacillus fermentum and lactobacillus acidophilus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1: identification of Lactobacillus helveticus

[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, 1.0 μL of upstream and downstream target DNA-specific primers 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 s, 7...

Embodiment 2

[0073] Embodiment 2: Identification of Lactobacillus fermentum

[0074] 1. Bacterial DNA extraction

[0075] 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.

[0076] 2. PCR amplification

[0077] PCR uses a 25 μL reaction system, 1.0 μL of upstream and downstream target DNA-specific primers 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 s, 72...

Embodiment 3

[0080] Example 3: Identification of Lactobacillus acidophilus

[0081] 1. Bacterial DNA extraction

[0082] 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.

[0083] 2. PCR amplification

[0084] PCR uses a 25 μL reaction system, 1.0 μL of upstream and downstream target DNA-specific primers 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 s, 72°...

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Abstract

The invention discloses a molecular marker, detection primer and detection method for identifying lactobacillus helveticus, lactobacillus fermentum and lactobacillus acidophilus. The nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, 2, 3, 4, 5 or 6. The molecular marker, detection primer and detection method in the invention can be used for effectively identifying lactobacillushelveticus, lactobacillus fermentum and lactobacillus acidophilus, have the advantages of being rapid, accurate, economic, simple in operation and the like, and are convenient for food enterprises andinspection institutions to use.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to molecular markers, detection primers and detection methods for identifying Lactobacillus helveticus, Lactobacillus fermentum and Lactobacillus acidophilus. Background technique: [0002] Lactobacillus helveticus is one of the most auxotrophic strains among lactic acid bacteria. It has strong proteolysis ability, and its proteolysis products have anti-hypertensive, anti-inflammatory and anti-cancer functions. It is a safe probiotic. The bacterium can adapt to various environments, including high temperature, low pH, low osmotic pressure, low oxygen, etc., and is more suitable for making probiotic preparations than other probiotics such as Bifidobacterium and Lactobacillus. Lactobacillus helveticus can produce many polypeptide substances and organic acids with antibacterial activity, which have a good inhibitory effect on pathogenic bacteria and play an important role in f...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/225C12R1/23
CPCC12Q1/689C12Q1/686Y02A50/30
Inventor 张淑红黄远斌吴清平张菊梅杨广珠丁郁薛亮陈谋通王涓曾海燕叶青华韦献虎古其会张友雄
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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