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Method and kit for screening/detecting lactobacillus

A Lactobacillus and kit technology, applied in the field of bacterial detection, can solve the problems of high cost, huge workload, strong primer specificity, etc., and achieve the effect of strong application value, cost saving and time saving.

Inactive Publication Date: 2011-11-23
CHENGDU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, traditional separation and identification methods need to detect morphological characteristics such as cell shape, size, arrangement, motility, and colony morphology, as well as physiological and biochemical characteristics such as nutritional requirements, enzymes, metabolites, and drug sensitivity. The amount is very large, and it requires high technical proficiency, so it is difficult to quickly screen out Lactobacillus
Although the method of identifying Lactobacillus by measuring 16s rDNA / RNA sequence is relatively fast, DNA sequencing is performed on any fungus in the sample to determine whether it is Lactobacillus, which is too expensive to be of practical application value
[0004] Dubernet S. et al. designed a pair of primers to amplify the region between the 3' end of the 16S rDNA sequence and the 23S rDNA sequence. According to whether a 250bp band appears in the electrophoresis, it can be determined whether the 23 bacteria are Lactobacillus, but the primers in this document The specificity is too strong, it can only be used to detect the 23 species of Lactobacillus recorded in the literature, and cannot detect other Lactobacillus and new species of Lactobacillus

Method used

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  • Method and kit for screening/detecting lactobacillus
  • Method and kit for screening/detecting lactobacillus
  • Method and kit for screening/detecting lactobacillus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Primer Design

[0025] As shown in Table 1, according to 14 kinds of Lactobacillus genome sequences in the Genome database of NCBI, look for the Lactobacillus conserved sequence, this conserved sequence and Lactobacillus belong to the same Lactobacillus Pediococcus (Pediococcus), and Lactobacillus belong to the same Lactobacillus order Enterococcus (Enterococcus), Lactococcus (Lactococcus) and Leuconostoc (Leuconostoc), and Bacillus (Bacillus) and Staphylococcus (Staphylococcus), which belong to the same genus of Lactobacillus, are aligned with the corresponding sequences, and Lactobacillus-specific primers are designed .

[0026] Table 1 List of 14 Lactobacillus species and 5 closely related species that have been sequenced

[0027]

[0028] In order to achieve the purpose of quickly screening out all lactobacilli from a sample containing a large number of microorganisms, a pair of primers needs to be designed in the present invention, and the primers sho...

Embodiment 2

[0032] Example 2 Primer Specificity Experiment

[0033] For Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus delbrueckii, Leuconostoc, Lactococcus lactis, Bacillus subtilis and Staphylococcus epidermidis, PCR amplification detection was carried out as follows :

[0034] 1. Primers

[0035] The primers prepared in Example 1 were used.

[0036] 2. Template preparation

[0037] Pick traces from single colonies of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus delbrueckii, Leuconostoc, Lactococcus lactis, Bacillus subtilis and Staphylococcus epidermidis, respectively The bacterial cells were placed in a PCR tube, the PCR tube was placed in a microwave oven, heated at 100% fire for 3 min, and then added to the PCR system.

[0038] 3. PCR amplification

[0039] Using the heat-treated cells in step 2 as temp...

Embodiment 3

[0053] Embodiment 3 The composition of the kit of the present invention and its use method

[0054] 1. Composition of the kit

[0055] 25μL system:

[0056] Primers provided in Example 1: 0.5 μl each

[0057] 10x PCR buffer: 2.5 μl

[0058] Magnesium Chloride: 2 µl

[0059] dNTP: 2 µl

[0060] exTaq enzyme: 0.25 μl

[0061] ddH 2 O: 17.25 µl

[0062] 2. How to use the kit

[0063] 1), isolate a single colony: isolate a single colony of strains from the sample to be tested;

[0064] 2), gene amplification:

[0065] Using the bacterial genome DNA obtained in step 1) as a template, gene amplification was performed with a kit. The reaction procedure was as follows: 94°C for 5 min; 95°C for 40s, 57°C for 30s, 72°C for 100s, 30 cycles; 72°C for 10 min.

[0066] 3), result detection:

[0067] 5 μL of the reaction solution was taken and subjected to agarose gel (0.7% W / V) electrophoresis to detect PCR amplification results.

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Abstract

The invention provides a kit for rapidly screening / detecting lactobacillus. The kit comprises a primer pair with the sequence shown in SEQ ID NO:1-2, wherein the primer pair is used for amplifying genes from the lactobacillus. The invention further relates to a method for rapidly screening / detecting lactobacillus. The kit provided by the invention can be used for rapidly screening out the lactobacillus from a sample to be detected which contains a large quantity of microbes so as to further screen and identify unknown lactobacillus, thus saving the time and the cost and having higher application value.

Description

technical field [0001] The invention relates to a detection technology for bacteria, in particular to a detection technology for Lactobacillus. Background technique [0002] Lactobacillus (Lactobacillus) is a kind of generally regarded-as-safe (GRAS) microorganisms, which can improve the intestinal environment and enhance the immunity of the host. At present, Lactobacillus is not only widely used in the food and feed industries, but also is being developed as a vaccine delivery carrier, a microecological corrector, and a whole enzyme preparation through genetic engineering, which is one of the current research hotspots. There are a large number of Lactobacillus in traditional fermented foods such as Sichuan kimchi, cheese, sausage, silage, and Korean kimchi. In order to improve the flavor of kimchi and collect microbial resources, L. plantarum, L. brevis, L. casei, and L. acidophilus have been isolated from Sichuan kimchi. acidophilus) and other lactobacilli. There are ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/225
Inventor 潘渠彭云杨维华杨四佳殷建华王广科余小平
Owner CHENGDU MEDICAL COLLEGE
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