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: 2012-12-12
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 the genome sequences of 14 kinds of Lactobacillus in the Genome database of NCBI, the conserved sequence of Lactobacillus was found, and the conserved sequence and Lactobacillus belonged to Pediococcus (Pediococcus) of Lactobacillus family, and Lactobacillus belonged to Lactobacillus order Enterococcus (Enterococcus), Lactococcus (Lactococcus) and Leuconostoc (Leuconostoc), and the corresponding sequence alignment of Bacillus (Bacillus) and Staphylococcus (Staphylococcus), which belong to the class Bacillus with Lactobacillus, design Lactobacillus-specific primers .

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

[0027]

[0028] In order to achieve the purpose of quickly screening out all Lactobacilli from samples to be tested containing a large number of microorganisms, the present invention needs to design a pair of primers, and the prime...

Embodiment 2

[0032] Embodiment 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 for PCR amplification detection according to the following steps :

[0034] 1. Primers

[0035] Primers prepared in Example 1 were used.

[0036] 2. Template preparation

[0037] Pick trace amounts from single colonies of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus delbrueckii, Leuconostoc, Lactococcus lactis, Bacillus subtilis and Staphylococcus epidermidis Put the bacteria into the PCR tube, put the PCR tube into the microwave oven, heat it at 100% power for 3 minutes, and then add the PCR system.

[0038] 3. PCR amplification

[0039] Using the heat-treated thalli in step 2 as a template, the...

Embodiment 3

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

[0054] 1. The composition of the kit

[0055] 25μL system:

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

[0057] 10× PCR buffer: 2.5 μl

[0058] Magnesium chloride: 2 µl

[0059] dNTPs: 2 µl

[0060] exTaq enzyme: 0.25 µl

[0061] wxya 2 O: 17.25 µl

[0062] 2. How to use the kit

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

[0064] 2), gene amplification:

[0065] Using the bacterial genomic DNA obtained in step 1) as a template, the kit was used for gene amplification. The reaction program was: 94°C for 5 minutes; 95°C for 40s, 57°C for 30s, 72°C for 100s, 30 cycles; 72°C for 10 minutes.

[0066] 3), result detection:

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

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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 of bacteria, in particular to a detection technology of lactobacillus. Background technique [0002] Lactobacillus (Lactobacillus) is a kind of microorganism generally regarded as safe (Generally regarded-as-safe, GRAS). At present, Lactobacillus is not only widely used in the food and feed industry, but also is being developed as a vaccine delivery carrier, microecological correction agent, and whole enzyme preparation through genetic engineering, which is one of the current research hotspots. There are a lot of Lactobacillus in traditional fermented foods such as Sichuan kimchi, cheese, sausage, silage, Korean kimchi, etc. Sichuan kimchi is as famous as Korean kimchi and Japanese kimchi, and is recognized as a healthy fermented vegetable product worldwide. In order to improve the flavor of pickles and collect microbial resources, Lactobacillus plantarum (L.plantarum), Lactobacillus brevis (L.brevis), L...

Claims

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Application Information

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Patent Type & Authority Patents(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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