Primer sequence for identifying lactobacillus brevis and application thereof

A primer sequence and Lactobacillus technology, applied in the field of molecular biology, can solve the problems of long detection period, poor accuracy, and low degree of automation, and achieve the effect of low detection cost, simple process, and rapid identification

Active Publication Date: 2011-06-15
福建大北农华有水产科技集团有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the domestic isolation and identification of Lactobacillus pumilus mostly adopts traditional inspection methods such as plate culture method, biochemical test, and gas growth test. The above methods have disadvantages such as long detection period, low degree of automation, and poor accuracy.

Method used

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  • Primer sequence for identifying lactobacillus brevis and application thereof
  • Primer sequence for identifying lactobacillus brevis and application thereof
  • Primer sequence for identifying lactobacillus brevis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Step 1 Design and synthesis of primers

[0022] Obtain the 16S rDNA sequence of Lactobacillus pumilus and its homologous Lactobacillus from the Genebank database, search for specific regions that are conserved in Lactobacillus pumilus but not conserved in other Lactobacillus species, design primers, and screen and optimize specific sequences. Finally, a pair of primers LABBF and LABBR with better parameters were obtained, and the above two primers were synthesized.

[0023] The specific sequences of the above primers and the corresponding relationship in the sequence listing are shown in Table 2 below:

[0024] Table 2

[0025] Primer name

sequence

serial number

LABBF

5`-GAGAGTAACTGTTCAAGGG-3`

SEQ ID NO: 1

LABBR

5`-TCTCCCAGTTTCCGATGC-3`

SEQ ID NO: 2

[0026] The physical positions of the above primers on the 16S rDNA sequence are as follows: figure 1 shown.

[0027] Step 2 Preparation of PCR template

[0028] 1)...

Embodiment 2

[0056] The design and synthesis of step 1 primers are the same as the primer sequences shown in the primer list in Example 1

[0057] Step 2 Preparation of PCR template

[0058] 1) Pick a single colony of each strain on the petri dish to a 1.5mL sterilized centrifuge tube with 50μL sterile water, and mix well;

[0059] 2) Boil the centrifuge tube in boiling water at 100°C for 5 minutes;

[0060] 3) Place the centrifuge tube in an ultra-low temperature freezer at -80°C for 15 minutes;

[0061] 4) Boil the centrifuge tube in boiling water at 100°C for 5 minutes;

[0062] 5) Centrifuge at 4000 rpm for 10 minutes, and take the supernatant as a PCR template.

[0063] Step 3PCR amplification

[0064] 1. PCR reaction system

[0065] Use the DNA prepared in step 2 as a template, and use the base sequence synthesized in step 1 as a primer to carry out a PCR reaction. The reaction system is:

[0066]Template 1μL

[0067] 10×PCR Buffer 2.5μL

[0068] dNTP (10mM) 0.5μL

[0069] L...

Embodiment 3

[0084] The design and synthesis of step 1 primers are the same as the primer sequences shown in the primer list in Example 1

[0085] Step 2 Preparation of PCR template

[0086] 1) Pick a single colony of each strain on the petri dish to a 1.5mL sterilized centrifuge tube with 100μL sterile water, and mix well;

[0087] 2) Boil the centrifuge tube in boiling water at 100°C for 15 minutes;

[0088] 3) Place the centrifuge tube in an ultra-low temperature freezer at -80°C for 40 minutes;

[0089] 4) Boil the centrifuge tube in boiling water at 100°C for 15 minutes;

[0090] 5) Centrifuge at 8000 rpm for 5 minutes, and take the supernatant as a PCR template.

[0091] Step 3PCR amplification

[0092] 3. PCR reaction system

[0093] Use the DNA prepared in step 2 as a template, and use the base sequence synthesized in step 1 as a primer to carry out a PCR reaction. The reaction system is:

[0094] Template 1μL

[0095] 10×PCR Buffer 2.5μL

[0096] dNTP (10mM) 0.5μL

[0097...

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Abstract

The invention discloses a primer sequence for identifying lactobacillus brevis and an application thereof, belonging to the field of molecular biology. The base sequences of a primer disclosed by the invention are shown in SEQ ID NO:1 and SEQ ID NO:2. The primer sequence disclosed by the invention can be used for identifying lactobacillus brevis. The primer can specifically amplify the partial sequence of lactobacillus brevis 16SrDNA, has high amplification specificity, and can accurately and quickly identify lactobacillus brevis. The process of identifying lactobacillus brevis by using the primer is simple, the identification method is stable and efficient, the detection time is shortened, and the detection cost is low. The invention provides a detection method for identifying germ plasmresources of lactobacillus brevis, and lays a good foundation for screening fine strains of lactobacillus brevis.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a primer sequence for identifying Lactobacillus pumilus and its application. Background technique [0002] Lactobacillus brevis (Lactobacillus brevis) is one of the important species of Lactobacillus, which has a wide range of sources and is found in pickles, fresh milk, and animal intestines. Lactobacillus pumilus has the characteristics of producing γ-aminobutyric acid, conjugated linoleic acid, malic acid, interferon and other functional products, and has broad application prospects in food and pharmaceutical production. At present, the domestic isolation and identification of Lactobacillus pumilus mostly adopts traditional inspection methods such as plate culture method, biochemical test, and gas growth test. The above methods have disadvantages such as long detection period, low degree of automation, and poor accuracy. Contents of the invention [0003] The in...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 张军田子罡闫秩洁王安如张广民
Owner 福建大北农华有水产科技集团有限公司
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