CN102094089B - Primer sequence for identifying lactobacillus brevis and application thereof - Google Patents
Primer sequence for identifying lactobacillus brevis and application thereof Download PDFInfo
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- CN102094089B CN102094089B CN 201010574429 CN201010574429A CN102094089B CN 102094089 B CN102094089 B CN 102094089B CN 201010574429 CN201010574429 CN 201010574429 CN 201010574429 A CN201010574429 A CN 201010574429A CN 102094089 B CN102094089 B CN 102094089B
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- bacterium lacticum
- small bacterium
- lactobacillus brevis
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- 240000001929 Lactobacillus brevis Species 0.000 title abstract description 9
- 235000013957 Lactobacillus brevis Nutrition 0.000 title abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 24
- 230000003321 amplification Effects 0.000 claims abstract description 13
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 241000894006 Bacteria Species 0.000 claims description 44
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 230000001580 bacterial effect Effects 0.000 claims description 13
- 238000009835 boiling Methods 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 238000012797 qualification Methods 0.000 claims description 4
- 238000001962 electrophoresis Methods 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 2
- 230000004087 circulation Effects 0.000 claims description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 claims description 2
- 229960005542 ethidium bromide Drugs 0.000 claims description 2
- 238000010186 staining Methods 0.000 claims description 2
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 2
- 239000012498 ultrapure water Substances 0.000 claims description 2
- 238000012216 screening Methods 0.000 abstract 1
- 241000186660 Lactobacillus Species 0.000 description 8
- 229940039696 lactobacillus Drugs 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000013461 design Methods 0.000 description 4
- 108010006785 Taq Polymerase Proteins 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 241001134659 Lactobacillus curvatus Species 0.000 description 2
- 241001468157 Lactobacillus johnsonii Species 0.000 description 2
- 241001438705 Lactobacillus rogosae Species 0.000 description 2
- 241000186869 Lactobacillus salivarius Species 0.000 description 2
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 2
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
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- 239000002253 acid Substances 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960004232 linoleic acid Drugs 0.000 description 1
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- 239000008267 milk Substances 0.000 description 1
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- 150000007523 nucleic acids Chemical class 0.000 description 1
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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
The invention belongs to biology field, be specifically related to primer sequence and application thereof for the identification of short and small Bacterium lacticum.
Background technology
Short and small Bacterium lacticum (Lactobacillus brevis) is one of important bacterial classification of lactobacillus, and its wide material sources all have discovery in pickles, fresh milk, animal intestinal.Short and small Bacterium lacticum has the characteristic of multiple function products such as producing gamma amino butyric acid, conjugated linolic acid, oxysuccinic acid, Interferon, rabbit, has broad application prospects in food and medicine production.Traditional methods of inspection such as the short and small Bacterium lacticum of the present domestic isolation identification plate cultures that adopt, biochemical test, gas growth test, the aforesaid method existence detects long, shortcomings such as level of automation is low, poor accuracy of cycle more.
Summary of the invention
The invention provides the primer sequence for the identification of short and small Bacterium lacticum, the partial sequence that this primer can the short and small Bacterium lacticum 16S of specific amplification rDNA, the specific amplification height can be accurately, the short and small Bacterium lacticum of Rapid identification.This primer is simple for the identification of short and small Bacterium lacticum process, and authentication method is stable, efficient, has not only shortened detection time, and it is low to detect cost.
The objective of the invention is to be achieved through the following technical solutions:
The invention provides the primer sequence for the identification of short and small Bacterium lacticum, it is characterized in that: forward primer has the described base sequence of SEQ ID NO:1 in the sequence table, and reverse primer has the described base sequence of SEQ IDNO:2 in the sequence table.
Primer sequence of the present invention can be used for identifying short and small Bacterium lacticum, and its qualification process is as follows: the template DNA of (1) preparation bacterial strain to be measured;
(2) right to use 1 described primer carries out pcr amplification to the template DNA of step (1) preparation;
(3) utilize agarose gel electrophoresis to detect the PCR product, produce the bacterial strain of specific amplification band at the 200bp place, then be accredited as short and small Bacterium lacticum.
Wherein, the template DNA process for preparing bacterial strain to be measured is as follows: single bacterium colony of the short and small Bacterium lacticum of picking is to the ultrapure water of 50-100 microlitre, placed the boiling water water-bath 5-15 minute, in-80 ℃ of frozen 15-40 minutes, to the boiling water water-bath 5-15 minute, 4000-8000 rev/min centrifugal 5-10 minute, get supernatant and namely get template DNA.
Wherein, the pcr amplification program is as follows: 95 ℃ of pre-sex change 5min, and 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 20s, carry out 30 circulations, and last 72 ℃ are extended 10min.
Electrophoresis detection PCR product of the present invention adopts is 2% agarose gel electrophoresis, and uses ethidium bromide staining, takes pictures in the gel imaging instrument at last.
Wherein, the short and small Bacterium lacticum that template DNA of the present invention relates in extracting only is experiment material, the present invention does not limit specific short and small lactobacterium strain, can be the short and small Bacterium lacticum of any strain that openly maybe can buy now both at home and abroad, also can be arbitrarily parting material voluntarily, the used short and small Bacterium lacticum of the present invention is bought in Chinese industrial microbial strains preservation administrative center, and deposit number is: CICC6239, and no matter the short and small Bacterium lacticum of any strain does not all influence enforcement of the present invention; The lactobacterium casei of using in embodiment 4 electrophoresis, Lactobacillus johnsonii, lactobacillus delbruckii, Lactobacillus rogosae, plant lactobacillus, lactobacillus salivarius, lactobacillus curvatus, bacterial strains such as lactobacterium helveticus are the control strain experiment material, must not be defined as certain strain bacterial strain, can be as required or available experiment material selected, enforcement of the present invention is not had influence.
Compared with prior art, technical scheme of the present invention has following advantage: the partial sequence that the primer sequence for the identification of short and small Bacterium lacticum of the present invention can the short and small Bacterium lacticum 16S of specific amplification rDNA, the specific amplification height can be accurately, the short and small Bacterium lacticum of Rapid identification.This primer is simple fast for the identification of short and small Bacterium lacticum process, and authentication method is stable, efficient, has not only shortened detection time, and detects cost low (seeing Table 1).The present invention identifies for short and small Bacterium lacticum germplasm resource provides detection method, and then is that the strain excellent that screens short and small Bacterium lacticum is laid a good foundation.
Table 1 prior art and qualification process of the present invention are relatively
| Qualification time | Appraisal cost | |
| Art methods | About one week | About 25 yuan |
| The inventive method | About 2 hours | About 3 yuan |
Description of drawings
Fig. 1 is the position collection of illustrative plates of primer sequence of the present invention on 16S rDNA gene;
Fig. 2 is the gel electrophoresis spectrum of each bacterial strain amplified production in the PCR specific amplification.
Among Fig. 2, M is the nucleic acid molecular weight standard; 1 short and small Bacterium lacticum, 2 lactobacterium caseis, 3 Lactobacillus johnsonii, 4 lactobacillus delbruckiis, 5 Lactobacillus rogosaes, 6 plant lactobacilluss, 7 lactobacillus salivarius, 8 lactobacillus curvatuses, 9 lactobacterium helveticus, the contrast of CK water
, it should be understood that described embodiment only is for the present invention is described, rather than limit the scope of the invention by any way more specific description the present invention by the following example.
Embodiment
In the Genebank database, obtain short and small Bacterium lacticum and reach the lactobacillus 16SrDNA sequence that has homology with it, search in the short and small lactobacillus species and guard and the conservative specific regions of all the other Bacterium lacticum, the design primer also screens and optimizes specific sequence, finally obtained a pair of parameter more excellent primer LABBF and LABBR, and synthetic above-mentioned two primers.
Corresponding relation is as shown in table 2 below in the concrete sequence of above-mentioned primer and the sequence table:
Table 2
| The primer title | Sequence | Sequence number |
| LABBF | 5`-GAGAGTAACTGTTCAAGGG-3` | SEQ ID NO：1 |
| LABBR | 5`-TCTCCCAGTTTCCGATGC-3` | SEQ ID NO：2 |
Physical location is as shown in Figure 1 on 16S rDNA sequence for above-mentioned primer.
The preparation of step 2PCR template
1) during single bacterium colony of each bacterial strain is sterilized centrifuge tube to the 1.5mL that is added with 50 μ L sterilized waters on the picking culture dish, mixing;
2) centrifuge tube is placed 100 ℃ boiling water boiled 10 minutes;
3) centrifuge tube is placed-80 ℃ freezing 30 minutes of Ultralow Temperature Freezer;
4) centrifuge tube is placed 100 ℃ boiling water boiled 10 minutes;
5) 5000 rev/mins centrifugal 10 minutes, get supernatant as pcr template.
Step 3PCR amplification
1, PCR reaction system
DNA with preparation in the step 2 is template, is that primer carries out the PCR reaction with base sequence synthetic in the step 1, and reaction system is:
Template 1μL
10×PCR Buffer 2.5μL
dNTP(10mM) 0.5μL
LABBF(20mM) 0.5μL
LABBR(20mM) 0.5μL
Taq Polymerase(5U) 0.25μL
Deionized water adds to 20 μ L
2, PCR response procedures
Sample is placed on carries out the PCR reaction in the centrifuge tube of 200 μ L, response procedures is:
Pre-95 ℃ of 5min of sex change
94 ℃ of 30s of sex change
58 ℃ of 30s anneal
Extend 72 ℃ of 20s
Extend 72 ℃ of 10min
Cycle number 30 times
Reaction finishes the back amplified production through 2% agarose gel electrophoresis result of determination.
The specificity of the short and small Bacterium lacticum PCR method checking of step 4
DNA with 9 kinds of lactobacilluss of short and small Bacterium lacticum and nearly edge thereof is template, with the water belongs with yin contrast, detects pcr amplification product by agarose gel electrophoresis, namely is judged to be positive findings at 200bp place discovery specific amplification band.
Experimental result shows: have only the pcr amplification product of short and small Bacterium lacticum positive amplification to occur, and amplified signal does not all appear in other nearly edge Bacterium lacticum and negative control, the results are shown in Figure 2.
The preparation of step 2PCR template
1) during single bacterium colony of each bacterial strain is sterilized centrifuge tube to the 1.5mL that is added with 50 μ L sterilized waters on the picking culture dish, mixing;
2) centrifuge tube is placed 100 ℃ boiling water boiled 5 minutes;
3) centrifuge tube is placed-80 ℃ freezing 15 minutes of Ultralow Temperature Freezer;
4) centrifuge tube is placed 100 ℃ boiling water boiled 5 minutes;
5) 4000 rev/mins centrifugal 10 minutes, get supernatant as pcr template.
Step 3PCR amplification
1, PCR reaction system
DNA with preparation in the step 2 is template, is that primer carries out the PCR reaction with base sequence synthetic in the step 1, and reaction system is:
Template 1μL
10×PCR Buffer 2.5μL
dNTP(10mM) 0.5μL
LABBF(20mM) 0.5μL
LABBR(20mM) 0.5μL
Taq Polymerase(5U) 0.25μL
Deionized water adds to 20 μ L
2, PCR response procedures
Sample is placed on carries out the PCR reaction in the centrifuge tube of 200 μ L, response procedures is:
Pre-95 ℃ of 5min of sex change
94 ℃ of 30s of sex change
58 ℃ of 30s anneal
Extend 72 ℃ of 20s
Extend 72 ℃ of 10min
Cycle number 30 times
Reaction finishes the back amplified production through 2% agarose gel electrophoresis result of determination.
The specificity checking of the short and small Bacterium lacticum PCR method checking of step 4 is with the method shown in embodiment 1 step 4
The preparation of step 2PCR template
1) during single bacterium colony of each bacterial strain is sterilized centrifuge tube to the 1.5mL that is added with 100 μ L sterilized waters on the picking culture dish, mixing;
2) centrifuge tube is placed 100 ℃ boiling water boiled 15 minutes;
3) centrifuge tube is placed-80 ℃ freezing 40 minutes of Ultralow Temperature Freezer;
4) centrifuge tube is placed 100 ℃ boiling water boiled 15 minutes;
5) 8000 rev/mins centrifugal 5 minutes, get supernatant as pcr template.
Step 3PCR amplification
3, PCR reaction system
DNA with preparation in the step 2 is template, is that primer carries out the PCR reaction with base sequence synthetic in the step 1, and reaction system is:
Template 1μL
10×PCR Buffer 2.5μL
dNTP(10mM) 0.5μL
LABBF(20mM) 0.5μL
LABBR(20mM) 0.5μL
Taq Polymerase(5U) 0.25μL
Deionized water adds to 20 μ L
4, PCR response procedures
Sample is placed on carries out the PCR reaction in the centrifuge tube of 200 μ L, response procedures is:
Pre-95 ℃ of 5min of sex change
94 ℃ of 30s of sex change
58 ℃ of 30s anneal
Extend 72 ℃ of 20s
Extend 72 ℃ of 10min
Cycle number 30 times
Reaction finishes the back amplified production through 2% agarose gel electrophoresis result of determination.
The specificity checking of the short and small Bacterium lacticum PCR method checking of step 4 is with the method shown in embodiment 1 step 4
Claims (5)
1. for the identification of the primer of short and small Bacterium lacticum, it is characterized in that: the forward primer sequence is shown in SEQ ID NO:1, and the reverse primer sequence is shown in SEQ ID NO:2.
2. the application of the described primer of claim 1 in identifying short and small Bacterium lacticum, it is characterized in that: qualification process is as follows:
(1) template DNA of preparation bacterial strain to be measured;
(2) right to use requires 1 described primer that the template DNA of step (1) preparation is carried out pcr amplification;
(3) utilize agarose gel electrophoresis to detect the PCR product, produce the bacterial strain of specific amplification band at the 200bp place, then be accredited as short and small Bacterium lacticum.
3. the application of primer as claimed in claim 2 in identifying short and small Bacterium lacticum, it is characterized in that: the template DNA process for preparing bacterial strain to be measured is as follows: single bacterium colony of the short and small Bacterium lacticum of picking is to the ultrapure water of 50-100 microlitre, placed the boiling water water-bath 5-15 minute, in-80 ℃ of frozen 15-40 minutes, to the boiling water water-bath 5-15 minute, 4000-8000 rev/min centrifugal 5-10 minute, get supernatant and namely get template DNA.
4. the application of primer as claimed in claim 2 in identifying short and small Bacterium lacticum, it is characterized in that: the pcr amplification program is as follows: 95 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 20s, carry out 30 circulations, and last 72 ℃ are extended 10min.
5. the application of primer as claimed in claim 2 in identifying short and small Bacterium lacticum is characterized in that: electrophoresis detection PCR product adopts is 2% agarose gel electrophoresis, and uses ethidium bromide staining.
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|---|---|---|---|
| CN 201010574429 CN102094089B (en) | 2010-12-06 | 2010-12-06 | Primer sequence for identifying lactobacillus brevis and application thereof |
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| CN102433385B (en) * | 2011-12-20 | 2014-10-15 | 北京大北农科技集团股份有限公司 | Primers for identifying leuconostoc mesenteroides and application thereof |
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Inventor after: Zhang Jun Inventor after: Tian Zigang Inventor after: Yan Diejie Inventor after: Wang Anru Inventor after: Zhang Guangmin Inventor after: Li Xiaoqing Inventor before: Zhang Jun Inventor before: Tian Zigang Inventor before: Yan Zhijie Inventor before: Wang Anru Inventor before: Zhang Guangmin |
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Effective date of registration: 20191209 Address after: 363502, Fujian, Zhangzhou province Zhaoan County gold industrial concentration zone Co-patentee after: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Patentee after: FUJIAN DABEINONG FISHERIES SCIENCE & TECHNOLOGY Co.,Ltd. Co-patentee after: SHANDONG FENGWO XINNONG AGRICULTURAL AND ANIMAL HUSBANDRY SCIENCE AND TECHNOLOGY CO.,LTD. Address before: 100080, Zhongguancun building, No. 27 Zhongguancun street, Beijing, Haidian District, China, 14 floor Co-patentee before: SHANDONG FENGWO XINNONG AGRICULTURAL AND ANIMAL HUSBANDRY SCIENCE AND TECHNOLOGY CO.,LTD. Patentee before: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. |
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Address after: 363502 Jindu Industrial Concentration Zone, Zhaoan County, Zhangzhou City, Fujian Province Patentee after: Fujian Dabei nonghuayou Aquatic Technology Group Co.,Ltd. Patentee after: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Patentee after: SHANDONG FENGWO XINNONG AGRICULTURAL AND ANIMAL HUSBANDRY SCIENCE AND TECHNOLOGY CO.,LTD. Address before: 363502 Jindu Industrial Concentration Zone, Zhaoan County, Zhangzhou City, Fujian Province Patentee before: FUJIAN DABEINONG FISHERIES SCIENCE & TECHNOLOGY Co.,Ltd. Patentee before: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Patentee before: SHANDONG FENGWO XINNONG AGRICULTURAL AND ANIMAL HUSBANDRY SCIENCE AND TECHNOLOGY CO.,LTD. |