CN104450949B - A kind of specificity molecular marker DNA sequence of Lactobacillus plantarum and application thereof - Google Patents
A kind of specificity molecular marker DNA sequence of Lactobacillus plantarum and application thereof Download PDFInfo
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- CN104450949B CN104450949B CN201410855051.2A CN201410855051A CN104450949B CN 104450949 B CN104450949 B CN 104450949B CN 201410855051 A CN201410855051 A CN 201410855051A CN 104450949 B CN104450949 B CN 104450949B
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Abstract
Specificity molecular marker DNA sequence that the invention discloses a kind of Lactobacillus plantarum (Lactobacillus plantarum) and application thereof and the detection method of Lactobacillus plantarum.This specificity molecular marker DNA sequence is as shown in SEQ ID NO.6.This detection method includes step: (1) extracts the genomic DNA of sample to be tested；(2) with the genomic DNA of step (1) gained as template, single-primed PCR is carried out with the semi-random primer shown in SEQ ID NO.1 for amplimer；(3) step (2) gained amplified production is carried out cloning and sequencing, and sequencing result is compared with sequence shown in SEQ ID NO.6, if homology degree is more than 99%, then show in sample to be tested containing Lactobacillus plantarum.
Description
Technical field
The invention belongs to biological technical field, particularly to a kind of Lactobacillus plantarum (Lactobacillus
Plantarum) specificity molecular marker DNA sequence and application thereof.
Background technology
The detection that a kind of antibacterial to carry out molecular level needs to know the specific sequence of this antibacterial, and obtains a kind of thin
The specific sequence of bacterium, generally requires and online substantial amounts of known array is analyzed comparison；But the sequence that some antibacterial has been surveyed
Less, analyze extremely difficult, it is difficult to obtain its specific sequence.It is of course also possible to collect all bacterium of a certain antibacterial
Strain, carries out comparison of checking order, but the method experimental period is longer, costly.
Lactobacillus plantarum (Lactobacillus plantarum) is the one of probiotic bacteria, is widely used in various function
Property food in, especially in the developmental research of milk product, be increasingly subject to pay attention to.At present, the plant breast that can obtain on the net
The sequence that bacillus passes through order-checking is less, thus is difficult to analyze the specific sequence obtaining Lactobacillus plantarum, and this gives plant breast
The Molecular Detection of bacillus is made troubles.Therefore, it is necessary to the specific sequence of Lactobacillus plantarum is known in research and development, right to facilitate
It carries out Molecular Detection.
Summary of the invention
The technical problem to be solved is aiming at present Lactobacillus plantarum (Lactobacillus
Plantarum) carry out the present situation that Molecular Detection is inconvenient, and the specificity molecular marker DNA sequence of a kind of Lactobacillus plantarum is provided
Row and application thereof.The specificity molecular marker DNA sequence of the Lactobacillus plantarum of the present invention and the Lactobacillus plantarum having surveyed sequence
Genetic homology more than 99%, may be conveniently used and Lactobacillus plantarum is carried out Molecular Detection.Before this, this molecular marker is not
Through reporting, it it is the specific sequence of a newfound Lactobacillus plantarum.
The present invention solves above-mentioned technical problem by following technical proposals.
One of technical scheme that the present invention provides is: a kind of Lactobacillus plantarum (Lactobacillus plantarum)
Specificity molecular marker DNA, its nucleotide sequence is as shown in SEQ ID NO.6.
The two of the technical scheme that the present invention provides are: nucleotide sequence composition plant breast bar as shown in SEQ ID NO.6
The specificity molecular marker DNA of bacterium (Lactobacillus plantarum) purposes in detection Lactobacillus plantarum.
The specificity molecular marker DNA of nucleotide sequence composition Lactobacillus plantarum as shown in SEQ ID NO.6, is plant
The species specificity sequence of lactobacillus, can be as molecular genetics labelling, for the Molecular Detection of Lactobacillus plantarum.
The three of the technical scheme that the present invention provides are: the detection method of a kind of Lactobacillus plantarum, it comprises the steps:
(1) genomic DNA of sample to be tested is extracted；
(2) with the genomic DNA of step (1) gained as template, with the semi-random primer shown in SEQ ID NO.1 for amplification
Primer carries out single-primed PCR；
(3) step (2) gained amplified production is carried out cloning and sequencing, and by shown in sequencing result and SEQ ID NO.6
Nucleotide sequence is compared, if homology degree is more than 99%, then shows in sample to be tested containing Lactobacillus plantarum.
In the present invention, step (1) is to extract the genomic DNA of sample to be tested.The described genome extracting sample to be tested
The method of DNA is that this area is conventional, as used frozen-thawed-CTAB method, it would however also be possible to employ lysozyme Method, it is also possible to use
Commercially available various genome DNA extracting reagent kits operate.
In the present invention, step (2) be the genomic DNA with step (1) gained as template, shown in SEQ ID NO.1
Semi-random primer is that amplimer carries out single-primed PCR.Described single-primed PCR is conventionally referred the containing in this area
Justice, i.e. amplification system only add an amplimer and carries out the synthesis of DNA fragmentation.It is preferred that described single-primed PCR
Reaction system include following component: the 0.5～2.0 semi-random primers of μm ol/L, 0.2～1.0mmol/L dNTP, 1.0～
The Mg of 2.5mmol/L2+, 0.02～0.10U/ μ LTaq archaeal dna polymerase, and 0.5～2.0ng/ μ L genomic DNA template.Institute
The response procedures of the single-primed PCR stated includes: 1. 93-96 DEG C, 4-6min；2. 93-96 DEG C, 20-40s；3. 45-58 DEG C,
20-40s；4. 70-72 DEG C, 30-120s；5. 70～72 DEG C, 5～10min；Wherein, step 2. to period 4. be 25-40.
More preferably, the reaction system of described single-primed PCR includes following component: the 1.0 semi-random primers of μm ol/L, 0.5mmol/
The Mg of L dNTP, 1.5mmol/L2+, 0.05U/ μ LTaq archaeal dna polymerase, and 1.0ng/ μ L genomic DNA template.Described
The response procedures of single-primed PCR includes: 1. 95 DEG C, 5min；2. 95 DEG C, 30s；3. 50 DEG C, 30s；4. 72 DEG C, 2min；⑤
72 DEG C, 5min；Wherein, step 2. to period 4. be 35.
In the present invention, step (3) is that step (2) gained amplified production carries out cloning and sequencing, and by sequencing result and SEQ
Nucleotide sequence shown in ID NO.6 is compared, if homology degree is more than 99%, then shows in sample to be tested containing plant breast bar
Bacterium.Wherein, described step (2) gained amplified production is carried out cloning and sequencing carry out the most in the following manner: by step (2)
Gained amplified production carries out electrophoresis (preferably agarose gel electrophoresis, it is also possible to be polyacrylamide gel electrophoresis), if at 500bp
There is amplified band in position, then this band is carried out cloning and sequencing.
On the basis of meeting common sense in the field, above-mentioned each optimum condition, can combination in any, obtain each preferable reality of the present invention
Example.
Agents useful for same of the present invention and raw material are the most commercially.
Compared to prior art, the most progressive effect of the present invention is:
The specificity molecular marker DNA sequence of the Lactobacillus plantarum in the present invention and the Lactobacillus plantarum having surveyed sequence
Genetic homology is more than 99%, may be conveniently used and Lactobacillus plantarum is carried out Molecular Detection.The detection method behaviour of the present invention
Make simple, fast and easy, high specificity, it is not necessary to being specifically designed PCR primer can detect sample to be tested, and cost is relatively
Low, experimental period is short, has the most wide application prospect.
Accompanying drawing explanation
Fig. 1 is the result of the specific PCR band screening of different lactobacillus plantarum strain.Numbering " M " in figure is DL2,
000DNA Marker Takara Biotechnology(Dalian)Co,Ltd.；" 1-5 " is respectively as follows: Lactobacillus
plantarumST-III、Lactobacillus plantarumATCC8014、Lactobacillus
plantarumACCC11095、Lactobacillus plantarumATCC14917、Lactobacillus
The PCR result of plantarumWCFS1.
Fig. 2 is the result of the specific PCR band acquisition of different lactobacillus plantarum strain.Numbering " M " in figure is DL2,
000DNA Marker Takara Biotechnology(Dalian)Co,Ltd.." 1-8 " is respectively as follows: Lactobacillus
plantarum CGMCC1.119、Lactobacillus plantarum MCC1.11、Lactobacillus plantarum
CICC22710、Lactobacillus plantarum CICC6234、Lactobacillus plantarum CGMCC1.3、
Lactobacillus plantarum CICC20871、Lactobacillus plantarum CICC23132、
The PCR result of Lactobacillus plantarumCICC23506.
Fig. 3 is the result of the acquisition of the specific PCR band of lactobacillus plantarum strain.Numbering " M " in figure is DL2,
000DNA Marker Takara Biotechnology(Dalian)Co,Ltd.；" 1-2 " is respectively as follows: Lactobacillus
The PCR result of plantarumATCC14917, Lactobacillus plantarumWCFS1.
Fig. 4 is the result of the specific PCR band acquisition of different lactobacillus plantarum strain.Numbering " M " in figure is DL2,
000DNA Marker Takara Biotechnology(Dalian)Co,Ltd.." 1-10 " is respectively as follows: Lactobacillus
plantarum CGMCC1.119、Lactobacillus plantarum MCC1.11、Lactobacillus plantarum
CICC22710、Lactobacillus plantarum CICC6234、Lactobacillus plantarum CGMCC1.3、
Lactobacillus plantarum CICC20871、Lactobacillus plantarum CICC23132、
Lactobacillus plantarumCICC23506、Lactobacillus plantarumATCC14917、
The PCR result of Lactobacillus plantarumWCFS1.
Fig. 5 is the effectiveness comparison of different semi-random primer.In figure, " M " is DL2,000DNA (Marker Takara
Biotechnology (Dalian) Co, Ltd), numbering " 1-4 " is the PCR result that semi-random primer " AP2-AP5 " is the most corresponding,
Numbering 5 is semi-random PCR result corresponding for primer AP1.
Detailed description of the invention
Further illustrate the present invention below by the mode of embodiment, but the most therefore limit the present invention to described reality
Execute among example scope.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or according to business
Product description selects.
In following embodiment, if not otherwise indicated, agents useful for same and material are conventional commercial and can obtain.
In following embodiment, the source of following experiment material is:
Lactobacillus plantarum:
Lactobacillus plantarumST-III takes from Shanghai Bright Dairy & Food Co., Ltd., and this is the special of applicant
Profit bacterial strain, is also purchased from CGMCC, and its deposit number is CGMCC NO.0847；
Lactobacillus plantarumATCC8014 is purchased from Bei Nuo bio tech ltd, Shanghai；
Lactobacillus plantarumACCC11095 is purchased from Bei Nuo bio tech ltd, Shanghai；
Lactobacillus plantarumATCC14917 is purchased from Bei Nuo bio tech ltd, Shanghai；
Lactobacillus plantarumWCFS1 is purchased from Danisco US Inc. Genencor Divisi；
Lactobacillus plantarum CGMCC1.119 is purchased from Beijing North Na Kaichuan Bioisystech Co., Ltd；
Lactobacillus plantarum MCC1.11 is purchased from Beijing North Na Kaichuan Bioisystech Co., Ltd；
Lactobacillus plantarum CICC22710 is purchased from Beijing North Na Kaichuan Bioisystech Co., Ltd；
Lactobacillus plantarum CICC6234 is purchased from Beijing North Na Kaichuan Bioisystech Co., Ltd；
Lactobacillus plantarum CGMCC1.3 is purchased from Beijing North Na Kaichuan Bioisystech Co., Ltd；
Lactobacillus plantarum CICC20871 is purchased from Beijing North Na Kaichuan Bioisystech Co., Ltd；
Lactobacillus plantarum CICC23132 is purchased from Beijing North Na Kaichuan Bioisystech Co., Ltd；
Lactobacillus plantarumCICC23506 is purchased from Beijing North Na Kaichuan Bioisystech Co., Ltd.
The screening of the specific PCR band of the different lactobacillus plantarum strain of embodiment 1
(1) prepared by template
By each bacterial strain activate separation and Culture, it is thus achieved that bacterium colony picking individual colonies, be inoculated in 1ml MRS culture medium, detest for 37 DEG C
Oxygen is cultivated 24 hours, centrifugal acquisition thalline.Extracting bacterial genomes, the test kit of use is: TaKaRaminibest
bacterial genomic DNA extraction kit ver.2.0(Takara Biotechnology(Dalian)Co.,
Ltd.)。
(2) PCR amplification
The system of PCR amplification consists of: the 1 semi-random primer of μm ol/L (its sequence is as shown in SEQ ID NO.1),
The Mg of 0.5mmol/L dNTP, 1.5mmol/L2+, 0.05U/ μ LTaq archaeal dna polymerase, and 500ng (please representing with concentration) base
Because of group DNA profiling, cumulative volume 50 μ L.
The program of PCR amplification is: 1. 95 DEG C, 5min；2. 95 DEG C, 30s；3. 50 DEG C, 30s；4. 72 DEG C, 2min；5. 72 DEG C,
5min；Wherein, step 2. to period 4. be 35.
(3) acquisition of specific band
PCR primer is carried out electrophoresis, and result is shown in Fig. 1, and numbering " M " in figure is DL2,000DNA Marker Takara
Biotechnology(Dalian)Co,Ltd.." 1-5 " be respectively as follows: Lactobacillus plantarumST-III,
Lactobacillus plantarumATCC8014、Lactobacillus plantarumACCC11095、
Lactobacillus plantarumATCC14917, the PCR result of Lactobacillus plantarumWCFS1.
Purification cloning and sequencing are about reclaimed at the band (at arrow indication) of 500bp in position total in swimming lane 1-5,
Sequencing result is as shown in SEQ ID NO.6.
The acquisition of the specific PCR band of the different lactobacillus plantarum strain of embodiment 2
(1) prepared by template
By each bacterial strain activate separation and Culture, it is thus achieved that bacterium colony picking individual colonies, be inoculated in 1ml MRS culture medium, detest for 37 DEG C
Oxygen is cultivated 24 hours, centrifugal acquisition thalline.Extracting bacterial genomes, the test kit of use is: TaKaRaminibest
bacterial genomic DNA extraction kit ver.2.0(Takara Biotechnology(Dalian)Co.,
Ltd.)。
(2) PCR amplification
The system of PCR amplification consists of: the 2 semi-random primers of μm ol/L (its sequence is as shown in SEQ ID NO.1), 1mmol/
The Mg of L dNTP, 2.5mmol/L2+, 0.10U/ μ LTaq archaeal dna polymerase, and 2ng/ μ L genomic DNA template, cumulative volume 50 μ
L。
The program of PCR amplification is: 1. 95 DEG C, 4min；2. 95 DEG C, 20s；3. 50 DEG C, 20s；4. 72 DEG C, 60s；5. 72 DEG C,
5min；Wherein, step 2. to period 4. be 40.
(3) acquisition of specific band
PCR primer is carried out electrophoresis, and result is shown in Fig. 2, and numbering " M " in figure is DL2,000DNA Marker Takara
Biotechnology(Dalian)Co,Ltd.." 1-8 " be respectively as follows: Lactobacillus plantarum CGMCC1.119,
Lactobacillus plantarum MCC1.11、Lactobacillus plantarum CICC22710、
Lactobacillus plantarum CICC6234、Lactobacillus plantarum CGMCC1.3、
Lactobacillus plantarum CICC20871、Lactobacillus plantarum CICC23132、
The PCR result of Lactobacillus plantarumCICC23506.
Purification cloning and sequencing are about reclaimed at the band (at arrow indication) of 500bp in position total in swimming lane 1-8,
Sequencing result is all as shown in SEQ ID NO.6.
The acquisition of the specific PCR band of embodiment 3 lactobacillus plantarum strain
(1) prepared by template
Bacterial strain is activated separation and Culture, it is thus achieved that bacterium colony picking individual colonies, be inoculated in 1ml MRS culture medium, 37 DEG C of anaerobism
Cultivate 24 hours, centrifugal acquisition thalline.Extracting bacterial genomes, the test kit of use is: TaKaRaminibest
bacterial genomic DNA extraction kit ver.2.0(Takara Biotechnology(Dalian)Co.,
Ltd.)。
(2) PCR amplification
The system of PCR amplification consists of: the 0.5 semi-random primer of μm ol/L (its sequence is as shown in SEQ ID NO.1),
The Mg of 0.2mmol/L dNTP, 1.0mmol/L2+, 0.02U/ μ LTaq archaeal dna polymerase, and 0.5ng/ μ L genomic DNA mould
Plate, cumulative volume 50 μ L.
The program of PCR amplification is: 1. 96 DEG C, 4min；2. 93 DEG C, 40s；3. 45 DEG C, 40s；4. 70 DEG C, 120s；5. 70 DEG C,
10min；Wherein, step 2. to period 4. be 25.
(3) acquisition of specific band
PCR primer is carried out electrophoresis, and result is shown in Fig. 3, and numbering " M " in figure is DL2,000DNA Marker Takara
Biotechnology(Dalian)Co,Ltd.." 1-2 " be respectively as follows: Lactobacillus plantarumATCC14917,
The PCR result of Lactobacillus plantarumWCFS1.
Purification cloning and sequencing are about reclaimed at the band (at arrow indication) of 500bp in the position having in swimming lane, surveys
Sequence result is all as shown in SEQ ID NO.6.
The acquisition of the specific PCR band of embodiment 4 lactobacillus plantarum strain
(1) prepared by template
Bacterial strain is activated separation and Culture, it is thus achieved that bacterium colony picking individual colonies, be inoculated in 1ml MRS culture medium, 37 DEG C of anaerobism
Cultivate 24 hours, centrifugal acquisition thalline.Extracting bacterial genomes, the test kit of use is: TaKaRaminibest
bacterial genomic DNA extraction kit ver.2.0(Takara Biotechnology(Dalian)Co.,
Ltd.)。
(2) PCR amplification
The system of PCR amplification consists of: the 1 semi-random primer of μm ol/L (its sequence is as shown in SEQ ID NO.1),
The Mg of 0.5mmol/L dNTP, 1.5mmol/L2+, 0.05U/ μ LTaq archaeal dna polymerase, and 2ng/ μ L genomic DNA template,
Cumulative volume 50 μ L.
The program of PCR amplification is: 1. 93 DEG C, 6min；2. 96 DEG C, 20s；3. 58 DEG C, 30s；4. 72 DEG C, 30s；5. 70 DEG C,
10min；Wherein, step 2. to period 4. be 35.
(3) acquisition of specific band
PCR primer is carried out electrophoresis, and result is shown in Fig. 4, and numbering " M " in figure is DL2,000DNA Marker Takara
Biotechnology(Dalian)Co,Ltd.." 1-10 " be respectively as follows: Lactobacillus plantarum CGMCC1.119,
Lactobacillus plantarum MCC1.11、Lactobacillus plantarum CICC22710、
Lactobacillus plantarum CICC6234、Lactobacillus plantarum CGMCC1.3、
Lactobacillus plantarum CICC20871、Lactobacillus plantarum CICC23132、
Lactobacillus plantarumCICC23506、Lactobacillus plantarumATCC14917、
The PCR result of Lactobacillus plantarumWCFS1.
Purification cloning and sequencing are about reclaimed at the band (at arrow indication) of 500bp in the position having in swimming lane, surveys
Sequence result is all as shown in SEQ ID NO.6.
Embodiment 5 sequence-specific is verified
The specific sequence (as shown in SEQ ID NO.6) embodiment 1～4 obtained carries out BLAST in NCBI website,
The genetic homology of specific sequence and Lactobacillus plantarum that result display obtains is more than 99%, with the homology of other species all
The highest or extremely low, concrete outcome sees table 1, thereby determines that the specific sequence that sequence is Lactobacillus plantarum of gained.
Table 1
| Bacterial strain | Homology |
| Lactobacillus plantarum (Lactobacillus plantarum 16) | 100.0% |
| Lactobacillus plantarum (Lactobacillus plantarum ZJ316) | 100.0% |
| Lactobacillus plantarum (Lactobacillus plantarum WCFS1) | 100.0% |
| Lactobacillus plantarum (Lactobacillus plantarum ST-III) | 100.0% |
| Lactobacillus plantarum (Lactobacillus plantarum JDM1) | 100.0% |
| Lactobacillus plantarum (Lactobacillus plantarum P-8) | 100.0% |
| Lactobacillus pentosus (Lactobacillus pentosus IG1) | 97.5% |
| Lactobacillus pentosus (Lactobacillus pentosus MP-10) | 97.5% |
| Lactobacillus brevis (Lactobacillus brevis KB290) | 64.3% |
| Lactobacillus brevis (Lactobacillus brevis ATCC 367) | 63.1% |
| Lactobacillus rhamnosus (Lactobacillus reuteri I5007) | 54.2% |
| Lactobacillus rhamnosus (Lactobacillus reuteri SD2112) | 54.2% |
| Lactobacillus rhamnosus (Lactobacillus reuteri TD1) | 53.9% |
| Enterococcus faecalis (Enterococcus faecium T110) | 44.3% |
The effectiveness comparison of the different semi-random primer of comparative example 1
(1) prepared by template
By Lactobacillus plantarumST-III bacterial strain activate separation and Culture, it is thus achieved that bacterium colony picking individual colonies,
It is inoculated in 1ml MRS culture medium, 37 DEG C of Anaerobic culturel 24 hours, centrifugal obtain thalline.Extract bacterial genomes, the reagent of use
Box is: TaKaRaminibest bacterial genomic DNA extraction kit ver.2.0 (Takara
Biotechnology(Dalian)Co.,Ltd.)。
(2) PCR amplification
Amplification reaction system and program are with embodiment 1.Semi-random primer therein is " AP1-AP5 ", and its sequence is the most such as
Shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 (being specifically shown in Table 2).
Table 2
| Primer | Primer sequence (5 ' → 3 ') |
| AP1 | GTCGGCGTTTATTCAGAAG(N)6GGACGAA |
| AP2 | GTCGGCGTTTATTCAGAAG(N)6CGCC |
| AP3 | GTCGGCGTTTATTCAGAAG(N)6ACGCC |
| AP4 | GTCGGCGTTTATTCAGAAG(N)6GACGCC |
| AP5 | GTCGGCGTTTATTCAGAAG(N)6GGACGGG |
(3) results contrast
The PCR primer that different semi-random primer amplifications obtain is carried out electrophoresis, and electrophoresis result is shown in Fig. 5, and in figure, " M " is DL2,
000DNA (Marker Takara Biotechnology (Dalian) Co, Ltd), numbering " 1-4 " is semi-random primer " AP2-
AP5 " distinguish corresponding PCR result, numbering 5 is semi-random PCR result corresponding for primer AP1.Wherein the band of AP1 amplification is the most
Clearly, show that semi-random primer used in the present invention has greater advantage on expanding effect.
The detection of Lactobacillus plantarum in application examples 1 testing sample
(1) preparation of template DNA: sample to be tested is the clear and lucid excellent plant lactobacillus beverage of light (original flavor, 340ml, commercially available), sample
Product draw plate isolation.To the single bacterium colony amplification cultivation grown, collect thalline, extract DNA.The preparation of template DNA uses liquid
Nitrogen freeze thawing-CTAB method extracting sample DNA.Extraction for genomic DNA, it is possible to use the business-like DNA extraction of equivalence
Test kit is also operated by its description.
(2) with the genomic DNA of step (1) gained as template, with the semi-random primer shown in SEQ ID NO.1 for amplification
Primer carries out single-primed PCR.The system of PCR amplification consists of: the 1 semi-random primer of μm ol/L (its sequence such as SEQ ID
Shown in NO.1), the Mg of 0.5mmol/L dNTP, 1.5mmol/L2+, 0.05U/ μ L Taq archaeal dna polymerase, and 2ng/ μ L gene
Group DNA profiling, cumulative volume 50 μ L.The program of PCR amplification is: 1. 93 DEG C, 6min；2. 96 DEG C, 20s；3. 58 DEG C, 30s；④72
DEG C, 30s；5. 70 DEG C, 10min；Wherein, step 2. to period 4. be 35.
(3) PCR primer being carried out electrophoresis, result shows there is clear band near 500bp, and rubber tapping is reclaimed this band and gone forward side by side
Row cloning and sequencing, and sequencing result is compared with sequence shown in SEQ ID NO.6.Comparison result shows, homology degree is more than
99%, show in sample to be tested containing Lactobacillus plantarum.
Using API 50CH reagent strip (France Mei Liai), the most traditional Physiology and biochemistry method is identified, result and above-mentioned point
The result of sub-authentication method is consistent.
The detection of Lactobacillus plantarum in application examples 2 testing sample
(1) preparation of template DNA: sample to be tested is the clear and lucid excellent Lactobacillus plantarum Yoghourt of light (125g, commercially available), and sample is drawn flat
Plate separation and Culture.To the single bacterium colony amplification cultivation grown, collect thalline, extract DNA.The preparation of template DNA uses lysozyme Method
Extracting sample DNA.
(2) with the genomic DNA of step (1) gained as template, with the semi-random primer shown in SEQ ID NO.1 for amplification
Primer carries out single-primed PCR.The system of PCR amplification consists of: the 2 semi-random primers of μm ol/L (its sequence such as SEQ ID
Shown in NO.1), the Mg of 1mmol/L dNTP, 2.5mmol/L2+, 0.10U/ μ LTaq archaeal dna polymerase, and 2ng/ μ L genome
DNA profiling, cumulative volume 50 μ L.The program of PCR amplification is: 1. 95 DEG C, 4min；2. 95 DEG C, 20s；3. 50 DEG C, 20s；4. 72 DEG C,
60s；5. 72 DEG C, 5min；Wherein, step 2. to period 4. be 40.
(3) PCR primer being carried out electrophoresis, result shows there is clear band near 500bp, and rubber tapping is reclaimed this band and gone forward side by side
Row cloning and sequencing, and sequencing result is compared with sequence shown in SEQ ID NO.6.Comparison result shows, homology degree is more than
99%, show in sample to be tested containing Lactobacillus plantarum.
Using API 50CH reagent strip (France Mei Liai), the most traditional Physiology and biochemistry method is identified, result and above-mentioned point
The result of sub-authentication method is consistent.
The detection of Lactobacillus plantarum in application examples 3 testing sample
(1) preparation of template DNA: sample to be tested is the clear and lucid excellent Lactobacillus plantarum fruit grain fermentation milk (210g, commercially available) of light, sample
Product draw plate isolation.To the single bacterium colony amplification cultivation grown, collect thalline, extract DNA.The preparation of template DNA uses liquid
Nitrogen freeze thawing-CTAB method extracting sample DNA.
(2) with the genomic DNA of step (1) gained as template, with the semi-random primer shown in SEQ ID NO.1 for amplification
Primer carries out single-primed PCR.The system of PCR amplification consists of: the 0.5 semi-random primer of μm ol/L (its sequence such as SEQ ID
Shown in NO.1), the Mg of 0.2mmol/L dNTP, 1.0mmol/L2+, 0.02U/ μ LTaq archaeal dna polymerase, and 0.5ng/ μ L base
Because of group DNA profiling, cumulative volume 50 μ L.The program of PCR amplification is: 1. 96 DEG C, 4min；2. 93 DEG C, 40s；3. 45 DEG C, 40s；④70
DEG C, 120s；5. 70 DEG C, 10min；Wherein, step 2. to period 4. be 25.
(3) PCR primer being carried out electrophoresis, result shows there is clear band near 500bp, and rubber tapping is reclaimed this band and gone forward side by side
Row cloning and sequencing, and sequencing result is compared with sequence shown in SEQ ID NO.6.Comparison result shows, homology degree is more than
99%, show in sample to be tested containing Lactobacillus plantarum.
Using API 50CH reagent strip (France Mei Liai), the most traditional Physiology and biochemistry method is identified, result and above-mentioned point
The result of sub-authentication method is consistent.
The detection of Lactobacillus plantarum in application examples 4 testing sample
(1) preparation of template DNA: sample to be tested is bright strong energy original flavor AB100 Yogurt (100g*6, commercially available), sample
Draw plate isolation.To the single bacterium colony amplification cultivation grown, collect thalline, extract DNA.The preparation of template DNA uses liquid nitrogen
Freeze thawing-CTAB method extracting sample DNA.Extraction for genomic DNA, it is possible to use the business-like DNA extraction examination of equivalence
Agent box is also operated by its description.
(2) with the genomic DNA of step (1) gained as template, with the semi-random primer shown in SEQ ID NO.1 for amplification
Primer carries out single-primed PCR.The system of PCR amplification consists of: the 1 semi-random primer of μm ol/L (its sequence such as SEQ ID
Shown in NO.1), the Mg of 0.5mmol/L dNTP, 1.5mmol/L2+, 0.05U/ μ L Taq archaeal dna polymerase, and 2ng/ μ L gene
Group DNA profiling, cumulative volume 50 μ L.The program of PCR amplification is: 1. 93 DEG C, 6min；2. 96 DEG C, 20s；3. 58 DEG C, 30s；④72
DEG C, 30s；5. 70 DEG C, 10min；Wherein, step 2. to period 4. be 35.
(3) PCR primer carrying out electrophoresis, result shows that surveyed bacterium colony does not all have 500bp band, shows in sample to be tested not
Containing Lactobacillus plantarum.
Using traditional Physiology and biochemistry method to identify, result is consistent with the result of above-mentioned method for identifying molecules.
Above-mentioned application examples shows, the specificity molecular marker DNA sequence of the present invention can be square in detection Lactobacillus plantarum
Just applying, simple to operate, result is accurately and reliably.
Should be understood that after the foregoing having read the present invention, the present invention can be made various by those skilled in the art
Changing or amendment, these equivalent form of values fall within the application appended claims limited range equally.
Claims (10)
1. the specificity molecular marker DNA of a Lactobacillus plantarum (Lactobacillus plantarum), it is characterised in that
Its nucleotide sequence is as shown in SEQ ID NO.6.
2. nucleotide sequence forms the Lactobacillus plantarum (Lactobacillus plantarum) as shown in SEQ ID NO.6
Specificity molecular marker DNA purposes in detection Lactobacillus plantarum.
3. the detection method of a Lactobacillus plantarum, it is characterised in that it comprises the steps:
(1) genomic DNA of sample to be tested is extracted；
(2) with the genomic DNA of step (1) gained as template, with the semi-random primer shown in SEQ ID NO.1 as amplimer
Carry out single-primed PCR；
(3) step (2) gained amplified production is carried out cloning and sequencing, and by sequencing result and the nucleoside shown in SEQ ID NO.6
Acid sequence is compared, if homology degree is more than 99%, then shows in sample to be tested containing Lactobacillus plantarum.
4. detection method as claimed in claim 3, it is characterised in that the side of the described genomic DNA extracting sample to be tested
Method is frozen-thawed-CTAB method or lysozyme Method.
5. detection method as claimed in claim 3, it is characterised in that the reaction system of described single-primed PCR includes
Following component: the 0.5～2.0 semi-random primers of μm ol/L, 0.2～1.0mmol/L dNTP, the Mg of 1.0～2.5mmol/L2+,
0.02～0.10U/ μ LTaq archaeal dna polymerase, and 0.5～2.0ng/ μ L genomic DNA template.
6. detection method as claimed in claim 3, it is characterised in that the response procedures of described single-primed PCR includes:
1. 93-96 DEG C, 4-6min；2. 93-96 DEG C, 20-40s；3. 45-58 DEG C, 20-40s；4. 70-72 DEG C, 30-120s；5. 70～72
DEG C, 5～10min；Wherein, step 2. to period 4. be 25-40.
7. detection method as claimed in claim 3, it is characterised in that the reaction system of described single-primed PCR includes
Following component: the 1.0 semi-random primers of μm ol/L, the Mg of 0.5mmol/L dNTP, 1.5mmol/L2+, 0.05U/ μ LTaq DNA gathers
Synthase, and 1.0ng/ μ L genomic DNA template.
8. detection method as claimed in claim 3, it is characterised in that the response procedures of described single-primed PCR includes:
1. 95 DEG C, 5min；2. 95 DEG C, 30s；3. 50 DEG C, 30s；4. 72 DEG C, 2min；5. 72 DEG C, 5min；Wherein, step is 2. to following 4.
Number of rings is 35.
9. detection method as claimed in claim 3, it is characterised in that described step (2) gained amplified production is carried out gram
Grand order-checking is carried out in the following manner: step (2) gained amplified production is carried out electrophoresis, if there is amplified band in 500bp position,
Then this band is carried out cloning and sequencing.
10. detection method as claimed in claim 9, it is characterised in that described electrophoresis is agarose gel electrophoresis.
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