CN104450949A - Specific molecular marker DNA sequence of lactobacillus plantarum and application of specific molecular marker DNA sequence - Google Patents
Specific molecular marker DNA sequence of lactobacillus plantarum and application of specific molecular marker DNA sequence Download PDFInfo
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Abstract
The invention discloses a specific molecular marker DNA sequence of lactobacillus plantarum, application of the specific molecular marker DNA sequence and a detection method of the lactobacillus plantarum. The specific molecular marker DNA sequence is shown in the SEQ ID NO.6. The detection method includes the following steps that (1) genomic DNA of a sample to be detected is extracted; (2) single-primer PCR amplification is conducted by taking the genomic DNA obtained in the step (1) as a template and taking a semi-random primer shown in the SEQ ID NO.1 as an amplification primer; (3) clone sequencing is conducted on amplification products obtained in the step (2), the sequencing result is compared with the sequence shown in the SEQ ID NO.6, and if the homologous degree is larger than 99%, it is indicated that the sample to be detected contains the lactobacillus plantarum.
Description
Technical field
The invention belongs to biological technical field, particularly the specificity molecular marker DNA sequence and uses thereof of a kind of plant lactobacillus (Lactobacillusplantarum).
Background technology
The detection will carrying out molecular level to a kind of bacterium needs to know the specific sequence of this bacterium, and obtains the specific sequence of a kind of bacterium, often needs to analyse and compare to known arrays a large amount of on the net; But the sequence that some bacterium has been surveyed is less, analyzes very difficult, be difficult to obtain its specific sequence.Certainly, also can collect all bacterial strains of a certain bacterium, carry out order-checking comparison, but the method experimental period is longer, costly.
Plant lactobacillus (Lactobacillus plantarum) is the one of probiotic bacterium, is widely used in, in various functional foodstuff, especially in the development research of milk-product, day by day coming into one's own.At present, the sequence of the plant lactobacillus that can obtain on the net through order-checking is less, is thus difficult to analyze the specific sequence obtaining plant lactobacillus, and this makes troubles to the Molecular Detection of plant lactobacillus.Therefore, be necessary to research and develop the specific sequence knowing plant lactobacillus, to facilitate, Molecular Detection carried out to it.
Summary of the invention
Technical problem to be solved by this invention is exactly the present situation for carrying out Molecular Detection inconvenience at present to plant lactobacillus (Lactobacillusplantarum), and provides specificity molecular marker DNA sequence of a kind of plant lactobacillus and uses thereof.The specificity molecular marker DNA sequence of plant lactobacillus of the present invention is greater than 99% with the genetic homology of the plant lactobacillus surveying sequence, can be advantageously used in carrying out Molecular Detection to plant lactobacillus.Before this, this molecule marker, without reporting, is the specific sequence of a newfound plant lactobacillus.
The present invention is solved the problems of the technologies described above by following technical proposals.
One of technical scheme provided by the invention is: the specificity molecular marker DNA of a kind of plant lactobacillus (Lactobacillusplantarum), and its nucleotide sequence is as shown in SEQ ID NO.6.
Two of technical scheme provided by the invention is: the specificity molecular marker DNA of the plant lactobacillus (Lactobacillus plantarum) of nucleotide sequence composition as shown in SEQ ID NO.6 is detecting the purposes in plant lactobacillus.
The specificity molecular marker DNA of the plant lactobacillus of nucleotide sequence composition as shown in SEQ ID NO.6, is the species specificity sequence of plant lactobacillus, can marks as molecular genetics, for the Molecular Detection of plant lactobacillus.
Three of technical scheme provided by the invention is: a kind of detection method of plant lactobacillus, it comprises the steps:
(1) genomic dna of sample to be tested is extracted;
(2) with the genomic dna of step (1) gained for template, with half random primer shown in SEQ ID NO.1 for amplimer carries out single-primed PCR;
(3) cloning and sequencing is carried out to step (2) gained amplified production, and the nucleotide sequence shown in sequencing result and SEQ IDNO.6 is compared, if homology degree is greater than 99%, then show in sample to be tested containing plant lactobacillus.
In the present invention, step (1) is extract the genomic dna of sample to be tested.The method of the genomic dna of described extraction sample to be tested is that this area is conventional, as adopted frozen-thawed-CTAB method, also can adopt lysozyme Method, commercially available various genome DNA extracting reagent kits can also be used to operate.
In the present invention, step (2) is with the genomic dna of step (1) gained for template, with half random primer shown in SEQID NO.1 for amplimer carries out single-primed PCR.Described single-primed PCR is the implication of the conventional indication in this area, namely only adds the synthesis that an amplimer carries out DNA fragmentation in amplification system.Preferably, the reaction system of described single-primed PCR comprises following component: 0.5 ~ 2.0 μm of ol/L half random primer, the Mg of 0.2 ~ 1.0mmol/L dNTP, 1.0 ~ 2.5mmol/L
2+, 0.02 ~ 0.10U/ μ LTaq archaeal dna polymerase, and 0.5 ~ 2.0ng/ μ L genomic DNA template.The response procedures of described single-primed PCR comprises: 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 cycle number be 4. 25-40.More preferably, the reaction system of described single-primed PCR comprises following component: 1.0 μm of ol/L half random primers, the Mg of 0.5mmol/L dNTP, 1.5mmol/L
2+, 0.05U/ μ LTaq archaeal dna polymerase, and 1.0ng/ μ L genomic DNA template.The response procedures of described single-primed PCR comprises: 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 cycle number be 4. 35.
In the present invention, step (3) is for carry out cloning and sequencing to step (2) gained amplified production, and the nucleotide sequence shown in sequencing result and SEQ ID NO.6 is compared, if homology degree is greater than 99%, then show in sample to be tested containing plant lactobacillus.Wherein, described cloning and sequencing is carried out to step (2) gained amplified production preferably carry out in the following manner: step (2) gained amplified production is carried out electrophoresis (preferred agarose gel electrophoresis, also can be polyacrylamide gel electrophoresis), if there is amplified band in 500bp position, then this band is carried out cloning and sequencing.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Compared to prior art, positive progressive effect of the present invention is:
The specificity molecular marker DNA sequence of the plant lactobacillus in the present invention is greater than 99% with the genetic homology of the plant lactobacillus surveying sequence, can be advantageously used in carrying out Molecular Detection to plant lactobacillus.Detection method of the present invention is simple, convenient fast, high specificity, and not needing to design PCR primer especially can detect sample to be tested, and cost is lower, and experimental period is short, has very wide application prospect.
Accompanying drawing explanation
Fig. 1 is the result of the specific PCR band screening of different lactobacterium plantarum strain.Numbering " M " in figure is DL2,000DNA Marker Takara Biotechnology (Dalian) Co, Ltd.; " 1-5 " is respectively: the PCR result of Lactobacillus plantarumST-III, Lactobacillus plantarumATCC8014, Lactobacillus plantarumACCC11095, Lactobacillus plantarumATCC14917, Lactobacillus plantarumWCFS1.
Fig. 2 is the result that the specific PCR band of different lactobacterium plantarum strain obtains.Numbering " M " in figure is DL2,000DNA Marker Takara Biotechnology (Dalian) Co, Ltd.." 1-8 " is respectively: the PCR result of Lactobacillus plantarum CGMCC1.119, Lactobacillus plantarumMCC1.11, Lactobacillus plantarum CICC22710, Lactobacillus plantarumCICC6234, Lactobacillus plantarum CGMCC1.3, Lactobacillus plantarumCICC20871, Lactobacillus plantarum CICC23132, LactobacillusplantarumCICC23506.
Fig. 3 is the result of the acquisition of the specific PCR band of lactobacterium plantarum strain.Numbering " M " in figure is DL2,000DNA Marker Takara Biotechnology (Dalian) Co, Ltd.; " 1-2 " is respectively: the PCR result of Lactobacillus plantarumATCC14917, Lactobacillus plantarumWCFS1.
Fig. 4 is the result that the specific PCR band of different lactobacterium plantarum strain obtains.Numbering " M " in figure is DL2,000DNA Marker Takara Biotechnology (Dalian) Co, Ltd.." 1-10 " is respectively: Lactobacillus plantarum CGMCC1.119, Lactobacillus plantarumMCC1.11, Lactobacillus plantarum CICC22710, Lactobacillus plantarumCICC6234, Lactobacillus plantarum CGMCC1.3, Lactobacillus plantarumCICC20871, Lactobacillus plantarum CICC23132, LactobacillusplantarumCICC23506, Lactobacillus plantarumATCC14917, the PCR result of LactobacillusplantarumWCFS1.
Fig. 5 is the effectiveness comparison of different half random primer.In figure, " M " is DL2,000DNA (MarkerTakara Biotechnology (Dalian) Co, Ltd), numbering " 1-4 " is the PCR result that half random primer " AP2-AP5 " is corresponding respectively, and numbering 5 is the PCR result that half random primer AP1 is corresponding.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
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:
Plant lactobacillus:
Lactobacillus plantarumST-III takes from Shanghai Bright Dairy & Food Co., Ltd., and this is the patented strain of applicant, also can 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 lactobacterium plantarum strain of embodiment 1
(1) Template preparation
By each bacterial strain activation separation and Culture, the bacterium colony picking individual colonies of acquisition, is inoculated in 1ml MRS substratum, 37 DEG C of Anaerobic culturel 24 hours, centrifugal acquisition thalline.Extract bacterial genomes, the test kit of use is: TaKaRaminibest bacterial genomic DNA extraction kit ver.2.0 (TakaraBiotechnology (Dalian) Co., Ltd.).
(2) pcr amplification
The system of pcr amplification consists of: 1 μm of ol/L half random primer (its sequence is as shown in SEQ ID NO.1), the Mg of 0.5mmol/L dNTP, 1.5mmol/L
2+, 0.05U/ μ LTaq archaeal dna polymerase, and 500ng (please representing with concentration) genomic DNA template, 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 cycle number be 4. 35.
(3) acquisition of specific band
PCR primer is carried out electrophoresis, and the results are shown in Figure and numbering " M " in 1, figure is DL2,000DNAMarker Takara Biotechnology (Dalian) Co, Ltd.." 1-5 " is respectively: the PCR result of LactobacillusplantarumST-III, Lactobacillus plantarumATCC8014, LactobacillusplantarumACCC11095, Lactobacillus plantarumATCC14917, LactobacillusplantarumWCFS1.
Purifying is reclaimed and cloning and sequencing about the band of 500bp (arrow indication place) greatly in position total in swimming lane 1-5, and sequencing result is as shown in SEQ ID NO.6.
The acquisition of the specific PCR band of the different lactobacterium plantarum strain of embodiment 2
(1) Template preparation
By each bacterial strain activation separation and Culture, the bacterium colony picking individual colonies of acquisition, is inoculated in 1ml MRS substratum, 37 DEG C of Anaerobic culturel 24 hours, centrifugal acquisition thalline.Extract bacterial genomes, the test kit of use is: TaKaRaminibest bacterial genomic DNA extraction kit ver.2.0 (TakaraBiotechnology (Dalian) Co., Ltd.).
(2) pcr amplification
The system of pcr amplification consists of: 2 μm of ol/L half random primers (its sequence is as shown in SEQ ID NO.1), the Mg of 1mmol/L dNTP, 2.5mmol/L
2+, 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 cycle number be 4. 40.
(3) acquisition of specific band
PCR primer is carried out electrophoresis, and the results are shown in Figure and numbering " M " in 2, figure is DL2,000DNAMarker Takara Biotechnology (Dalian) Co, Ltd.." 1-8 " is respectively: the PCR result of Lactobacillusplantarum CGMCC1.119, Lactobacillus plantarum MCC1.11, Lactobacillusplantarum CICC22710, Lactobacillus plantarum CICC6234, Lactobacillusplantarum CGMCC1.3, Lactobacillus plantarum CICC20871, Lactobacillusplantarum CICC23132, Lactobacillus plantarumCICC23506.
Purifying is reclaimed and cloning and sequencing about the band of 500bp (arrow indication place) greatly in position total in swimming lane 1-8, and sequencing result is all as shown in SEQ ID NO.6.
The acquisition of the specific PCR band of embodiment 3 lactobacterium plantarum strain
(1) Template preparation
Bacterial strain is activated separation and Culture, the bacterium colony picking individual colonies of acquisition, be inoculated in 1ml MRS substratum, 37 DEG C of Anaerobic culturel 24 hours, centrifugal acquisition thalline.Extract bacterial genomes, the test kit of use is: TaKaRaminibest bacterial genomic DNA extraction kit ver.2.0 (TakaraBiotechnology (Dalian) Co., Ltd.).
(2) pcr amplification
The system of pcr amplification consists of: 0.5 μm of ol/L half random primer (its sequence is as shown in SEQ ID NO.1), the Mg of 0.2mmol/L dNTP, 1.0mmol/L
2+, 0.02U/ μ LTaq archaeal dna polymerase, and 0.5ng/ μ L genomic DNA template, 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 cycle number be 4. 25.
(3) acquisition of specific band
PCR primer is carried out electrophoresis, and the results are shown in Figure and numbering " M " in 3, figure is DL2,000DNAMarker Takara Biotechnology (Dalian) Co, Ltd.." 1-2 " is respectively: the PCR result of LactobacillusplantarumATCC14917, Lactobacillus plantarumWCFS1.
Purifying is reclaimed and cloning and sequencing about the band of 500bp (arrow indication place) greatly in the position had in swimming lane, and sequencing result is all as shown in SEQ ID NO.6.
The acquisition of the specific PCR band of embodiment 4 lactobacterium plantarum strain
(1) Template preparation
Bacterial strain is activated separation and Culture, the bacterium colony picking individual colonies of acquisition, be inoculated in 1ml MRS substratum, 37 DEG C of Anaerobic culturel 24 hours, centrifugal acquisition thalline.Extract bacterial genomes, the test kit of use is: TaKaRaminibest bacterial genomic DNA extraction kit ver.2.0 (TakaraBiotechnology (Dalian) Co., Ltd.).
(2) pcr amplification
The system of pcr amplification consists of: 1 μm of ol/L half random primer (its sequence is as shown in SEQ ID NO.1), the Mg of 0.5mmol/L dNTP, 1.5mmol/L
2+, 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 cycle number be 4. 35.
(3) acquisition of specific band
PCR primer is carried out electrophoresis, and the results are shown in Figure and numbering " M " in 4, figure is DL2,000DNAMarker Takara Biotechnology (Dalian) Co, Ltd.." 1-10 " is respectively: Lactobacillusplantarum CGMCC1.119, Lactobacillus plantarum MCC1.11, Lactobacillusplantarum CICC22710, Lactobacillus plantarum CICC6234, Lactobacillusplantarum CGMCC1.3, Lactobacillus plantarum CICC20871, Lactobacillusplantarum CICC23132, Lactobacillus plantarumCICC23506, LactobacillusplantarumATCC14917, the PCR result of Lactobacillus plantarumWCFS1.
Purifying is reclaimed and cloning and sequencing about the band of 500bp (arrow indication place) greatly in the position had in swimming lane, and sequencing 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 the specific sequence that result display obtains and plant lactobacillus is greater than 99%, not high with the homology of other species, or it is extremely low, concrete outcome, see table 1, determines that the sequence of gained is the specific sequence of plant lactobacillus thus.
Table 1
| Bacterial strain | Homology |
| Plant lactobacillus (Lactobacillus plantarum 16) | 100.0％ |
| Plant lactobacillus (Lactobacillus plantarum ZJ316) | 100.0％ |
| Plant lactobacillus (Lactobacillus plantarum WCFS1) | 100.0％ |
| Plant lactobacillus (Lactobacillus plantarum ST-III) | 100.0％ |
| Plant lactobacillus (Lactobacillus plantarum JDM1) | 100.0％ |
| Plant lactobacillus (Lactobacillus plantarum P-8) | 100.0％ |
| Lactobacillus pentosus (Lactobacillus pentosus IG1) | 97.5％ |
| Lactobacillus pentosus (Lactobacillus pentosus MP-10) | 97.5％ |
| Short lactobacillus (Lactobacillus brevis KB290) | 64.3％ |
| Short lactobacillus (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％ |
| Faecium (Enterococcus faecium T110) | 44.3％ |
The effectiveness comparison of different half random primer of comparative example 1
(1) Template preparation
By Lactobacillus plantarumST-III bacterial strain activation separation and Culture, the bacterium colony picking individual colonies of acquisition, is inoculated in 1ml MRS substratum, 37 DEG C of Anaerobic culturel 24 hours, centrifugal acquisition thalline.Extract bacterial genomes, the test kit of use is: TaKaRaminibest bacterial genomic DNAextraction kit ver.2.0 (Takara Biotechnology (Dalian) Co., Ltd.).
(2) pcr amplification
Amplification reaction system and program are with embodiment 1.Half random primer is wherein " AP1-AP5 ", and its sequence is respectively as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 (specifically 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 half random primer amplification obtains is carried out electrophoresis, electrophoresis result is shown in Fig. 5, in figure, " M " is DL2,000DNA (Marker Takara Biotechnology (Dalian) Co, Ltd), numbering " 1-4 " is the PCR result that half random primer " AP2-AP5 " is corresponding respectively, and numbering 5 is the PCR result that half random primer AP1 is corresponding.Wherein the band of AP1 amplification is the most clear, shows that half random primer used in the present invention has greater advantage on expanding effect.
The detection of plant lactobacillus in application examples 1 testing sample
(1) preparation of template DNA: sample to be tested is bright smooth excellent plant lactobacillus drink (original flavor, 340ml, commercially available), and plate isolation drawn by sample.To the single bacterium colony amplification cultivation grown, collect thalline, extract DNA.The preparation of template DNA adopts frozen-thawed-CTAB method extracting sample DNA.For the extraction of genomic dna, also can use the business-like DNA extraction kit of equivalence and operate by its specification sheets.
(2) with the genomic dna of step (1) gained for template, with half random primer shown in SEQ ID NO.1 for amplimer carries out single-primed PCR.The system of pcr amplification consists of: 1 μm of ol/L half random primer (its sequence is as shown in SEQ ID NO.1), the Mg of 0.5mmol/L dNTP, 1.5mmol/L
2+, 0.05U/ μ L Taq 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 cycle number be 4. 35.
(3) PCR primer is carried out electrophoresis, result is presented near 500bp clear band, and rubber tapping is reclaimed this band and carried out cloning and sequencing, and sequence shown in sequencing result and SEQ ID NO.6 is compared.Comparison result shows, and homology degree is greater than 99%, shows in sample to be tested containing plant lactobacillus.
Adopt API 50CH reagent strip (French Mei Liai), namely traditional Physiology and biochemistry method qualification, result is consistent with the result of above-mentioned molecular assay method.
The detection of plant lactobacillus in application examples 2 testing sample
(1) preparation of template DNA: sample to be tested is bright smooth excellent plant lactobacillus Yoghourt (125g, commercially available), and plate isolation drawn by sample.To the single bacterium colony amplification cultivation grown, collect thalline, extract DNA.The preparation of template DNA adopts lysozyme Method extracting sample DNA.
(2) with the genomic dna of step (1) gained for template, with half random primer shown in SEQ ID NO.1 for amplimer carries out single-primed PCR.The system of pcr amplification consists of: 2 μm of ol/L half random primers (its sequence is as shown in SEQ ID NO.1), the Mg of 1mmol/L dNTP, 2.5mmol/L
2+, 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 cycle number be 4. 40.
(3) PCR primer is carried out electrophoresis, result is presented near 500bp clear band, and rubber tapping is reclaimed this band and carried out cloning and sequencing, and sequence shown in sequencing result and SEQ ID NO.6 is compared.Comparison result shows, and homology degree is greater than 99%, shows in sample to be tested containing plant lactobacillus.
Adopt API 50CH reagent strip (French Mei Liai), namely traditional Physiology and biochemistry method qualification, result is consistent with the result of above-mentioned molecular assay method.
The detection of plant lactobacillus in application examples 3 testing sample
(1) preparation of template DNA: sample to be tested is bright smooth excellent plant lactobacillus fruit grain fermented-milk (210g, commercially available), and plate isolation drawn by sample.To the single bacterium colony amplification cultivation grown, collect thalline, extract DNA.The preparation of template DNA adopts frozen-thawed-CTAB method extracting sample DNA.
(2) with the genomic dna of step (1) gained for template, with half random primer shown in SEQ ID NO.1 for amplimer carries out single-primed PCR.The system of pcr amplification consists of: 0.5 μm of ol/L half random primer (its sequence is as shown in SEQ ID NO.1), the Mg of 0.2mmol/L dNTP, 1.0mmol/L
2+, 0.02U/ μ LTaq archaeal dna polymerase, and 0.5ng/ μ L genomic DNA template, 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 cycle number be 4. 25.
(3) PCR primer is carried out electrophoresis, result is presented near 500bp clear band, and rubber tapping is reclaimed this band and carried out cloning and sequencing, and sequence shown in sequencing result and SEQ ID NO.6 is compared.Comparison result shows, and homology degree is greater than 99%, shows in sample to be tested containing plant lactobacillus.
Adopt API 50CH reagent strip (French Mei Liai), namely traditional Physiology and biochemistry method qualification, result is consistent with the result of above-mentioned molecular assay method.
The detection of plant lactobacillus 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), and plate isolation drawn by sample.To the single bacterium colony amplification cultivation grown, collect thalline, extract DNA.The preparation of template DNA adopts frozen-thawed-CTAB method extracting sample DNA.For the extraction of genomic dna, also can use the business-like DNA extraction kit of equivalence and operate by its specification sheets.
(2) with the genomic dna of step (1) gained for template, with half random primer shown in SEQ ID NO.1 for amplimer carries out single-primed PCR.The system of pcr amplification consists of: 1 μm of ol/L half random primer (its sequence is as shown in SEQ ID NO.1), the Mg of 0.5mmol/L dNTP, 1.5mmol/L
2+, 0.05U/ μ L Taq 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 cycle number be 4. 35.
(3) PCR primer is carried out electrophoresis, result display institute surveys bacterium colony and does not all have 500bp band, shows not contain plant lactobacillus in sample to be tested.
Adopt traditional Physiology and biochemistry method qualification, result is consistent with the result of above-mentioned molecular assay method.
Above-mentioned application examples shows, specificity molecular marker DNA sequence of the present invention can be applied easily in detection plant lactobacillus, and simple to operate, result accurately and reliably.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a specificity molecular marker DNA for plant lactobacillus (Lactobacillus plantarum), is characterized in that, its nucleotide sequence is as shown in SEQ ID NO.6.
2. the specificity molecular marker DNA of the plant lactobacillus (Lactobacillusplantarum) of nucleotide sequence composition as shown in SEQ ID NO.6 is detecting the purposes in lactobacterium casei.
3. a detection method for plant lactobacillus, is characterized 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 for template, with half random primer shown in SEQ ID NO.1 for amplimer carries out single-primed PCR;
(3) cloning and sequencing is carried out to step (2) gained amplified production, and the nucleotide sequence shown in sequencing result and SEQ IDNO.6 is compared, if homology degree is greater than 99%, then show in sample to be tested containing plant lactobacillus.
4. detection method as claimed in claim 3, it is characterized in that, the method for the genomic dna of described extraction sample to be tested is frozen-thawed-CTAB method or lysozyme Method.
5. detection method as claimed in claim 3, it is characterized in that, the reaction system of described single-primed PCR comprises following component: 0.5 ~ 2.0 μm of ol/L half random primer, the Mg of 0.2 ~ 1.0mmol/LdNTP, 1.0 ~ 2.5mmol/L
2+, 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 characterized in that, the response procedures of described single-primed PCR comprises: 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 cycle number be 4. 25-40.
7. detection method as claimed in claim 3, it is characterized in that, the reaction system of described single-primed PCR comprises following component: 1.0 μm of ol/L half random primers, the Mg of 0.5mmol/L dNTP, 1.5mmol/L
2+, 0.05U/ μ LTaq archaeal dna polymerase, and 1.0ng/ μ L genomic DNA template.
8. detection method as claimed in claim 3, it is characterized in that, the response procedures of described single-primed PCR comprises: 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 cycle number be 4. 35.
9. detection method as claimed in claim 3, it is characterized in that, described cloning and sequencing is carried out to step (2) gained amplified production carry 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 characterized in that, described electrophoresis is agarose gel electrophoresis.
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| CN117512152A (en) * | 2023-12-08 | 2024-02-06 | 华中农业大学 | Molecular marker combination and primer combination for rapidly detecting lactobacillus plantarum Liang and detection method thereof |
| CN117512152B (en) * | 2023-12-08 | 2024-05-28 | 华中农业大学 | Molecular marker combination and primer combination for rapidly detecting lactobacillus plantarum Liang and detection method thereof |
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| CN101712987A (en) * | 2009-09-21 | 2010-05-26 | 内蒙古农业大学 | Method for quickly, qualitatively and quantitatively measuring Lactobacillus plantarum in probiotic dairy products |
-
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Non-Patent Citations (2)
| Title |
|---|
| 孙志宏等: "分子标记技术用于乳酸菌分类鉴定研究进展", 《中国乳品工业》, vol. 34, no. 5, 31 December 2006 (2006-12-31), pages 35 - 38 * |
| 胡哲: "两种标签引物RT-PCR结合Sanger测序检测登革病毒的研究", 《中国博士学位论文全文数据库 医药卫生科技辑》, no. 7, 15 July 2010 (2010-07-15), pages 059 - 7 * |
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| CN117512152A (en) * | 2023-12-08 | 2024-02-06 | 华中农业大学 | Molecular marker combination and primer combination for rapidly detecting lactobacillus plantarum Liang and detection method thereof |
| CN117512152B (en) * | 2023-12-08 | 2024-05-28 | 华中农业大学 | Molecular marker combination and primer combination for rapidly detecting lactobacillus plantarum Liang and detection method thereof |
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