CN104531695B - A kind of specificity molecular marker DNA sequence of Lactobacillus casei and application thereof - Google Patents
A kind of specificity molecular marker DNA sequence of Lactobacillus casei and application thereof Download PDFInfo
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Abstract
The detection method of specificity molecular marker DNA sequence the invention discloses a kind of Lactobacillus casei (Lactobacillus casei) and application thereof and Lactobacillus casei.The specificity molecular marker DNA sequence is as shown in SEQ ID NO.6.The detection method includes step：(1) genomic DNA of sample to be tested is extracted；(2) genomic DNA obtained by step (1) is as template, and the semi-random primer shown in SEQ ID NO.1 carries out single-primed PCR as amplimer；(3) cloning and sequencing is carried out to step (2) gained amplified production, and sequencing result is compared with sequence shown in SEQ ID NO.6, if homologous degree is more than 99%, show to contain Lactobacillus casei in sample to be tested.
Description
Technical field
The invention belongs to biological technical field, more particularly to a kind of Lactobacillus casei (Lactobacillus casei)
Specificity molecular marker DNA sequence and application thereof.
Background technology
The detection of molecular level is carried out to a kind of bacterium to be needed to know the specific sequence of the bacterium, and obtains a kind of thin
The specific sequence of bacterium, generally requires to be analyzed comparison to online substantial amounts of known array；But the sequence that some bacteriums have been surveyed
Less, analysis is got up extremely difficult, it is difficult to obtain its specific sequence.It is of course also possible to collect all bacterium of a certain bacterium
Strain, carries out sequencing comparison, but the method experimental period is more long, high cost.
Lactobacillus casei (Lactobacillus casei) is one kind of probiotics, is widely used in various functions food
In product, especially in the developmental research of dairy products, it is increasingly subject to pay attention to.At present, the Lactobacillus casei that can be obtained on the net
The sequence for passing through sequencing is less, thus is difficult to analyze the specific sequence for obtaining Lactobacillus casei, and this gives Lactobacillus casei
Molecular Detection make troubles.Therefore, it is necessary to research and develop the specific sequence for knowing Lactobacillus casei, it is entered with facilitating
Row Molecular Detection.
The content of the invention
The technical problems to be solved by the invention are aiming at present to Lactobacillus casei (Lactobacillus casei)
The inconvenient present situation of Molecular Detection is carried out, and a kind of specificity molecular marker DNA sequence of Lactobacillus casei and application thereof is provided.
The specificity molecular marker DNA sequence of Lactobacillus casei of the invention with surveyed sequence Lactobacillus casei genetic homology
More than 99%, may be conveniently used carries out Molecular Detection to Lactobacillus casei.Before this, the molecular labeling is without reporting
One specific sequence of newfound Lactobacillus casei.
The present invention solves above-mentioned technical problem by following technical proposals.
One of technical scheme that the present invention is provided is：A kind of Lactobacillus casei (Lactobacillus casei) it is special
Property molecular marker DNA, its nucleotide sequence is as shown in SEQ ID NO.6.
In the present invention, described Lactobacillus casei (Lactobacillus casei) includes belonging to Lactobacillus casei
The lactobacillus paracasei (Lactobacillus paracasei) of (Lactobacillus casei) group.Lactobacillus paracasei
(Lactobacillus paracasei) is also lactobacillus paracasei, because at present for the taxonomy of lactobacillus paracasei
There is dispute always, many scholars advocate to cancel the title of lactobacillus paracasei, are classified to Lactobacillus casei and (refer to
" the application study progress of lactobacillus paracasei ", village sea ceases raining or snowing,《Biotechnology communications》, in November, 2006, the 6th phase of volume 17).
To exempt to obscure, signified Lactobacillus casei (Lactobacillus casei) is included and belongs to Lactobacillus casei in the application
The lactobacillus paracasei (Lactobacillus paracasei) of group, clearly states herein.
Technical scheme that the present invention is provided two is：Nucleotide sequence constitutes the cheese breast bar as shown in SEQ ID NO.6
Purposes of the specificity molecular marker DNA of bacterium (Lactobacillus casei) in Lactobacillus casei is detected.
Nucleotide sequence constitutes the spy of the Lactobacillus casei (Lactobacillus casei) as shown in SEQ ID NO.6
Opposite molecule marker DNA, is the species specificity sequence of Lactobacillus casei, can be marked as molecular genetics, for cheese breast
The Molecular Detection of bacillus.
Technical scheme that the present invention is provided three is：A kind of detection method of Lactobacillus casei, it comprises the following steps：
(1) genomic DNA of sample to be tested is extracted；
(2) as template, the semi-random primer shown in SEQ ID NO.1 is amplification to the genomic DNA obtained by step (1)
Primer carries out single-primed PCR；
(3) cloning and sequencing is carried out to step (2) gained amplified production, and by shown in sequencing result and SEQ ID NO.6
Nucleotide sequence is compared, if homologous degree is more than 99%, shows to contain Lactobacillus casei in sample to be tested.
In the present invention, step (1) is to extract the genomic DNA of sample to be tested.The genome of described extraction sample to be tested
The method of DNA is this area routine, can such as use frozen-thawed-CTAB methods, it would however also be possible to employ lysozyme Method, can also be used
Commercially available various genome DNA extracting reagent kits are operated.
In the present invention, step (2) be genomic DNA obtained by step (1) as template, with shown in SEQ ID NO.1
Semi-random primer carries out single-primed PCR for amplimer.Described single-primed PCR contains for this area is conventionally referred
An amplimer is only added in justice, i.e. amplification system carries out the synthesis of DNA fragmentation.It is preferred that described single-primed PCR
Reaction system include following component：0.5~2.0 μm of semi-random primer of ol/L, 0.2~1.0mmol/L dNTP, 1.0~
The Mg of 2.5mmol/L2+, 0.02~0.10U/ μ LTaq archaeal dna polymerases, and 0.5~2.0ng/ μ L genomic DNA templates.Institute
The response procedures of the single-primed PCR stated include：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, 2. step is 25-40 to period 4..
More preferably, the reaction system of described single-primed PCR includes following component：1.0 μm of semi-random primers of ol/L, 0.5mmol/
The Mg of L dNTP, 1.5mmol/L2+, 0.05U/ μ LTaq archaeal dna polymerases, and 1.0ng/ μ L genomic DNA templates.Described
The response procedures of single-primed PCR include：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, 2. step is 35 to period 4..
In the present invention, step (3) is to carry out cloning and sequencing to step (2) gained amplified production, and by sequencing result and SEQ
Nucleotide sequence shown in ID NO.6 is compared, if homologous degree is more than 99%, shows to contain cheese breast bar in sample to be tested
Bacterium.Wherein, it is described cloning and sequencing is carried out to step (2) gained amplified production preferably to carry out in the following manner：By step (2)
Gained amplified production carries out electrophoresis (preferably agarose gel electrophoresis, or polyacrylamide gel electrophoresis), if in 800bp
There is amplified band in position, then the band is carried out into cloning and sequencing.
On the basis of common sense in the field is met, above-mentioned each optimum condition can be combined, and obtain final product each preferable reality of the present invention
Example.
Agents useful for same of the present invention and raw material are commercially available.
Compared to prior art, positive effect of the invention is：
The specificity molecular marker DNA sequence of the Lactobacillus casei in the present invention and the Lactobacillus casei for having surveyed sequence
Genetic homology is more than 99%, and may be conveniently used carries out Molecular Detection to Lactobacillus casei.Detection method behaviour of the invention
Make simple, fast and easy, high specificity, it is not necessary to sample to be tested is detected by being specifically designed PCR primer, cost compared with
Low, experimental period is short, with very wide application prospect.
Brief description of the drawings
Fig. 1 is the selection result of the specific PCR band of different lactobacillus casei bacterial strains.It is DL2 that " M " is numbered in figure,
000DNA Marker Takara Biotechnology(Dalian)Co,Ltd.；" 1-4 " is respectively：Lactobacillus
casei 431、Lactobacillus casei LC2W、Lactobacillus casei01、Lactobacillus casei
Shirota、PCR results.
Fig. 2 is the acquisition result of the specific PCR band of different lactobacillus casei bacterial strains.It is DL2 that " M " is numbered in figure,
000DNA Marker Takara Biotechnology(Dalian)Co,Ltd.." 1-6 " is respectively：Lactobacillus
caseiATCC393、Lactobacillus caseiCICC21001、Lactobacillus caseiACCC10639、
Lactobacillus caseiCICC23185、Lactobacillus caseiCICC20994、Lactobacillus
The PCR results of caseiCCTCCM94011.
Fig. 3 is the acquisition result of the specific PCR band of lactobacillus casei bacterial strain.It is DL2,000DNA that " M " is numbered in figure
Marker Takara Biotechnology(Dalian)Co,Ltd.." 1-2 " is respectively：Lactobacillus casei
01st, the PCR results of Lactobacillus casei Shirota.
Fig. 4 is the acquisition result of the specific PCR band of lactobacillus casei bacterial strain.It is DL2,000DNA that " M " is numbered in figure
Marker Takara Biotechnology(Dalian)Co,Ltd.." 1-10 " is respectively：Lactobacillus
caseiATCC393、Lactobacillus caseiCICC21001、Lactobacillus caseiACCC10639、
Lactobacillus caseiCICC23185、Lactobacillus caseiCICC20994、Lactobacillus
caseiCCTCCM94011、Lactobacillus casei 431、Lactobacillus casei LC2W、
The PCR results of Lactobacillus casei 01, Lactobacillus casei Shirota.
Fig. 5 is that the effect of different semi-random primers compares." M " is DL2,000DNA (Marker Takara in figure
Biotechnology (Dalian) Co, Ltd), numbering " 1-4 " is semi-random primer " AP2-AP5 " corresponding PCR results respectively,
Numbering 5 is the corresponding PCR results of semi-random primer AP1.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to described reality
Apply among a scope.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or according to business
Product specification is selected.
In following embodiments, if not otherwise indicated, agents useful for same and material are conventional commercial and can obtain.
In following embodiments, the source of following experiment materials is：
Lactobacillus casei：
Lactobacillus casei 431 are purchased from Danisco US Inc. Genencor Divisi；
Lactobacillus casei LC2W take from Shanghai Bright Dairy ＆ Food Co., Ltd., and this is the patent bacterium of applicant
Strain, is also purchased from CGMCC, and its deposit number is CGMCC NO.0828；
Lactobacillus casei 01 are purchased from Shanghai Bei Nuo bio tech ltd；
Lactobacillus casei Shirota are purchased from Shanghai Bei Nuo bio tech ltd；
Lactobacillus caseiATCC393 are purchased from Shanghai Bei Nuo bio tech ltd；
Lactobacillus caseiCICC21001 are purchased from Beijing North Na Kaichuan Bioisystech Co., Ltd；
Lactobacillus caseiACCC10639 are purchased from Beijing North Na Kaichuan Bioisystech Co., Ltd；
Lactobacillus caseiCICC23185 are purchased from Beijing North Na Kaichuan Bioisystech Co., Ltd；
Lactobacillus caseiCICC20994 are purchased from Beijing North Na Kaichuan Bioisystech Co., Ltd；
Lactobacillus caseiCCTCCM94011 are purchased from Beijing North Na Kaichuan Bioisystech Co., Ltd.
The screening of the specific PCR band of the different lactobacillus casei bacterial strains of embodiment 1
(1) prepared by template
The activation of each bacterial strain is separately cultured, the bacterium colony picking individual colonies of acquisition are inoculated in 1ml MRS culture mediums, and 37 DEG C are detested
Oxygen culture 24 hours, centrifugation obtains thalline.Bacterial genomes are extracted, the kit for using is：TaKaRa minibest
bacterial genomic DNA extraction kit ver.2.0(Takara Biotechnology(Dalian)Co.,
Ltd.)。
(2) PCR amplifications
The system of PCR amplifications constitutes and is：1 μm of semi-random primer of 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 L Taq archaeal dna polymerases, and 2ng/ μ L genomic DNA templates,
The μ L of cumulative volume 50.
PCR amplification program be：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, 2. step is 35 to period 4..
(3) acquisition of specific band
PCR primer is carried out into electrophoresis, Fig. 1 is as a result seen, it is DL2,000DNA Marker Takara that " M " is numbered in figure
Biotechnology(Dalian)Co,Ltd.." 1-4 " is respectively：Lactobacillus casei431、Lactobacillus
casei LC2W、Lactobacillus casei01、Lactobacillus caseiShirota、PCR results.
Will in swimming lane 1-4 have position about 800bp band (at arrow meaning) recovery purifying simultaneously cloning and sequencing,
Sequencing result is as shown in SEQ ID NO.6.
The acquisition of the specific PCR band of the different lactobacillus casei bacterial strains of embodiment 2
(1) prepared by template
The activation of each bacterial strain is separately cultured, the bacterium colony picking individual colonies of acquisition are inoculated in 1ml MRS culture mediums, and 37 DEG C are detested
Oxygen culture 24 hours, centrifugation obtains thalline.Bacterial genomes are extracted, the kit for using is：TaKaRaminibest
bacterial genomic DNA extraction kit ver.2.0(Takara Biotechnology(Dalian)Co.,
Ltd.)。
(2) PCR amplifications
The system of PCR amplifications constitutes and is：2 μm of semi-random primers of 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 polymerases, and 2ng/ μ L genomic DNA templates, the μ of cumulative volume 50
L。
PCR amplification program be：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, 2. step is 40 to period 4..
(3) acquisition of specific band
PCR primer is carried out into electrophoresis, Fig. 2 is as a result seen, it is DL2,000DNA Marker Takara that " M " is numbered in figure
Biotechnology(Dalian)Co,Ltd.." 1-6 " is respectively：Lactobacillus caseiATCC393、
Lactobacillus caseiCICC21001、Lactobacillus caseiACCC10639、Lactobacillus
CaseiCICC23185, Lactobacillus caseiCICC20994, Lactobacillus caseiCCTCCM94011's
PCR results.
Will in swimming lane 1-6 have position about 800bp band (at arrow meaning) recovery purifying simultaneously cloning and sequencing,
Sequencing result is all as shown in SEQ ID NO.6.
The acquisition of the specific PCR band of the lactobacillus casei bacterial strain of embodiment 3
(1) prepared by template
Bacterial strain activation is separately cultured, the bacterium colony picking individual colonies of acquisition are inoculated in 1ml MRS culture mediums, 37 DEG C of anaerobism
Culture 24 hours, centrifugation obtains thalline.Bacterial genomes are extracted, the kit for using is：TaKaRaminibest
bacterial genomic DNA extraction kit ver.2.0(Takara Biotechnology(Dalian)Co.,
Ltd.)。
(2) PCR amplifications
The system of PCR amplifications constitutes and is：0.5 μm of semi-random primer of 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 polymerases, and 0.5ng/ μ L genomic DNA moulds
Plate, the μ L of cumulative volume 50.
PCR amplification program be：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, 2. step is 25 to period 4..
(3) acquisition of specific band
PCR primer is carried out into electrophoresis, Fig. 3 is as a result seen, it is DL2,000DNA Marker Takara that " M " is numbered in figure
Biotechnology(Dalian)Co,Ltd.." 1-2 " is respectively：Lactobacillus casei01、Lactobacillus
The PCR results of casei Shirota.
The position that will be had in swimming lane about in band (at the arrow meaning) recovery purifying and cloning and sequencing of 800bp, is surveyed
Sequence result is all as shown in SEQ ID NO.6.
The acquisition of the specific PCR band of the lactobacillus casei bacterial strain of embodiment 4
(1) prepared by template
Bacterial strain activation is separately cultured, the bacterium colony picking individual colonies of acquisition are inoculated in 1ml MRS culture mediums, 37 DEG C of anaerobism
Culture 24 hours, centrifugation obtains thalline.Bacterial genomes are extracted, the kit for using is：TaKaRaminibest
bacterial genomic DNA extraction kit ver.2.0(Takara Biotechnology(Dalian)Co.,
Ltd.)。
(2) PCR amplifications
The system of PCR amplifications constitutes and is：1 μm of semi-random primer of 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 L Taq archaeal dna polymerases, and 2ng/ μ L genomic DNA templates,
The μ L of cumulative volume 50.
PCR amplification program be：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, 2. step is 35 to period 4..
(3) acquisition of specific band
PCR primer is carried out into electrophoresis, Fig. 4 is as a result seen, it is DL2,000DNA Marker Takara that " M " is numbered in figure
Biotechnology(Dalian)Co,Ltd.." 1-10 " is respectively：Lactobacillus caseiATCC393、
Lactobacillus caseiCICC21001、Lactobacillus caseiACCC10639、Lactobacillus
caseiCICC23185、Lactobacillus caseiCICC20994、Lactobacillus caseiCCTCCM94011、
Lactobacillus casei 431、Lactobacillus casei LC2W、Lactobacillus casei 01、
The PCR results of Lactobacillus casei Shirota.
The position that will be had in swimming lane about in band (at the arrow meaning) recovery purifying and cloning and sequencing of 800bp, is surveyed
Sequence result is all as shown in SEQ ID NO.6.
The sequence-specific of embodiment 5 is verified
The specific sequence (as shown in SEQ ID NO.6) that embodiment 1~4 is obtained carries out BLAST in NCBI websites,
The specific sequence that result display is obtained only (also includes belonging to Lactobacillus casei group, classification together with Lactobacillus casei in the present invention
Learn status exist dispute lactobacillus paracasei) genetic homology be more than 99%, the homology with other species is not high, or
Person is extremely low, and concrete outcome is the specific sequence of Lactobacillus casei referring to table 1, the sequence for thereby determining that gained.
Table 1
Note：Lactobacillus paracasei (Lactobacillus paracasei), is named as lactobacillus paracasei again, belongs to
Lactobacillus casei (Lactobacillus casei) group in lactobacillus.
The effect of the different semi-random primers of comparative example 1 compares
(1) prepared by template
The activation of the bacterial strains of Lactobacillus casei 01 is separately cultured, the bacterium colony picking individual colonies of acquisition are inoculated in
1ml MRS culture mediums, 37 DEG C of Anaerobic culturels 24 hours, centrifugation obtains thalline.Bacterial genomes are extracted, the kit for using is：
TaKaRa minibest bacterial genomic DNA extraction kit ver.2.0(Takara
Biotechnology(Dalian)Co.,Ltd.)。
(2) PCR amplifications
Amplification reaction system and program are with embodiment 1.Semi-random primer therein is " AP1-AP5 ", and its sequence is respectively such as
(2 are specifically shown in Table shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5).
Table 2
| Primer | Primer sequence (5 ' → 3 ') |
| AP1 | |
| AP2 | |
| AP3 | |
| AP4 | |
| AP5 |
(3) results contrast
The PCR primer that different semi-random primer amplifications are obtained is carried out into electrophoresis, electrophoresis result is shown in Fig. 5, and " M " is DL2 in figure,
000DNA (Marker Takara Biotechnology (Dalian) Co, Ltd), numbering " 1-4 " is semi-random primer " AP2-
The corresponding PCR results of AP5 " difference, numbering 5 is the corresponding PCR results of semi-random primer AP1.Wherein, numbering 4 is drawn for semi-random
The corresponding PCR results of thing AP5, although show band, but band is weaker less and sequence of cloning and sequencing is more miscellaneous；And AP1
The band of amplification is the most clear, shows that semi-random primer used in the present invention has greater advantage on expanding effect.
The detection of Lactobacillus casei in the testing sample of application examples 1
(1) preparation of template DNA：Sample to be tested is the clear and lucid excellent plant lactobacillus drink (original flavor, 340ml is commercially available) of light, sample
Product draw plate isolation.To the single bacterium colony amplification cultivation for growing, collects thalline extracts DNA.The preparation of template DNA uses liquid
Nitrogen freeze thawing-CTAB methods extract sample DNA.For the extraction of genomic DNA, it is also possible to extracted using equivalent commercialized DNA
Kit is simultaneously operated by its specification.
(2) as template, the semi-random primer shown in SEQ ID NO.1 is amplification to the genomic DNA obtained by step (1)
Primer carries out single-primed PCR.The system of PCR amplifications constitutes and is：The 1 μm of semi-random primers of 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 polymerases, and 2ng/ μ L genes
Group DNA profiling, the μ L of cumulative volume 50.PCR amplification program be：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, 2. step is 35 to period 4..
(3) PCR primer is carried out into electrophoresis, being as a result displayed near 800bp has clear band, rubber tapping is reclaimed the 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 that homologous degree is more than
99%, show to contain Lactobacillus casei in sample to be tested.
Using API 50CH reagent strips (French Mei Liai), i.e., traditional Physiology and biochemistry method identification, as a result with above-mentioned point
The result of sub- authentication method is consistent.
The detection of Lactobacillus casei in the testing sample of application examples 2
(1) preparation of template DNA：Sample to be tested is light e+ probiotics fermentions breast (950g, commercially available), and sample draws flat board point
From culture.To the single bacterium colony amplification cultivation for growing, collects thalline extracts DNA.The preparation of template DNA is extracted using lysozyme Method
Sample DNA.
(2) as template, the semi-random primer shown in SEQ ID NO.1 is amplification to the genomic DNA obtained by step (1)
Primer carries out single-primed PCR.The system of PCR amplifications constitutes and is：The 2 μm of semi-random primers of 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 polymerases, and 2ng/ μ L genomes
DNA profiling, the μ L of cumulative volume 50.PCR amplification program be：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, 2. step is 40 to period 4..
(3) PCR primer is carried out into electrophoresis, being as a result displayed near 800bp has clear band, rubber tapping is reclaimed the 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 that homologous degree is more than
99%, show to contain Lactobacillus casei in sample to be tested.
Using API 50CH reagent strips (French Mei Liai), i.e., traditional Physiology and biochemistry method identification, as a result with above-mentioned point
The result of sub- authentication method is consistent.
The detection of Lactobacillus casei in the testing sample of application examples 3
(1) preparation of template DNA：Sample to be tested is bright strong energy original flavor AB100 Yogurts (100g*6, commercially available), sample
Draw plate isolation.To the single bacterium colony amplification cultivation for growing, collects thalline extracts DNA.The preparation of template DNA uses liquid nitrogen
Freeze thawing-CTAB methods extract sample DNA.
(2) as template, the semi-random primer shown in SEQ ID NO.1 is amplification to the genomic DNA obtained by step (1)
Primer carries out single-primed PCR.The system of PCR amplifications constitutes and is：The 0.5 μm of semi-random primers of 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 polymerases, and 0.5ng/ μ L bases
Because of a group DNA profiling, the μ L of cumulative volume 50.PCR amplification program be：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, 2. step is 25 to period 4..
(3) PCR primer is carried out into electrophoresis, being as a result displayed near 800bp has clear band, rubber tapping is reclaimed the 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 that homologous degree is more than
99%, show to contain Lactobacillus casei in sample to be tested.
Using API 50CH reagent strips (French Mei Liai), i.e., traditional Physiology and biochemistry method identification, as a result with above-mentioned point
The result of sub- authentication method is consistent.
The detection of Lactobacillus casei in the testing sample of application examples 4
(1) preparation of template DNA：Sample to be tested is Mongolia Ox's big fruit yogurt (mulberries+coconut palm fruit, commercially available) of triangle cup, sample
Draw plate isolation.To the single bacterium colony amplification cultivation for growing, collects thalline extracts DNA.The preparation of template DNA uses liquid nitrogen
Freeze thawing-CTAB methods extract sample DNA.For the extraction of genomic DNA, it is also possible to extract examination using equivalent commercialized DNA
Agent box is simultaneously operated by its specification.
(2) as template, the semi-random primer shown in SEQ ID NO.1 is amplification to the genomic DNA obtained by step (1)
Primer carries out single-primed PCR.The system of PCR amplifications constitutes and is：The 1 μm of semi-random primers of 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 polymerases, and 2ng/ μ L genes
Group DNA profiling, the μ L of cumulative volume 50.PCR amplification program be：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, 2. step is 35 to period 4..
(3) PCR primer is carried out into electrophoresis, as a result shows that surveyed bacterium colony does not all have 800bp bands, shown in sample to be tested not
Containing Lactobacillus casei.
Identify that as a result the result with above-mentioned method for identifying molecules is consistent using traditional Physiology and biochemistry method.
Above-mentioned application examples shows that specificity molecular marker DNA sequence of the invention can be square in Lactobacillus casei is detected
Just apply, it is simple to operate, as a result accurately and reliably.
It should be understood that after the above of the invention has been read, those skilled in the art can make various to the present invention
Change or change, these equivalent form of values equally fall within the application appended claims limited range.
Claims (10)
1. a kind of specificity molecular marker DNA of Lactobacillus casei (Lactobacillus casei), it is characterised in that its core
Nucleotide sequence is as shown in SEQ ID NO.6.
2. Lactobacillus casei (Lactobacillus casei) of the nucleotide sequence composition as shown in SEQ ID NO.6 is special
Property molecular marker DNA detect Lactobacillus casei in purposes.
3. a kind of detection method of Lactobacillus casei, it is characterised in that it comprises the following steps：
(1) genomic DNA of sample to be tested is extracted；
(2), as template, the semi-random primer shown in SEQ ID NO.1 is as amplimer for the genomic DNA obtained by step (1)
Carry out single-primed PCR；
(3) cloning and sequencing carried out to step (2) gained amplified production, and by the nucleosides shown in sequencing result and SEQ ID NO.6
Acid sequence is compared, if homologous degree is more than 99%, shows to contain Lactobacillus casei in sample to be tested.
4. detection method as claimed in claim 3, it is characterised in that the side of the genomic DNA of described extraction sample to be tested
Method is frozen-thawed-CTAB methods 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：0.5~2.0 μm of semi-random primer of ol/L, the Mg of 0.2~1.0mmol/L dNTP, 1.0~2.5mmol/L2+,
0.02~0.10U/ μ LTaq archaeal dna polymerases, and 0.5~2.0ng/ μ L genomic DNA templates.
6. detection method as claimed in claim 3, it is characterised in that the response procedures of described single-primed PCR include：
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, 2. step is 25-40 to period 4..
7. detection method as claimed in claim 3, it is characterised in that the reaction system of described single-primed PCR includes
Following component：1.0 μm of semi-random primers of ol/L, the Mg of 0.5mmol/L dNTP, 1.5mmol/L2+, 0.05U/ μ LTaq DNA gather
Synthase, and 1.0ng/ μ L genomic DNA templates.
8. detection method as claimed in claim 3, it is characterised in that the response procedures of described single-primed PCR include：
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 to be carried out to step (2) gained amplified production gram
Grand sequencing is carried out in the following manner：Step (2) gained amplified production is carried out into electrophoresis, if there is amplified band in 800bp positions,
The band is then carried out into 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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| CN102757960A (en) * | 2012-07-30 | 2012-10-31 | 光明乳业股份有限公司 | Semi-random polymerase chain reaction (PCR) technique for amplifying unknown sequence and primer and kit used for technique |
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| CN101712989A (en) * | 2009-09-21 | 2010-05-26 | 内蒙古农业大学 | Method for quickly, qualitatively and quantitatively measuring Lactobacillus casei in probiotic dairy products |
| CN102757960A (en) * | 2012-07-30 | 2012-10-31 | 光明乳业股份有限公司 | Semi-random polymerase chain reaction (PCR) technique for amplifying unknown sequence and primer and kit used for technique |
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