CN104480204A - Primer, method and kit for rapidly detecting lactobacillus plantarum ST-III - Google Patents
Primer, method and kit for rapidly detecting lactobacillus plantarum ST-III Download PDFInfo
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- CN104480204A CN104480204A CN201410747460.0A CN201410747460A CN104480204A CN 104480204 A CN104480204 A CN 104480204A CN 201410747460 A CN201410747460 A CN 201410747460A CN 104480204 A CN104480204 A CN 104480204A
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Abstract
The invention aims to provide a specific labeled molecule of lactobacillus plantarum ST-III. The specific labeled molecule is characterized by being a gene used for coding the protein KdpA of lactobacillus plantarum ST-III and placed on a plasmid pST-III from lactobacillus plantarum ST-III. The invention also aims to provide a primer pair for detecting lactobacillus plantarum ST-III. The primer pair is characterized in that one primer in the primer pair is the same as the specific labeled molecule of lactobacillus plantarum ST-III in sequence and has a length of 15-40bp, and the other primer in the primer pair is complementary to the specific labeled molecule of lactobacillus plantarum ST-III in sequence and has a length of 15-40bp, and the amplified fragment of the primer pair has a length of 200-1773bp. The specific labeled molecule of lactobacillus plantarum ST-III has the advantages of high detection speed, high specificity, high sensitivity and low cost.
Description
Technical field
The present invention relates to the primer of a kind of rapid detection plant lactobacillus ST-III and detection method thereof and test kit, belong to biological technical field.
Background technology
Plant lactobacillus ST-III is the typical probiotic bacterium of a strain, self has multiple prebiotic functions such as reducing cholesterol, reducing blood-fat, raising body immunity.The genome of this bacterial strain measured in 2010 and announces, and this bacterial strain is applied to industrial production as starter simultaneously, but existing market is relatively chaotic, and existence is mixed the spurious with the genuine in a large number, shoddy phenomenon.Determined at present a lot of prebiotic function of plant lactobacillus ST-III and the pST-III plasmid of himself closely related, and pST-III plasmid determines some special propertys of plant lactobacillus ST-III.PST-III plasmid size is 53.6kbp, multiple copied, and nucleotide sequence has also come forth mensuration.The announcement of these nucleotide sequences can make us understand genetic construction and the feature of different probiotic bacterium, finds out the proprietary feature of different strains, thus lays the foundation for the differentiation between bacterium different strains of the same race and qualification.Find that pST-III plasmid exists a coding kalium ion transport gene cluster Kdp by analysis, this gene cluster gives the extremely strong salt tolerance of plant lactobacillus ST-III, and is in Bacterium lacticum, only find this kalium ion transport bunch at plant lactobacillus ST-III so far.This carries out molecule marker for plant lactobacillus ST-III, and provides specific detection method to provide volume may.Molecular Detection mainly PCR method detection has the advantage that detection speed is fast, highly sensitive, high specificity, cost are low, plant lactobacillus ST-III and other lactobacterium plantarum strain can make a distinction by PCR method fast that therefore set up plant lactobacillus ST-III, thus mix the spurious with the genuine in detection market, shoddy milk-product, to the supervision of milk-product, there is very strong using value.
Summary of the invention
For solving the problem, present invention employs following technical scheme:
An object of the present invention is to provide a kind of specific marker molecule of plant lactobacillus ST-III bacterial strain, it is characterized in that:
Specific marker molecule is the gene of the KdpA protein of coded plant Bacterium lacticum ST-III, is positioned on the pST-III plasmid in plant lactobacillus ST-III source.
Specific marker molecule provided by the present invention, can also have such feature:
The nucleotide sequence of specific marker molecule as the 720th of SEQ ID NO.1 the to shown in the 1200th.
Another object of the present invention is to provide a kind of primer pair detecting plant lactobacillus ST-III, it is characterized in that:
In this primer pair, a primer is identical with a section in the sequence of the specific marker molecule of plant lactobacillus ST-III bacterial strain, and length is 15-40bp; One section of complementation in the sequence of the specific molecular marker of another primer and plant lactobacillus ST-III bacterial strain, length is 15-40bp, and the fragment length that this primer pair amplifies goes out is 200-1773bp.
Primer provided by the present invention, can also have such feature, primer pair in, the sequence of a primer is as shown in SEQ ID NO.2, and the sequence of another primer is as shown in SEQ IDNO.3.
Another object of the present invention is to provide a kind of test kit detecting plant lactobacillus ST-III bacterial strain, it is characterized in that: this test kit comprises the primer pair provided in above-mentioned purpose.
According to test kit provided by the present invention, it is characterized in that:
Test kit also comprise dNTP, 10 × PCR damping fluid and Taq archaeal dna polymerase.
Further object of the present invention is the detection method providing a kind of plant lactobacillus ST-III bacterial strain, it is characterized in that, comprises the steps:
Step one, extracts the plasmid DNA in testing sample bacterial strain;
Step 2, with step one gained plasmid DNA for template, carries out PCR reaction with the primer pair provided in above-mentioned purpose;
Step 3, carries out electrophoresis detection by step 2 gained PCR reaction product, the fragment that the primer amplification whether detection exists claim 3 or 4 goes out.
In addition, the detection method of plant lactobacillus ST-III bacterial strain provided by the present invention, can also have such feature:
Wherein, the reaction system of the PCR reaction in step 2 comprises primer pair, and the concentration of two kinds of primers in this primer pair is 0.5mmol/L; The dNTP of 0.2mmol/L; 1 × PCRbuffer; The Mg of 1.5mmol/L
2+; The Taq archaeal dna polymerase of 0.02U/ μ L; And the DNA profiling of 1.0-2.0ng/ μ L.
In addition, the detection method of plant lactobacillus ST-III bacterial strain provided by the present invention, can also have such feature:
PCR response procedures in step 2 is: 95 DEG C, 3min; 45s at 95 DEG C, 45s at 55 DEG C, 1min at 72 DEG C, carry out 30 circulations; 72 DEG C, 5min; 4 DEG C of preservations.
Another object of the present invention is to provide a kind of above-mentioned specific marker molecule detecting the application in plant lactobacillus ST-III bacterial strain as the characteristic sequence of plant lactobacillus ST-III bacterial strain.
Invention effect and effect
The primer of specific detection plant lactobacillus ST-III bacterial strain provided by the present invention and detection method thereof and test kit, tool has the following advantages:
1. detection speed is fast, specificity is high
With need the whole qualification cycle of the biochemical culture authenticate technology of traditional microorganism compared with 5-7 days, detection method provided by the present invention only needs 3-4 hour, and detection speed obviously accelerates.Primer pair designed by the present invention designs according to the specific marker fragment of coded plant Bacterium lacticum ST-IIIKdpA protein gene, this primer can amplify corresponding DNA fragmentation in coded plant Bacterium lacticum ST-III bacterial strain, and the only gene of specific amplification plant lactobacillus ST-III, and respective segments can not be amplified in other Bacterium lacticum, comprise the lactobacterium strain checked order through full-length genome (containing plasmid) that NCBI has reported, illustrate that the PCR reactive system designed based on this gene fragment has good specificity.
2. highly sensitive, cost is low
The growing amount of PCR primer exponentially increases, can by the template amplification initial to be measured of pieck stage to microgram level, therefore highly sensitive.And the template in the present invention is the plasmid (about 10 copies) of multiple copied, therefore with general with genome be template PCR method compared with, method in the present invention can improve the sensitivity of an order of magnitude again, and the method sensitivity therefore in the present invention is higher.In addition, compared with being checked order by whole genome sequence and distinguishing with different strains between bacterioid method, expense greatly reduces, experimental period also shortens dramatically, and can drop into practical application easily.
Embodiment
Below the specific embodiment of the present invention is described
The detection of plant lactobacillus ST-III in embodiment 1 Yoghourt
Get 10ml Yoghourt, the centrifugal 10min of 8000g, abandons supernatant, gets precipitation.With deionization water washing and precipitating 2 times, extract the plasmid DNA of thalline with plasmid extraction kit (SanPrep pillar plasmid DNA extraction agent box, purchased from Sheng Gong biotechnology company limited).Then with extraction DNA carry out PCR reaction for template.
PCR reaction system is: the upstream primer of 0.5mmol/L, the downstream primer of 0.5mmol/L, the dNTP of 0.2mmol/L, 1XPCR buffer, the Taq archaeal dna polymerase of the Mg2+ of 1.5mmol/L, 0.02U/uL, and the DNA profiling of 1uL adds example water to 20ul.PCR response procedures is: 95 DEG C, 3min; By 45s at 95 DEG C, 45s at 55 DEG C, 1min at 72 DEG C, 30 circulations are carried out to above three steps; 72 DEG C of 5min; 4 DEG C of preservations.
Finally carry out electrophoresis detection, get 5ul PCR primer electrophoresis in 1% sepharose, electrophoretic voltage is 100V, and electrophoresis time is 40min.Be labeled as contrast with DNA molecular amount to detect.
There is the nucleic acid fragment of 480bp in result, illustrates in testing sample to there is plant lactobacillus ST-III under being presented at ultraviolet lamp.
Embodiment 2 specificity verification
Select different lactobacterium plantarum strain 20 strains, comprise 5 lactobacillus plantarum plant subspecies, the plant lactobacillus ST-III of 5 strain different sourcess, 3 lactobacillus plantarum P8 bacterial strains, 2 lactobacillus plantarum 299v bacterial strains, 1 lactobacillus plantarum ATCC8014,1 lactobacillus plantarum CCFM8610,1 lactobacillus plantarum CCFM8724,1 lactobacillus plantarum CCFM8661,1 lactobacillus plantarum CW006.The strain of other kind of Bacterium lacticum 50, comprise 10 strain Lactobacterium acidophilums, 5 strain Lactobacillus animalis, 5 strain short lactobacillus, 5 strain Lactobacillus buchneri, 5 strain solution starch milk bacillus, 5 strain lactobacillus crispatus, 5 strain lactobacillus delbruckiis, 5 strain lactobacillus fermentums, 5 strain lactobacterium caseis, utilize primer pair provided by the present invention to carry out PCR reaction, and the concrete operations of PCR amplification system and program and electrophoresis are with reference to embodiment 1.
Under result is presented at ultraviolet lamp, only have plant lactobacillus ST-III to have brighter 480bp electrophoretic band, other bacterial strain and bacterial classification all do not have 480bp electrophoretic band.Illustrate that the PCR primer detection specificity of design is strong, can accurately, easily plant lactobacillus ST-III and other different bacterium be made a distinction.
Present embodiment provides a kind of specific marker molecule of plant lactobacillus ST-III bacterial strain, and this specific marker molecule is the gene of coded plant Bacterium lacticum ST-III KdpA protein.
KdpA protein is one of protein participating in kalium ion transport, and the GenBank Accession No. of the gene of coded plant Bacterium lacticum ST-III bacterial strain KdpA protein is LPST_P0009.Preferably, specific marker molecule is the gene of coded plant Bacterium lacticum ST-IIIKdpA protein.Preferred, specific marker molecule be coded plant Bacterium lacticum ST-III KdpA protein gene 720bp to 1200bp shown in the Nucleotide of sequence.Specific marker molecule's its nucleotide sequence most preferred is as shown in the 720bp to 1200bp of SEQ ID NO.1.
Above-described embodiment also provides a kind of primer pair detecting plant lactobacillus ST-III bacterial strain.In this primer pair, a primer is identical with the front terminal sequence of the specific marker molecule of plant lactobacillus ST-III bacterial strain, and primer length is 15-40bp, it is still further preferred that 23bp; The complementary of the specific molecular marker of another primer and plant lactobacillus ST-III bacterial strain, primer length 15-40bp, it is still further preferred that 23bp; The fragment length that this primer pair amplifies goes out is 200-1773bp., preferred 480bp.Preferably, in primer pair, the sequence of a primer is as shown in SEQ ID NO.2, and the sequence of another primer is as shown in SEQ ID NO.3.
The primer of the specific amplification plant lactobacillus ST-III bacterial strain that above-described embodiment provides, the sequence according to specific marker molecule of the present invention designs, and is obtained by conventional DNA synthetic method.
The present embodiment also provides a kind of test kit detecting plant lactobacillus ST-III bacterial strain, the primer pair that this test kit comprises, and this test kit preferably also comprises dNTP, 10XPCR damping fluid and Taq archaeal dna polymerase.Better, test kit also comprises positive control or gene extraction agent.
Testing sample of the present invention is any material that may contain plant lactobacillus ST-III, preferably such as milk or milk-product.
The sequence of the Bacterium lacticum that the present invention has reported according to NCBI, uses blast program to find out nucleotide sequence kdpA gene specific to plant lactobacillus ST-III, designs corresponding primer according to these nucleotide sequences.Pass through the screening to these primers and checking, obtain the Auele Specific Primer that can be used in plant identification Bacterium lacticum ST-III.The present invention provides the method and test kit that use this primer in the lump.Should be understood that above embodiment only for illustration of the present invention but not for limiting scope of the present invention.The experimental technique of unreceipted actual conditions in above-mentioned example, conveniently condition is carried out usually.
Claims (10)
1. a specific marker molecule for plant lactobacillus ST-III bacterial strain, is characterized in that:
Described specific marker molecule is the gene of the KdpA protein of coded plant Bacterium lacticum ST-III, is positioned on the pST-III plasmid in plant lactobacillus ST-III source.
2. the specific marker molecule of plant lactobacillus ST-III bacterial strain as claimed in claim 1, is characterized in that:
The nucleotide sequence of described specific marker molecule is as the 720th of SEQ ID NO.1 the to shown in the 1200th.
3. detect a primer pair for plant lactobacillus ST-III bacterial strain, it is characterized in that:
In this primer pair, a primer is identical with a section in the sequence of the specific marker molecule of plant lactobacillus ST-III bacterial strain, and length is 15-40bp; One section of complementation in the sequence of the specific molecular marker of another primer and plant lactobacillus ST-III bacterial strain, length is 15-40bp, and the fragment length that this primer pair amplifies goes out is 200-1773bp.
4. the primer pair detecting plant lactobacillus ST-III bacterial strain as claimed in claim 3, is characterized in that:
In described primer pair, the sequence of a primer is as shown in SEQ ID NO.2, and the sequence of another primer is as shown in SEQ ID NO.3.
5. detect a test kit for plant lactobacillus ST-III bacterial strain, it is characterized in that:
This test kit comprises the primer pair as described in claim 3 or 4.
6. the test kit detecting plant lactobacillus ST-III bacterial strain as claimed in claim 5, is characterized in that, also comprise:
DNTP; 10 × PCR damping fluid and Taq archaeal dna polymerase.
7. a detection method for plant lactobacillus ST-III bacterial strain, is characterized in that, comprises the steps:
Step one, extracts the plasmid DNA in testing sample bacterial strain;
Step 2, with the described plasmid DNA of step one gained for template, carries out PCR reaction with the primer pair described in claim 3 or 4;
Step 3, carries out electrophoresis detection to the PCR reaction product of step 2 gained, detects the fragment that whether there is the primer amplification described in claim 3 or 4 and go out.
8. the detection method of plant lactobacillus ST-III bacterial strain as claimed in claim 7, is characterized in that:
Wherein, the reaction system of the PCR reaction described in step 2 comprises
Described primer pair, the concentration of two kinds of primers in this primer pair is 0.5mmol/L; The dNTP of 0.2mmol/L; 1 × PCR buffer; The Mg of 1.5mmol/L
2+; The Taq archaeal dna polymerase of 0.02U/ μ L; And the DNA profiling of 1.0-2.0ng/ μ L.
9. the detection method of plant lactobacillus ST-III bacterial strain as claimed in claim 7, is characterized in that:
Wherein, the response procedures of the PCR reaction described in step 2 is followed successively by, 95 DEG C, 3min; 45s at 95 DEG C, 45s at 55 DEG C, 1min at 72 DEG C, carry out 30 circulations; 72 DEG C, 5min; 4 DEG C of preservations.
10. specific marker molecule as claimed in claim 1 is detecting the application in plant lactobacillus ST-III bacterial strain as the characteristic sequence of plant lactobacillus ST-III bacterial strain.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112143820A (en) * | 2020-09-29 | 2020-12-29 | 广东省微生物研究所（广东省微生物分析检测中心） | Molecular marker, detection primer and detection method for identifying lactobacillus plantarum and lactobacillus pentosus |
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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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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
Non-Patent Citations (3)
| Title |
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| 吕嘉枥等: "乳品中活性益生菌选择性检测方法的研究", 《陕西科技大学学报：自然科学版》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112143820A (en) * | 2020-09-29 | 2020-12-29 | 广东省微生物研究所（广东省微生物分析检测中心） | Molecular marker, detection primer and detection method for identifying lactobacillus plantarum and lactobacillus pentosus |
| CN112143820B (en) * | 2020-09-29 | 2022-05-06 | 广东省微生物研究所（广东省微生物分析检测中心） | Molecular marker, detection primer and detection method for identifying lactobacillus plantarum and lactobacillus pentosus |
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