CN109468397A - The gene and its amplification primers of one specific detection Lactococcus lactis bacterial strain IMAU11823 is to, kit and method - Google Patents
The gene and its amplification primers of one specific detection Lactococcus lactis bacterial strain IMAU11823 is to, kit and method Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The present invention provides the gene of specific detection Lactococcus lactis bacterial strain IMAU11823 and its amplification primers to, kit and method, belongs to gene engineering technology field.The nucleotide sequence of gene of the present invention such as SEQ ID No.3.Its amplification primers is to including upstream primer and downstream primer, and the nucleotide sequence of upstream primer is as shown in SEQ ID No.1；The nucleotide sequence of downstream primer is as shown in SEQ ID No.2.Gene provided by the invention has good specificity, is detected using primer pair provided by the invention to the Lactococcus lactis bacterial strain IMAU11823 in fermented dairy product sample, and specificity is good, can mutually distinguish with higher 22 strains of homology.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a specific detection Lactococcus lactis bacterial strain
The gene and its amplification primers of IMAU11823 is to, kit and method.
Background technique
Lactococcus lactis (Lactococcus lactis) is used as typical lactic acid bacteria, it is considered to be " generally recognized as safe
(GRAS) " bacterial strain.Lactococcus lactis is often applied to Dairy fermentation (especially cheesemaking) with its excellent fermentation character
In.Further, since Lactococcus lactis can generate unique metabolite during the fermentation, in the sense organ product of fermented product
It plays an important role in terms of matter.China's traditional zymotic dairy product type is abundant and with a long history, wherein containing abundant
Lactic acid bacteria culturers resource.These lactic acid bacterias pass through long-term natural selection and domestication, are no lack of the bacterial strain with excellent industry characteristics.
However the excellent starter culture in China falls behind relatively, the excellent fermenting microbe for possessing independent intellectual property right more lacks.Especially
With it is unique produce junket fragrance and the excellent cream of fermenting property to dissipate galactococcus strain more rare, therefore screen and protect excellent fermentation
Agent strain is extremely urgent.
Lactococcus lactis (Lactococcus lactis) IMAU11823 is from China Inner Mongolia Autonomous Region Xilinhot City
Mongols's traditional zymotic dairy product, which is chewed an isolated plant height in mouth and produced, glutinous produces junket fragrance and the excellent one plant of cream of fermenting property
Yogurt coccus.Lactococcus lactis bacterial strain IMAU11823 fermentation yogurt, which reaches fermentation termination only, to be needed 8 hours, and rate of producing acid is very fast.This
Outside, Lactococcus lactis bacterial strain IMAU11823 can stick production junket fragrance by high yield during the fermentation, assign the unique mouthfeel of fermentation yogurt
And flavor.Lactococcus lactis bacterial strain IMAU11823 fermenting property is excellent, is suitable as leavening for industrial production, needs one kind
It can be in the method for the bacterial strain in qualitative detection acidified milk.
Summary of the invention
The purpose of the present invention is to provide the gene of specific detection Lactococcus lactis bacterial strain IMAU11823 and its amplifications
With primer pair, kit and method.
The present invention provides one be used for specific detection Lactococcus lactis bacterial strain IMAU11823 gene, the gene
Nucleotide sequence is as shown in SEQ ID No.3.
The present invention provides a kind of for detecting the primer pair of Lactococcus lactis bacterial strain IMAU11823, including upstream primer and
Downstream primer, the nucleotide sequence of upstream primer is as shown in SEQ ID No.1；The nucleotide sequence of downstream primer such as SEQ ID
Shown in No.2.
The present invention provides a kind of for detecting the kit of Lactococcus lactis bacterial strain IMAU11823, and the kit includes
Above-mentioned primer pair.
Preferably, the kit further includes 10 × PCRmix.
Preferably, the kit further includes positive reference substance and negative controls；The positive reference substance is lactic acid cream
The genomic DNA of coccus strain IMAU11823；The negative controls are distilled water.
The present invention provides a kind of methods for detecting Lactococcus lactis bacterial strain IMAU11823, include the following steps:
(1) genomic DNA is extracted from sample to be tested, obtains PCR template DNA；
(2) PCR amplification is carried out to step (1) template DNA using above-mentioned primer pair, obtains pcr amplification product；
(3) pcr amplification product is judged using agargel electrophoresis: if occurring characteristic bands at 284bp, really
Determine to contain Lactococcus lactis bacterial strain IMAU11823 in sample to be tested.
Preferably, step (1) described sample to be tested includes fermented dairy product.
Preferably, step (2) PCR amplification is 50 μ L reaction systems: 10 upstream μ L, 10mol/L 10 × PCRmix
0.4 μ L, 10mol/L downstream primer of primer 0.4 μ L, DNA profiling 100ng, distilled water are supplemented to 50 μ L.
Preferably, the response procedures of step (2) described PCR amplification are as follows: 95 DEG C of denaturation 3min；95 DEG C of denaturation 1min, 58 DEG C
Anneal 45s, 72 DEG C of extension 90S, recycles 30 times；Last 72 DEG C of extensions 7min, 4 DEG C of preservations.
The utility model has the advantages that the present invention provides the gene of a specific detection Lactococcus lactis bacterial strain IMAU11823, the base
The nucleotide sequence of cause such as SEQ ID No.3.Its amplification primers is to including upstream primer and downstream primer, the core of upstream primer
Nucleotide sequence is as shown in SEQ ID No.1；The nucleotide sequence of downstream primer is as shown in SEQ ID No.2.It is provided by the invention
Gene has good specificity, using primer pair provided by the invention to the Lactococcus lactis bacterial strain in fermented dairy product sample
IMAU11823 is detected, and specificity is good, can mutually be distinguished with higher 22 strains of homology.
Detailed description of the invention
Fig. 1 is agargel electrophoresis figure described in the embodiment of the present invention 1；
Fig. 2 is agargel electrophoresis figure described in the embodiment of the present invention 2, wherein sample 1 is Lactococcus lactis
The yoghurt example of (Lactococcus lactis) IMAU11823 fermentation, sample 2 are containing the bacterial strain DNA for having purpose amplified fragments
As the positive control of template, sample 3 is the unknown sample of detection, and sample 4 is the negative control using aqua sterilisa as template.
Specific embodiment
The present invention provides a kind of for detecting the primer pair of Lactococcus lactis bacterial strain IMAU11823.The primer pair includes
Upstream primer LL408F:TTGCTTTTCACCATCAATGACATAA (SEQ ID No.1) and downstream primer LL408R:
AGGCAATTAAACCAATGCAAGAT(SEQ ID No.2).The present invention is with Lactococcus lactis (Lactococcus lactis)
The one section of specific segment (nucleotide sequence of the segment such as SEQ ID No.3 institute in the whole genome sequence of IMAU11823
Show) it is foundation, devise the specific primer for the bacterium.PCR analysis is carried out using the bacterium primer pair purpose bacterial strain, it can be fast
The fast qualitative research for accurately completing Lactococcus lactis bacterial strain IMAU11823, Lactococcus lactis bacterial strain especially in fermented dairy product
The qualitative research of IMAU11823.
The present invention provides a kind of for detecting the kit of Lactococcus lactis bacterial strain IMAU11823, and the kit includes
Above-mentioned primer pair.In the present invention, it is also preferable to include 10 × PCRmix for the kit.The present invention to described 10 ×
The source of PCRmix is not particularly limited, commercial product of this field conventionally used for PCR.In an embodiment of the present invention,
The PCRmix is bought from Takara company.In the present invention, it is also preferable to include positive reference substance and feminine gender are right for the kit
According to product；The positive reference substance is the genomic DNA of Lactococcus lactis bacterial strain IMAU11823；The negative controls are distilled water.
The positive reference substance and negative controls are used to examine the accuracy of testing result.
The present invention also provides a kind of methods for detecting Lactococcus lactis bacterial strain IMAU11823, include the following steps:
(1) genomic DNA is extracted from sample to be tested, obtains PCR template DNA；
(2) PCR amplification is carried out to step (1) template DNA using above-mentioned primer pair, obtains pcr amplification product；
(3) pcr amplification product is judged using agargel electrophoresis: if occurring characteristic bands at 284bp, really
Determine to contain Lactococcus lactis bacterial strain IMAU11823 in sample to be tested.
The present invention first extracts genomic DNA from sample to be tested, obtains PCR template DNA.In the present invention, it is described to
Sample preferably includes fermented dairy product.The present invention is not particularly limited the extracting method of the genomic DNA, and this field is normal
Rule method.
After obtaining template DNA, the present invention carries out PCR amplification to step (1) template DNA using above-mentioned primer pair, obtains
To pcr amplification product.In the present invention, the system of step (2) described PCR amplification is preferred are as follows: 50 μ L:10 × PCRmix, 10 μ L,
0.4 μ L, 10mol/L downstream primer of 10mol/L upstream primer 0.4 μ L, DNA profiling 100ng, distilled water are supplemented to 50 μ L.Step
(2) program of the PCR amplification is preferred are as follows: 95 DEG C of denaturation 3min；95 DEG C of denaturation 1min, 58 DEG C of annealing 45s, 72 DEG C extend
90S is recycled 30 times；Last 72 DEG C of extensions 7min, 4 DEG C of preservations.
After obtaining pcr amplification product, the present invention judges pcr amplification product using agargel electrophoresis: if
Occur characteristic bands at 284bp, it is determined that contain Lactococcus lactis bacterial strain IMAU11823 in sample to be tested.The present invention is to the fine jade
The specific method of rouge gel electrophoresis is not particularly limited, conventional method in that art.
The present invention pick on taxonomy with Lactococcus lactis (Lactococcus lactis subsp.
Lactis) higher 22 strains (the Lactococcus lactis subsp.lactis of IMAU11823 homology
ATCC19842T、Lactococcus lactis subsp.cremoris ATCC19257T、Lactococcus lactis
subsp.tructae DSM2152T、Lactococcus lactis subsp.hordniae DSM20450T、
Streptococcus thoraltensis ATCC700865T、Streptococcus tigurinus DSM24864T、
Streptococcus suis ATCC43765T、Streptococcus sobrinus ATCC33478T、Streptococcus
sinensis DSM14990T、Streptococcus shiloi ATCC51499T、Streptococcus sanguinis
ATCC10556T、 Streptococcus saliviloxodontae JSM19288T、Streptococcus
salivariusATCC7073 T、Streptococcus saccharolyticusATCC43076T、
Bacteroidesfragilis JCM 11019 T、Bifidobacterium animalis DSM 10140T、
Bifidobacterium breve ATCC 15700、 Bifidobacterium longum ATCC 15697、
Enterococcusfaecalis ATCC 19433T、 Enterococcusfaecium ATCC 19434、Escherichia
Coli JCM 1649 and Lactobacillus acidophilus ATCC4356T).Using primer pair provided by the invention and
Detection method carries out PCR amplification comparison analysis determination: primer pair and detection method provided by the invention to above-mentioned 22 bacterium clocks
Lactococcus lactis (Lactococcus lactis) IMAU11823 can only be expanded, remaining 22 plants of bacterium cannot be amplified.Show this hair
The primer pair and detection method of bright offer have good specificity.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
Lactococcus lactis (Lactococcus lactis) IMAU11823 specific primer specific assay
(1) macro genome DNA extracts in fermented dairy product sample
1.0g acidified milk sample carrying out washing treatment is taken, the thallus of washing is placed in 1.5mL centrifuge tube, is immediately placed in liquid nitrogen
Fully charge is put into 65 DEG C of water-bath 5min, multigelation 3 times, adds the SDS and 10.0 μ L of 10% volumetric concentration of 0.1mL after taking-up
200r/min shakes 2h in 10mg/mL Proteinase K, with 37 DEG C of constant-temperature tables, and 12000g is centrifuged 10min at room temperature, collects supernatant
Liquid is transferred in another centrifuge tube, and in 12000g centrifugation 10min, Aspirate supernatant is transferred to separately for supernatant and isometric chloroform
It is carried out phenol chloroform 2 times in one centrifuge tube, the ice isopropyl acid through 0.1 times of volume precipitates genomic DNA, and then 70% ethyl alcohol is washed
Wash precipitating twice, back dissolving is spare.
(2) Lactococcus lactis (Lactococcus lactis) IMAU11823 specific primer specific detection
1. on sort status with Lactococcus lactis (Lactococcus lactis subsp.lactis)
Higher 22 strains of IMAU11823 homology (Lactococcus lactis subsp.lactis ATCC19842T、
Lactococcus lactis subsp.cremoris ATCC19257T、Lactococcus lactis subsp.tructae
DSM2152T、Lactococcus lactis subsp.hordniae DSM20450T、Streptococcus
thoraltensis ATCC700865T、Streptococcus tigurinus DSM24864T、Streptococcus suis
ATCC43765T、Streptococcus sobrinus ATCC33478T、Streptococcus sinensis DSM14990T、
Streptococcus shiloi ATCC51499T、Streptococcus sanguinis ATCC10556T、
Streptococcussaliviloxodontae JSM19288T、Streptococcus salivariusATCC7073 T、
Streptococcus saccharolyticusATCC43076T、Bacteroidesfragilis JCM 11019 T、
Bifidobacterium animalis DSM 10140T、Bifidobacterium breve ATCC 15700、
Bifidobacterium longum ATCC 15697、Enterococcusfaecalis ATCC 19433T、
Enterococcusfaecium ATCC 19434, Escherichia coli JCM 1649 and Lactobacillus
acidophilus ATCC4356T).Using primer pair provided by the invention (with LL408F:
TTGCTTTTCACCATCAATGACATAA is upstream primer；Draw by downstream of LL408R:AGGCAATTAAACCAATGCAAGAT
Object) PCR amplification comparison analysis is carried out to above-mentioned 22 kinds of bacterial strains.
2. amplification system:
50 μ L:10 × PCRmix of reaction system, 10 μ L, 10mol/L upstream primer, 0.4 μ L, 10mol/L downstream primer 0.4
μ L, DNA profiling 100ng, ddH2O are supplemented to 50 μ L；
3. amplification condition:
95 DEG C of denaturation 3min；95 DEG C of denaturation 1min, 58 DEG C of annealing 45s, 72 DEG C of extension 90S are recycled 30 times；Last 72 DEG C
Extend 7min, 4 DEG C of preservations.
4. agarose gel electrophoresis:
By, with the voltage of 5V/cm, electrophoresis 20-30min, dyeing is ultraviolet to take pictures, inspection in PCR product and 1% Ago-Gel
Survey expanding effect；Wherein there is sun of the Lactococcus lactis bacterial strain IMAU11823 bacterial strain DNA of purpose amplified fragments as template to contain
Property control, using aqua sterilisa as the negative control of template, according to whether occurring expected characteristic bands at 284bp, determine lactic acid
Whether specificity is good for galactococcus (Lactococcus lactis) IMAU11823 specific primer.
Experimental result is as shown in Figure 1, Fig. 1 shows: primer pair provided by the invention can only expand Lactococcus lactis
(Lactococcus lactis) IMAU11823 cannot amplify remaining 22 plants of bacterium.Illustrate that primer pair provided by the invention has
Good specificity.
Embodiment 2
The qualitative detection of Lactococcus lactis (Lactococcus lactis) IMAU11823
(1) in fermented dairy product (or excrement) sample macro genome DNA extraction
The yoghurt example of Lactococcus lactis (Lactococcus lactis) IMAU11823 fermentation is acquired as sample 1, sample
Product 2 is, containing there is positive control of the bacterial strain DNA of purpose amplified fragments as template, sample 3 are the unknown sample of detection, sample 4
For the negative control using aqua sterilisa as template.
Using frozen-thawed-CTAB method: 1.0g sample carrying out washing treatment is taken, by the thallus of elution in 1.5mL centrifuge tube,
It is immediately placed on fully charge in liquid nitrogen, thawing (about 5min), multigelation 3 in 65 DEG C of water-baths are put into after taking-up and is determined, 0.1mL is added
10%SDS and 10.0 μ L 10mg/mL Proteinase Ks, in 37 DEG C of constant-temperature tables 200r/min shake 2h, at room temperature 10000g from
Heart 10min collects supernatant and is transferred in another centrifuge tube.Supernatant and isometric chloroform in 10000g be centrifuged 10min (for
Make precipitating, water phase and organic phase layering), Aspirate supernatant is transferred in another centrifuge tube and carries out phenol chloroform 2 times, passes through
The sodium acetate of 0.1 times of volume, the ice isopropanol precipitating genomic DNA of 1 times of volume, then 70% ethanol washing precipitates 2 times, back dissolving
It is spare.
(2) Lactococcus lactis (Lactococcus lactis) IMAU11823 specific primer specific amplification
1. amplification system:
50 μ L:10 × PCRmix of reaction system, 10 μ L, 10mol/L upstream primer, 0.4 μ L, 10mol/L downstream primer 0.4
μ L, DNA profiling 100ng, ddH2O are supplemented to 50 μ L；
Wherein, the upstream primer is LL408F:ttgcttttcaccatcaatgacataa；The downstream primer is
LL408R:aggcaattaaaccaatgcaagat；
2. amplification condition:
95 DEG C of denaturation 3min；95 DEG C of denaturation 1min, 58 DEG C of annealing 45s, 72 DEG C of extension 90S are recycled 30 times；Last 72 DEG C
Extend 7min, 4 DEG C of preservations.
3. agarose gel electrophoresis
By, with the voltage of 5V/cm, electrophoresis 20-30min, dyeing is ultraviolet to take pictures, inspection in PCR product and 1% Ago-Gel
Survey expanding effect；Wherein there is sun of the Lactococcus lactis bacterial strain IMAU11823 bacterial strain DNA of purpose amplified fragments as template to contain
Property control, using aqua sterilisa as the negative control of template, according to whether occurring expected characteristic bands at 284bp, determine sample
In whether contain Lactococcus lactis (Lactococcus lactis) IMAU11823.
Experimental result is as shown in Figure 2, wherein sample 1 is Lactococcus lactis (Lactococcus lactis)
The yoghurt example of IMAU11823 fermentation, sample 2 are containing having positive control of the bacterial strain DNA of purpose amplified fragments as template, sample
Product 3 are the unknown sample of detection, and sample 4 is the negative control using aqua sterilisa as template.As can be seen that sun from Fig. 2 result
Property control can amplify the purpose band that band clearly becomes clear, and negative control contains cream in sample 1 without corresponding amplified production
Yogurt coccus (Lactococcus lactis) IMAU11823, and Lactococcus lactis (Lactococcus is not contained in sample 3
lactis) IMAU11823。
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Agricultural University of the Inner Mongol
The gene and its amplification primers of<120>specific detection Lactococcus lactis bacterial strain IMAU11823 to, kit and
Method
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ttgcttttca ccatcaatga cataa 25
<210> 2
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aggcaattaa accaatgcaa gat 23
<210> 3
<211> 563
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atgagtgaag ttattgaaaa ccaagaagta aaagatattc aaattgagtt taagccggct 60
gctatcaata ttcttgaaga agaaaaattc aaagaatata ttgataaagt tgttgctgag 120
tataaggaca tgttccaaaa gcagacaatc taacagttga tagaaaaact cgtgcaaaac 180
taaacggact tatgactaat cttgaagctc gtcgtaaaga aataaaaaaa gaaatcaatg 240
ttcctatact gagtttgaat cttggtataa gaaggcaatt aaaccaatgc aagatgttac 300
atcaacaatt gatgcaggaa tcaaaaaaat tgaagctgag caaaaagaag caagaaaaaa 360
agttgttcat gaattattgg ttgacctgac aacagacaca gaagtagatt cacgaatctt 420
tgaaagcttt gtggatgact gggccaaatc atcaaacttt aatgataata agcctaaaaa 480
acagcttatt gattctatta cttatgtcat tgatggtgaa agcaaaagat gatgaatacn 540
nnnnnnnnnc ggaaacaata taa 563
Claims (9)
1. one is used for the gene of specific detection Lactococcus lactis bacterial strain IMAU11823, which is characterized in that the nucleosides of the gene
Acid sequence is as shown in SEQ ID No.3.
2. a kind of primer pair for specific detection Lactococcus lactis bacterial strain IMAU11823, including upstream primer and downstream primer,
It is characterized by:
The nucleotide sequence of upstream primer is as shown in SEQ ID No.1；
The nucleotide sequence of downstream primer is as shown in SEQ ID No.2.
3. a kind of for detecting the kit of Lactococcus lactis bacterial strain IMAU11823, it is characterised in that: the kit includes right
It is required that primer pair described in 2.
4. kit according to claim 3, which is characterized in that the kit further includes 10 × PCRmix.
5. kit according to claim 4, which is characterized in that the kit further includes that positive reference substance and feminine gender are right
According to product；The positive reference substance is the genomic DNA of Lactococcus lactis bacterial strain IMAU11823；The negative controls are distilled water.
6. a kind of method for detecting Lactococcus lactis bacterial strain IMAU11823, which comprises the steps of:
(1) genomic DNA is extracted from sample to be tested, obtains PCR template DNA；
(2) PCR amplification is carried out to step (1) template DNA using primer pair described in claim 2, obtains PCR amplification production
Object；
(3) pcr amplification product is judged using agargel electrophoresis: if occurring characteristic bands at 284bp, it is determined that
Contain Lactococcus lactis bacterial strain IMAU11823 in sample.
7. according to the method described in claim 6, it is characterized in that, step (1) described sample to be tested includes fermented dairy product.
8. according to the method described in claim 6, it is characterized in that, step (2) PCR amplification is 50 μ L reaction systems: 10
10 μ L, 10mol/L upstream primer of × PCRmix, 0.4 μ L, 10mol/L downstream primer 0.4 μ L, DNA profiling 100ng, distilled water are mended
It is charged to 50 μ L.
9. according to the method described in claim 6, it is characterized in that, the response procedures of step (2) described PCR amplification are as follows: 95 DEG C
It is denaturalized 3min；95 DEG C of denaturation 1min, 58 DEG C of annealing 45s, 72 DEG C of extension 90S are recycled 30 times；Last 72 DEG C of extensions 7min, 4 DEG C of guarantors
It deposits.
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| CN112029784A (en) * | 2020-09-16 | 2020-12-04 | 内蒙古农业大学 | Gene related to proteolytic ability and screening method of lactobacillus helveticus with high proteolytic ability based on gene |
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| CN104480203A (en) * | 2014-12-08 | 2015-04-01 | 上海理工大学 | Primer, method and kit for rapidly detecting lactococcus lactis |
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| CN112029784A (en) * | 2020-09-16 | 2020-12-04 | 内蒙古农业大学 | Gene related to proteolytic ability and screening method of lactobacillus helveticus with high proteolytic ability based on gene |
| CN114277160A (en) * | 2020-09-27 | 2022-04-05 | 上海市质量监督检验技术研究院 | Primer pair and probe for detecting lactococcus lactis in bactericidal dairy product, and method and application thereof |
| CN112899386A (en) * | 2021-03-19 | 2021-06-04 | 上海应用技术大学 | PCR primer pair and PCR method for screening lactococcus lactis with high 3-methylbutyraldehyde yield and application |
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