CN103014160B - Method and primer for detecting lactobacilli in food - Google Patents
Method and primer for detecting lactobacilli in food Download PDFInfo
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- CN103014160B CN103014160B CN201210532379.1A CN201210532379A CN103014160B CN 103014160 B CN103014160 B CN 103014160B CN 201210532379 A CN201210532379 A CN 201210532379A CN 103014160 B CN103014160 B CN 103014160B
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- 238000000034 method Methods 0.000 title claims abstract description 32
- 235000013305 food Nutrition 0.000 title claims abstract description 19
- 241000186660 Lactobacillus Species 0.000 title abstract description 9
- 241000894006 Bacteria Species 0.000 claims abstract description 32
- 238000001514 detection method Methods 0.000 claims abstract description 28
- 238000012408 PCR amplification Methods 0.000 claims abstract description 25
- 238000001962 electrophoresis Methods 0.000 claims abstract description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 52
- 235000014655 lactic acid Nutrition 0.000 claims description 26
- 230000001580 bacterial effect Effects 0.000 claims description 14
- 235000013618 yogurt Nutrition 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 4
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 3
- 230000004087 circulation Effects 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 241000901050 Bifidobacterium animalis subsp. lactis Species 0.000 claims description 2
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 claims description 2
- 241000186672 Lactobacillus delbrueckii subsp. bulgaricus Species 0.000 claims description 2
- 241000194020 Streptococcus thermophilus Species 0.000 claims description 2
- 229940009289 bifidobacterium lactis Drugs 0.000 claims description 2
- 229940004208 lactobacillus bulgaricus Drugs 0.000 claims description 2
- 238000012163 sequencing technique Methods 0.000 abstract description 8
- 230000003321 amplification Effects 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 102000053602 DNA Human genes 0.000 abstract 2
- 108020004414 DNA Proteins 0.000 abstract 2
- 238000003752 polymerase chain reaction Methods 0.000 abstract 2
- 239000000047 product Substances 0.000 description 13
- 229940039696 lactobacillus Drugs 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 241001478240 Coccus Species 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 241001135265 Cronobacter sakazakii Species 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 101100489867 Mus musculus Got2 gene Proteins 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
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- 239000004310 lactic acid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 244000005700 microbiome Species 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 235000021262 sour milk Nutrition 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/14—Streptococcus; Staphylococcus
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Abstract
The invention discloses a method for detecting lactobacilli in food, which comprises the following steps: (1) extracting the genome DNA (deoxyribonucleic acid) of bacteria in food; (2) performing PCR (polymerase chain reaction) amplification, wherein the used primer pair is a degenerate primer pair; (3) performing electrophoresis detection on the PCR amplification product, wherein if a unique PCR amplification band is detected, the detection result is positive, otherwise the detection result is negative; if the detection result is positive, step (4) does not need to be performed; and if the detection result is negative, the step (4) needs to be performed; and (4) optionally sequencing the PCR amplification product. The invention also provides a primer and the like used in the method. According to the method disclosed by the invention, products of various manufacturers, containing single or multiple lactobacilli, can be directly detected, and the detection result can effectively eliminate false positive, thereby ensuring that the method is convenient to popularize and implement; and sequencing operation can be further performed on the product of which the detection result is negative, thereby ensuring the correctness of the detection result.
Description
Technical field
The invention belongs to the detection of nucleic acids field, particularly, the present invention relates to the method for edible milk-acid bacteria detection and the primer equity wherein used.
Background technology
Milk-acid bacteria is a group form, metabolism performance and the incomplete same gram-positive microorganism of physiologic character.Owing to thering are many useful metabolic characteristicss, be widely used on many industries such as light industry, food, medicine and fodder industry.The milk-acid bacteria of having found at occurring in nature at present is divided into 23 genus on systematic bacteriology, and the bacterial classification that is usually used in probiotics has bifidus bacillus, lactobacillus, lactic acid coccus, faecalis, suis, leukonid, sheet coccus and Bacillus licheniformis etc.
Most milk-acid bacterias are considered to safe usually, and therefore corresponding examination criteria imperfection, so unanimously ignored by the people the detection of milk-acid bacteria.Sell in the market containing lactobacillus product, only in label, list strain name, lack some concrete bacterial classification information, even the part strain name is that producer names voluntarily, with the information of international standard database can't be corresponding, also just can't trace to the source, more need not put forward the stability of corresponding product quality.Especially, some recent researchs show, the part milk-acid bacteria also may cause a disease (referring to Liu little Qing, etc. the safety research of milk-acid bacteria. Chinese microecology magazine, 21 (10): 952-955).Therefore, the inventor thinks, the today day by day drawn attention in food safety, determine foodstuff production producer milk-acid bacteria used bacterial classification, bacterial strain and inherent stability and the security determined be necessary.
Guarantee security and the quality of milk-acid bacteria and preparation thereof, need to carry out the detection of biology, genetics characteristics before operation to its bacterium " strain ", in order to examine its security.Because even bacterial strain carries out preservation, cultivation, also indistinguishable usually.And the most effective detection method is DNA sequencing, but due to containing the lactobacillus product complicated component, the mixture of various lactobacillus often, the serious interference of direct Sequencing; After isolated strains cultivation, check order, the time of expending is long again.More seriously, the cost of DNA sequencing is higher, and producer and food supervisory organ cost used in everyday is too high, is difficult for implementing, even formulate correlation method, also is difficult to big area enforcement, makes the method perform practically no function.
Summary of the invention
The method of milk-acid bacteria in the detection food that the technical problem to be solved in the present invention is to provide new, it can be detected the goods containing one or more milk-acid bacterias, and detected result can effectively be got rid of false positive.In addition, the present invention also provides primer or primer pair and application thereof etc. used in the method.
Particularly, in first aspect, the invention provides the method that detects milk-acid bacteria in food, it comprises,
(1) extract milk-acid bacteria genomic dna in food;
(2) DNA step (1) extracted carries out pcr amplification, and the primer pair that wherein used is degenerate primer pair;
(3) product of the pcr amplification of step (2) is carried out to electrophoresis detection; As unique pcr amplification band detected, and detected result is positive, otherwise detected result is negative; As positively as detected result do not need to carry out step (4), as negatively as detected result need to carry out step (4);
(4) optionally the product of the pcr amplification of step (2) is checked order.
In the specific embodiment of the present invention, food is Yoghourt.
Preferably, in the method for a first aspect of the present invention, milk-acid bacteria is one or more in Lactobacterium acidophilum, bifidus bacillus, streptococcus thermophilus, lactobacillus bulgaricus, bifidus, lactobacterium casei, lactobacterium acidophilus, bifidumbacterium bifidum, bifidobacterium lactis and bifidus longum bb.
In the present invention, degenerate primer has at least one to be degenerate primer to referring in forward in primer pair and/or reverse primer.In the present invention, degenerate primer is that those skilled in the art know, and having at least on a position of primer has two or more Nucleotide.In the process of nucleic acid synthesizer order synthetic primer, by the time will synthesizing the position with two or more Nucleotide, add this two or more Nucleotide to get final product simultaneously.This can synthesize to those skilled in the art fully, and has business-like company that Composite service is provided.Preferably, in the method for a first aspect of the present invention, the primer in primer pair is selected from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQID NO.10.
More preferably, in the method for a first aspect of the present invention, the forward primer in primer pair is selected from SEQ ID NO.1, SEQ IDNO.2, SEQ ID NO.3 and SEQ ID NO.4; And/or the reverse primer in primer pair is selected from SEQ ID NO.5, SEQID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.
Preferably, in step (2), the system of described pcr amplification adopts Takara DRR006A Ex Taq Hot Start Version test kit, as follows:
TaKaRa Ex Taq HS（5U/μl）0.25μl
10×Ex Taq Buffer（Mg
2+Plus）5μl
Each 2.5mM of dNTP Mixture() 4 μ l
Each 0.5 μ l of forward and reverse primer (each 20 μ M)
DNA50ng in the bacterial genomes extracting solution
Add water and be settled to 50 μ l;
Described pcr amplification condition is: 95 ℃ of 2min denaturations; 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 40s, 45 circulations; 72 ℃ are extended 5min.
Preferably, in the method for a first aspect of the present invention, in step (3), if the product of pcr amplification is that size is the unique pcr amplification band of 250-300bp through electrophoresis detection, detected result is positive, does not carry out step (4); Other bands also occurred in the time of if there is this band, detected result is negative, carries out step (4).
Preferably, in the method for a first aspect of the present invention, order-checking is illumina genome analyzer order-checking.Illuminagenome analyzer order-checking is a kind of method checked order while synthesize based on unit molecule bunch.Particularly, during order-checking, the random fragment of genomic dna is attached to optically transparent glass surface (Flow cell), these DNA fragmentations are after extension and bridge-type amplification, formed hundreds of millions of Cluster on Flow cell, each Cluster is the unit molecule bunch with thousands of parts of same template.Then utilize four kinds of special deoxyribonucleotides with fluorophor, the SBS(stopped by reversibility checks order while synthesizing) technology checked order to template DNA to be measured.IIllumina Genome Analyzer sequencing technologies has been avoided expending a large amount of human and material resources as traditional sequencing technologies and has been carried out the work such as fragment clone, conversion, plasmid extraction, and sequencing throughput has the raising of several orders of magnitude.
In second aspect, the invention provides primer, it is selected from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.
In addition, the present invention also provides primer pair, primer pair is comprised of forward primer and reverse primer, wherein the forward primer in primer pair is selected from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and wherein the reverse primer in primer pair is selected from SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.
In the third aspect, the invention provides the application in the method for the primer of second aspect present invention or primer pair milk-acid bacteria in detecting food.In preferred detection food, the method for milk-acid bacteria is the method for a first aspect of the present invention.
Beneficial effect of the present invention is, the present invention is in conjunction with the characteristics containing milk-acid bacteria advantage in lactobacillus product, the PCR-based method has been set up the method that milk-acid bacteria is identified in a set of simple and easy to do detection, directly can the goods containing one or more milk-acid bacterias of each producer be detected, detected result can effectively be got rid of false positive, thereby is convenient to promotion and implementation, and for detecting negative goods, coordinate again order-checking, can guarantee the exactness of detected result.While being applied to foodstuffs industry, can detect the goods containing various lactobacillus, adaptability is good; Detection speed is fast, and the overwhelming majority can complete detection in 2 ~ 3 hours; Easy and simple to handle, cost is low, most without employing expensive order-checking operation; Detection accuracy is high, and especially false positive rate is very low, thereby easy to utilize.
Below will to the present invention, be described in detail by specific embodiment and accompanying drawing.It needs to be noted, these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention.According to the discussion of this specification sheets, many variations of the present invention, change have been all obviously concerning one of ordinary skill in the art.
The accompanying drawing explanation
Fig. 1 is the electrophoresis detection collection of illustrative plates of the pcr amplification product in the embodiment of the present invention 2.
Embodiment
Below carry out example by specific embodiment, wherein agents useful for same all can be bought by market channel, if any part to the greatest extent not, can be with reference to the laboratory manual of corresponding PCR and gene sequencing.
Embodiment 1 food comprises the extraction of the bacterial genomes of milk-acid bacteria
24, the import and export Yoghourt sample that can buy from market, wherein imported product is 11 kinds, 13 kinds of domestic or joint brands, adopt the Tiangen DNA of bacteria to extract test kit (goods number DP302-02) and extract respectively the complete genome DNA of bacterium in 24 kinds of sour-milk product.Get different sample Yoghourt liquid 1ml, with 10000rpm centrifugal 1 minute, supernatant discarded liquid, the manufacturers instruction that then will precipitate the reference reagent box is extracted, and obtains the bacterial genomes extracting solution.By measuring extracting solution OD260/280 ratio, determine DNA concentration.
The PCR of embodiment 2 lactic bacterium strains detects
According to the inventor, early stage bacterial strain security and the order-checking of each manufacturer are studied, designed degenerate primer pair as shown in table 1 below, entrust Shanghai English fine horse bio tech ltd synthetic, the bacterial genomes extracting solution extracted from each manufacturer's Yoghourt for embodiment 1 record carries out pcr amplification respectively, separately gets intestinal bacteria, the genome extracting solution in contrast for Enterobacter sakazakii (claiming again slope Qi Shi enterobacteria).
Table 1
Annotate: in table 1, W means A and the T of equimolar amount, and Y means T and the C of equimolar amount, and R means A and the G of equimolar amount; The sequence of primer A2 is as shown in SEQ ID NO.1, the sequence of primer A3 is as shown in SEQ ID NO.2, the sequence of primer A4 is as shown in SEQ ID NO.3, the sequence of primer A5 is as shown in SEQ ID NO.4, and the sequence of primer S2 is as shown in SEQ ID NO.5, and the sequence of primer S3 is as shown in SEQ ID NO.6, the sequence of primer S4 is as shown in SEQ ID NO.7, the sequence of primer S5 is as shown in SEQID NO.8, and the sequence of primer S6 is as shown in SEQ ID NO.9, and the sequence of primer S7 is as SEQ ID NO.10.
The pcr amplification system adopts Takara DRR006A Ex Taq Hot Start Version test kit (goods number DRR006A), as follows:
TaKaRa Ex Taq HS（5U/μl）0.25μl
10×Ex Taq Buffer（Mg
2+Plus）5μl
Each 2.5mM of dNTP Mixture() 4 μ l
Each 0.5 μ l of forward and reverse primer (each 20 μ M)
DNA50ng in the bacterial genomes extracting solution
Add water and be settled to 50 μ l
The pcr amplification condition is: 95 ℃ of 2min denaturations; 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 40s, 45 circulations; 72 ℃ are extended 5min.
Amplified production is respectively got 2 μ L and is detected with 2% agarose gel electrophoresis, partial results as shown in Figure 1, all 24 lactobacteria-containing preparations have all detected apparent unique pcr amplification band, size is about 1450bp, and contrast this band not, also occur other bands in the time of in addition if there is this band, also regarded as feminine gender (living contaminants is arranged).
Embodiment 3PCR detects the sequence verification of the result of lactic bacterium strains
Manufacturer is expressed to each batch products of stable above-mentioned 24 products of bacterial strain and carry out the detection as embodiment 2,99.2% all presents positive findings, and 0.8% presents negative findings.For further checking, product to pcr amplification used in positive findings and negative findings, conventional 16S rRNA gene sequencing (utilizing Illumina genome analyzer to use the 100bppaired-end pattern to carry out high-flux sequence) is carried out in sampling respectively, as a result in positive findings without 1 official holiday positive findings, and 25% false negative result is arranged in negative findings.Owing to utilizing, method false positive rate of the present invention is low, even the stack order-checking also can be got rid of the workload that the needs more than 99% check order.
Claims (8)
1. the method for milk-acid bacteria in detection food, is characterized in that, comprises the steps:
(1) extract bacterial genomes DNA in food;
(2) DNA step (1) extracted carries out pcr amplification, and the primer pair that wherein used is degenerate primer pair, wherein said primer pair is SEQ ID NO.2 and SEQ ID NO.5, SEQ ID NO.2 and SEQ ID NO.6, SEQ ID NO.2 and SEQ IDNO.7, SEQ ID NO.2 and SEQ ID NO.8, SEQ ID NO.2 and SEQ ID NO.9, SEQ ID NO.2 and SEQ ID NO.10, SEQ ID NO.3 and SEQ ID NO.5, SEQ ID NO.3 and SEQ ID NO.6, SEQ ID NO.3 and SEQ IDNO.7, SEQ ID NO.3 and SEQ ID NO.8, SEQ ID NO.3 and SEQ ID NO.9, SEQ ID NO.3 and SEQ ID NO.10, SEQ ID NO.1 and SEQ ID NO.5, SEQ ID NO.1 and SEQ ID NO.6, SEQ ID NO.1 and SEQ IDNO.7, SEQ ID NO.1 and SEQ ID NO.8, SEQ ID NO.1 and SEQ ID NO.9, SEQ ID NO.1 and SEQ ID NO.10, SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.4 and SEQ ID NO.6, SEQ ID NO.4 and SEQ IDNO.7, SEQ ID NO.4 and SEQ ID NO.8, SEQ ID NO.4 and SEQ ID NO.9, SEQ ID NO.4 and SEQ ID NO.10,
(3) product of the pcr amplification of step (2) is carried out to electrophoresis detection; As unique pcr amplification band detected, and detected result is positive, otherwise detected result is negative; As positively as detected result do not need to carry out step (4), as negatively as detected result need to carry out step (4);
(4) optionally the product of the pcr amplification of step (2) is checked order.
2. the method for claim 1, is characterized in that, described food is Yoghourt.
3. the method for claim 1, it is characterized in that, described milk-acid bacteria is one or more in Lactobacterium acidophilum, bifidus bacillus, streptococcus thermophilus, lactobacillus bulgaricus, bifidus, lactobacterium casei, lactobacterium acidophilus, bifidumbacterium bifidum, bifidobacterium lactis and bifidus longum bb.
4. the method for claim 1, is characterized in that, in step (2), the system of described pcr amplification adopts Takara DRR006A Ex Taq Hot Start Version test kit, as follows:
TaKaRa Ex Taq HS（5U/μl） 0.25μl
10×Ex Taq Buffer（Mg
2+Plus） 5μl
Each 2.5mM of dNTP Mixture() 4 μ l
Each 0.5 μ l of forward and reverse primer (each 20 μ M)
DNA50ng in the bacterial genomes extracting solution
Add water and be settled to 50 μ l;
Described pcr amplification condition is: 95 ℃ of 2min denaturations; 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 40s, 45 circulations; 72 ℃ are extended 5min.
5. the method for claim 1, is characterized in that, in step (3), if the product of pcr amplification is that size is the unique pcr amplification band of 250-300bp through electrophoresis detection, detected result is positive, does not carry out step (4); Other bands also occurred in the time of if there is this band, detected result is negative, carries out step (4).
6. the method for claim 1, is characterized in that, in step (4), described order-checking is illumina genome analyzer order-checking.
7. for detection of the primer pair of milk-acid bacteria in food, it is characterized in that, described primer pair is SEQ ID NO.2 and SEQ ID NO.5, SEQ ID NO.2 and SEQ ID NO.6, SEQ ID NO.2 and SEQ IDNO.7, SEQ ID NO.2 and SEQ ID NO.8, SEQ ID NO.2 and SEQ ID NO.9, SEQ ID NO.2 and SEQ ID NO.10, SEQ ID NO.3 and SEQ ID NO.5, SEQ ID NO.3 and SEQ ID NO.6, SEQ ID NO.3 and SEQ IDNO.7, SEQ ID NO.3 and SEQ ID NO.8, SEQ ID NO.3 and SEQ ID NO.9, SEQ ID NO.3 and SEQ ID NO.10, SEQ ID NO.1 and SEQ ID NO.5, SEQ ID NO.1 and SEQ ID NO.6, SEQ ID NO.1 and SEQ IDNO.7, SEQ ID NO.1 and SEQ ID NO.8, SEQ ID NO.1 and SEQ ID NO.9, SEQ ID NO.1 and SEQ ID NO.10, SEQ ID NO.4 and SEQ ID NO.5, SEQ ID NO.4 and SEQ ID NO.6, SEQ ID NO.4 and SEQ IDNO.7, SEQ ID NO.4 and SEQ ID NO.8, SEQ ID NO.4 and SEQ ID NO.9, SEQ ID NO.4 and SEQ ID NO.10.
8. the application in the method for primer pair as claimed in claim 7 milk-acid bacteria in detecting food.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310739069.1A CN103667509B (en) | 2012-12-11 | 2012-12-11 | Detect method and the detection primer of lactic acid bacteria in food |
| CN201210532379.1A CN103014160B (en) | 2012-12-11 | 2012-12-11 | Method and primer for detecting lactobacilli in food |
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| CN201210532379.1A CN103014160B (en) | 2012-12-11 | 2012-12-11 | Method and primer for detecting lactobacilli in food |
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| CN103014160B true CN103014160B (en) | 2014-01-08 |
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| CN201210532379.1A Expired - Fee Related CN103014160B (en) | 2012-12-11 | 2012-12-11 | Method and primer for detecting lactobacilli in food |
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| CN103667509A (en) * | 2012-12-11 | 2014-03-26 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Method for detecting lactobacilli in foods and primer for detection |
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| CN103614469B (en) * | 2013-11-20 | 2016-03-23 | 南通市产品质量监督检验所 | Plant source protein composition discrimination method in a kind of raw dairy and milk |
| CN104278086A (en) * | 2014-09-20 | 2015-01-14 | 中山鼎晟生物科技有限公司 | Method for detecting lactic acid bacteria in fermented dairy product |
| CN104450941B (en) * | 2014-12-24 | 2017-05-10 | 光明乳业股份有限公司 | Method for detecting lactobacillus casei strain and kit and primer pair thereof |
| CN104531692B (en) * | 2014-12-24 | 2017-10-24 | 光明乳业股份有限公司 | A kind of method and its kit and primer pair for detecting lactobacillus casei bacterial strain |
| CN106676162B (en) * | 2015-11-09 | 2020-04-10 | 中国农业科学院农产品加工研究所 | Primer group for identifying lactobacillus bulgaricus with quorum sensing system and application thereof |
| CN105349669B (en) * | 2015-11-30 | 2019-02-22 | 武汉轻工大学 | Fast qualitative, immue quantitative detection reagent box and the detection method of Bifidobacterium for being added in feed and application |
| CN110804665A (en) * | 2018-08-06 | 2020-02-18 | 中国科学院微生物研究所 | Primer and method for identifying lactic acid bacteria in environmental sample at species level |
| CN112813139A (en) * | 2021-02-18 | 2021-05-18 | 中国农业科学院北京畜牧兽医研究所 | Nucleic acid extraction method of microbial feed additive for high-throughput sequencing |
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| CN101717828B (en) * | 2009-12-14 | 2012-05-30 | 内蒙古农业大学 | Method for rapidly detecting kinds of lactobacillus in fermented dairy product |
| CN102242193A (en) * | 2011-05-09 | 2011-11-16 | 南昌大学 | Application of DGGE (Denaturing Gradient Gel Electrophoresis) method to microorganism quick sort |
| CN103667509B (en) * | 2012-12-11 | 2016-05-18 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Detect method and the detection primer of lactic acid bacteria in food |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103667509A (en) * | 2012-12-11 | 2014-03-26 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Method for detecting lactobacilli in foods and primer for detection |
| CN103667509B (en) * | 2012-12-11 | 2016-05-18 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Detect method and the detection primer of lactic acid bacteria in food |
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| CN103667509A (en) | 2014-03-26 |
| CN103667509B (en) | 2016-05-18 |
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