CN104531873B - Method for detecting lactobacillus casei strain and kit and primer pair used by method - Google Patents
Method for detecting lactobacillus casei strain and kit and primer pair used by method Download PDFInfo
- Publication number
- CN104531873B CN104531873B CN201410840051.5A CN201410840051A CN104531873B CN 104531873 B CN104531873 B CN 104531873B CN 201410840051 A CN201410840051 A CN 201410840051A CN 104531873 B CN104531873 B CN 104531873B
- Authority
- CN
- China
- Prior art keywords
- lactobacillus casei
- pcr
- seq
- primer
- sequence table
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting a lactobacillus casei strain and a kit and a primer pair used by the method. The nucleotide sequences of the primer pair are as shown in SEQ ID NO. 1 in a sequence table and as shown in SEQ ID NO. 2 in the sequence table respectively. The method comprises the following steps: (1) extracting DNA (deoxyribonucleic acid) of a genome of a sample to be detected as a template, taking the primer pair as shown in SEQ ID NO. 1 and SEQ ID NO. 2 in the sequence table as primers, and performing PCR (polymerase chain reaction) amplification; and (2) detecting whether a single amplification product exists in the position of 750bp in PCR amplification products in the step (1). The method has the advantages of short detection time, low detection cost and reliable detection results, and can be applied to detection of lactobacillus casei in dairy products.
Description
Technical field
The present invention relates to biological field, and in particular to the method and its test kit of a kind of detection lactobacillus casei bacterial strain and draw
Thing.
Background technology
Lactobacillus casei (Lactobacillus casei) belongs to Lactobacillus (Lactobacillus), is gram sun
Property bacterium, does not produce spore, and atrichia is not moved, facultatively heterofermentative Lactose, and do not liquefy gelatin；Optimum growth temperature is 37 DEG C, G+
C content is 45.6%~47.2%；Thalline is different in size, and two ends are square, Chang Chenglian；Bacterium colony is coarse, canescence, sometimes in micro-
Yellow, can ferment various sugar.Lactobacillus casei is present in the oral cavity of people, intestinal content and stool and vagina, also usually goes out
Now in milk and cheese, milk product, feedstuff, dough and rubbish.Lactobacillus casei is a kind of to be beneficial to the prebiotic of health
Bacterium.Calendar year 2001 Ministry of Public Health discloses the probiotic bacteria strain list that can be used in health food, including lactobacillus casei and dry
Lactobacillus paracasei cheese subspecies (being called Lactobacillus paracasei).At present research of the China to lactobacillus casei is very few.
Lactobacillus casei is resistant to organic defense mechanism, including in oral cavity as one kind of probiotic bacteria
Bile acid of low ph value and small intestinal etc. in enzyme, gastric juice.So lactobacillus casei enter human body after can in intestinal large number of viable,
Play regulation intestinal flora balance, promote the effects such as human consumption's absorption.Meanwhile, lactobacillus casei has efficient blood pressure lowering, drop
Cholesterol, promotion cell division, produce antibody mediated immunity, strengthen human immunity and prophylaxis of cancer and suppress the functions such as tumour growth；
Also have and alleviate the prebiotic health-care effects such as lactose intolerance, allergy.In recent years, due to lactobacillus casei to its host's nutrition, exempt from
Epidemic disease, diseases prevention etc. have significant beneficial function, increasingly become people's research, exploitation, the focus of production.And Hypertensive Population
Increase and cause the research to lactobacillus casei and the meaning with its development functionality milk product to seem further important.
Lactobacillus casei is used as the fermentation of the milk product such as milk, yogurt, bean milk, butter and cheese as one of probiotic bacteria
Agent and assisted fermentation agent, using more especially in cheese, in order to adapt to cheese in high-load salt and low ph value, need to pass through
The metabolism of some important amino acids is increasing local flavor and promote the maturation of cheese.In recent years, lactobacillus casei is more and more
In for milk product, but whether it really adds and whether can survive in milk product then needs milk testing agency to enter
Row Rapid identification.Traditional detection method wastes time and energy, it usually needs 3~5 talentes can determine that.Additionally, traditional detection method
According to the requirement of GB GB4789.35-2010, required sample size is at least 25g or 25mL, therefore the sensitivity for detecting is usual
＞ 4CFU/g or ＞ 4CFU/mL, from the foregoing, it will be observed that in traditional milk product its detection efficiency of the detection method of lactobacillus casei compared with
Low, testing cost is higher.
The content of the invention
The technical problem to be solved in the present invention be overcome existing conventional art detection time length, waste time and energy, high cost
Defect, there is provided a kind of method and its test kit and primer of detection lactobacillus casei bacterial strain.It is dry using described method detection
Lactobacillus paracasei, detection speed is fast, low cost, and sensitivity is high, and testing result specificity is good, judges simple, and detects food
Also result is accurately and reliably for lactobacillus casei bacterial strain in product.
The purpose of the present invention is realized by following technical proposal.
One of the technical solution used in the present invention is：The primer pair of one species-specific amplification lactobacillus casei, its nucleotide
Sequence is respectively as shown in SEQ ID NO.1 in sequence table and as shown in SEQ ID NO.2 in sequence table.
Wherein, the primer in nucleotide sequence such as sequence table shown in SEQ ID NO.1 is referred to as VicR-L；Nucleotide sequence is such as
Primer in sequence table shown in SEQ ID NO.2 is referred to as VicR-R.The primer VicR-L and the primer VicR-R are expanded
Sequence be VicR genes, VicR genes play a role in bi-component regulating system.
The two of the technical solution used in the present invention are：A kind of test kit for specific amplification lactobacillus casei, its bag
Include the primer pair shown in SEQ ID NO.1 and as shown in SEQ ID NO.2 in sequence table in nucleotide sequence such as sequence table.
It is preferred that also including dNTP, PCR buffer, Mg in described test kit2+, Taq archaeal dna polymerases and ddH2In O
One or more；More preferably, described test kit also includes genome DNA extraction reagent.
The three of the technical solution used in the present invention are：A kind of method of detection lactobacillus casei bacterial strain, it includes following step
Suddenly：
(1) genomic DNA of measuring samples is extracted as template, nucleotide sequence is respectively such as SEQ ID in sequence table
NO.1 is shown and the primer pair as shown in SEQ ID NO.2 in sequence table is used as primer, enters performing PCR amplification；
(2) whether there is single amplified production in 750bp positions in detecting step (1) pcr amplification product.
Step (1) be the genomic DNA for extracting measuring samples as template, nucleotide sequence is respectively as in sequence table
SEQ ID NO.1 are shown and the primer pair as shown in SEQ ID NO.2 in sequence table is used as primer, enter performing PCR amplification.Wherein,
The extracting method of the genomic DNA is the conventional extracting method in this area；Preferably CTAB methods；More preferably, the CTAB
Method is extracted genomic DNA and is comprised the following steps：EDTA (500mM, pH8.0) is added in measuring samples；At 4 DEG C 6500g from
After the heart 30 minutes, -20 DEG C stand 10 minutes；Deionized water is added after dissolving under room temperature, 12000r/min centrifugation 5min are abandoned
Clearly；Add TE buffer, 37 DEG C of water-bath 1-2h；Add 10%SDS, 68 DEG C of drying 15min；Add 5M NaCl, 1%CTAB, 68
DEG C drying 15min；Add phenol, chloroform-isoamyl alcohol (24:1, v/v), stand, 12000r/min centrifugation 10min；Take and reset and add
Enter equal-volume chloroform-isoamyl alcohol (24:1, v/v), 12000r/min centrifugations 5min, takes supernatant, adds ice dehydrated alcohol, -20 DEG C
Place 2h；10000r/min is centrifuged 2min, abandons supernatant, adds 70% ethanol purge, 12000r/min centrifugation 2min, after abandoning supernatant
Air-dry under room temperature, you can.
The reaction system of the PCR amplifications is the conventional reaction system in this area；Preferably 1 × PCR reaction buffers,
10-15mmol/L Mg2+, 0.2-0.3mmol/L dNTP, the 0.1-0.3 μM of primer, Taq enzyme 0.01-0.1U/ μ L, the mould
Plate 10-100ng/ μ L；It is more preferably 1 × PCR reaction buffers, 12.5mmol/L Mg2+, 0.25mmol/L dNTP, 0.2 μM of institute
State primer, Taq enzyme 0.04U/ μ L, the template 40ng/ μ L.The response procedures of the PCR amplifications are the conventional reaction in this area
Program, preferably 92-95 DEG C denaturation 3-6min, starts afterwards following circulation, and the program of each circulation is：92-95 DEG C of change
Property 20-40s, 58-63 DEG C of annealing 20-40s, 68-74 DEG C of extension 20-40s, it is individual to circulate common 30-35, after loop ends, 70-74 DEG C
Extend 8-12min, be cooled to 4-15 DEG C, terminate；More preferably it is 4 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s；Circulation
Totally 30；After loop ends, 72 DEG C of extension 10min are cooled to 12 DEG C, terminate.
Whether in 750bp positions to there is single amplified production in detecting step (1) pcr amplification product in step (2).Its
In, described is detected as the conventional detection in this area, can observe the amplified production.Preferably gel electrophoresiss inspection
Survey；It is more preferably agarose gel electrophoresiies detection or polyacrylamide gel electrophoresis detection；Most preferably it is 1.5% agarose gel
Electrophoresis detection.It is preferred that the determination methods of described detection are as follows：If there is the single amplified production of 750bp positions, then say
Contain lactobacillus casei bacterial strain in bright measuring samples；If there is no the single amplified production of 750bp positions, then in measuring samples
Lactobacillus casei bacterial strain is not contained.
Room temperature of the present invention is 10~30 DEG C.
Lactobacillus casei of the present invention is Lactobacillus casei, including lactobacillus casei and lactobacillus casei
Subspecies (also known as Lactobacillus paracasei).
Measuring samples of the present invention are the conventional measuring samples in this area, preferably cheese or Yoghourt.
On the basis of common sense in the field is met, above-mentioned each optimum condition, can combination in any, 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.
The present invention positive effect be：Detection method of the present invention detects lactobacillus casei bacterial strain, detection
Time most only needs 24 hours soon, improves detection efficiency；Simple, testing cost is low；Testing result reliability, result judgement letter
It is single, can specifically determine whether lactobacillus casei.The present invention provides a kind of simple fast for milk detection technique field
The method of fast sensitive detection lactobacillus casei antibacterial, to whether being added with lactobacillus casei with more careless in differentiation milk
Justice.
Description of the drawings
Fig. 1 is the agarose gel electrophoresiies experimental result of PCR primer Jing 1.5% in embodiment 2.Swimming lane 1~17 is followed successively by：It is dry
Lactobacillus paracasei ATCC334, lactobacillus casei LC2W, lactobacillus casei BD-II, lactobacillus casei str-zhang, lactobacillus casei
BD0059, lactobacillus casei BD0090, lactobacillus casei BD1649, lactobacillus casei BD1803, lactobacillus casei BD3552, do
Lactobacillus paracasei BD3553, lactobacillus casei BD3554, lactobacillus casei BD3555, lactobacillus casei BDlb-01, lactobacillus casei
BDlb-02, Lactobacillus paracasei BDlb-03, negative control (sterile deionized water), 100bp DNA Marker.
Fig. 2 is the agarose gel electrophoresiies experimental result of PCR primer Jing 1.5% in embodiment 2.Swimming lane 18~21 is followed successively by：
Lactobacillus paracasei BD00095, Lactobacillus paracasei BD00116, Lactobacillus paracasei BD01950, Lactobacillus paracasei
BD02004, M are 100bp DNA Marker.
Fig. 3 is the agarose gel electrophoretogram of PCR primer Jing 1.5% in embodiment 2.Swimming lane 1~17 is followed successively by：Cheese breast
Bacillus ATCC334, Lactobacillus reuteri JCM112, lactobacillus rhamnosuss LGG, Lactobacillus plantarum ATCC14917, Lactobacillus plantarum
ST-III, streptococcus thermophiluss BDST001, lactobacillus helveticuss BDLH001, Deshi Lactobacilluss BDLD6240, bacillus acidophilus
BDLA6075, Lactobacillus plantarum BDLP6102, Lactobacillus bulgaricus L99, series bacillus BD3526, leuconostoc mesenteroides
BD1710, bifidobacterium longum BDBL001, bifidobacterium bifidum BDBB001, negative control (sterile deionized water), 100bp DNA
Marker。
Fig. 4 is the agarose gel electrophoresiies of PCR primer Jing 1.5% checking primer sensitivity experiment collection of illustrative plates in embodiment 3.Swimming lane
1~10 is followed successively by：100bp DNA Marker, 105.3ng/PCR, 10.5ng/PCR, 1.05ng/PCR, 105pg/PCR,
10.5pg/PCR, 10.5pg/PCR, 105fg/PCR, ddH2O。
Fig. 5 is actual sample in embodiment 4 Jing after PCR detections, the agarose gel electrophoretogram of PCR primer Jing 1.5%.Swimming
Road 1~11 is followed successively by：Lactobacillus casei ATCC334, cheese samples 1, cheese samples 2, yogurt sample 1, yogurt sample 2, cheese
Sample 3, cheese samples 4, the yogurt sample 3 of cheese samples 5., yogurt sample 4, ddH2O, M are 100bp DNA Marker.
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
Among applying a scope.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or according to business
Product description is selected.
Described room temperature refers to 10~30 DEG C.
The primer synthesis and PCR detections of the detection lactobacillus casei bacterial strain of embodiment 1
(1) primer synthesis
Synthesis can PCR amplification lactobacillus casei histidine kinase gene order in conserved sequence primer (by Shanghai
Sheng Gong biotechnologies Services Co., Ltd synthesizes), primer sequence is as follows：
VicR-L：5 '-CATTGCTCGCTGCCATTCTC-3 ' (its nucleotide sequence such as sequence table SEQ ID NO:1 institute
Show)；
VicR-R：5 '-CCGGTCGACTCAAGGTCAAA T-3 ' (its nucleotide sequence such as sequence table SEQ ID NO:2 institutes
Show).
(2) PCR detections
Using above-mentioned primer, the genomic DNA with lactobacillus casei reference culture ATCC334 enters performing PCR reaction as template
The foundation and optimization of system and response procedures, it is found that following reaction system and response procedures can obtain the expansion of single 750p or so
Volume increase thing.
Wherein, the acquisition of the genomic DNA of lactobacillus casei reference culture ATCC334 includes that mobile phone thalline and CTAB methods are carried
The step of taking genomic DNA, specifically includes the steps：
1), collects thalline：Lactobacillus casei reference culture ATCC334 is seeded in the MRS fluid mediums of 5mL, 37
DEG C increase bacterium 2h after, 1mL bacterium solutions are taken, in being put into 1.5mL centrifuge tubes；Then 3000r/min centrifugations 10min, takes supernatant, then
12000r/min is centrifuged 5min, collects thalline.Aseptic double-distilled water suspension thalline is used, 400 μ L TE are added after centrifuge washing, obtain bacterium
Liquid suspension；
2), genomic DNA is extracted using CTAB methods：
(1) 25mL steps 1) obtained by thallus suspension liquid in add 5mL EDTA (500mM, pH8.0)；
After 6500g is centrifuged 30 minutes at (2) 4 DEG C, -20 DEG C stand 10 minutes；
(3) the resuspended cleaning thalline of sterile deionized water, 12000r/min centrifugation 5min is used to abandon after dissolving under room temperature again
Clearly；
(4) 456 μ L TE buffer and 24 μ L lysozyme, 37 DEG C of water-bath 1-2h are added；Add 53 μ L10%SDS, 68 DEG C of bakings
Dry 15min；Add 87 μ L 5M NaCl, 69 μ L 1%CTAB, 68 DEG C of drying 15min；
(5) the μ L of phenol 250, chloroform-isoamyl alcohol (24 are added:1, v/v) 250 μ L, stand, 12,000r/min centrifugations
10min；
(6) the μ L of supernatant 400 are taken, equal-volume (400 μ L) chloroform-isoamyl alcohol (24 is added:1, v/v), 12,000r/min centrifugations
5min, takes the μ L of supernatant 300, adds 600 μ ice dehydrated alcohol, -20 DEG C of placement 2h；
(7) 10000r/min centrifugations 2min, abandons supernatant, adds 70% ethanol purge, 12000r/min centrifugation 2min to abandon
Room temperature leeward is done after clear.50~100 μ L sterile deionized water or TE dissolving DNAs are added, -20 DEG C of preservations are stand-by, can be as after
The template of continuous PCR reaction systems.
PCR reaction systems are：1 × PCR reaction buffers, 10-15mmol/L Mg2+, 0.2-0.3mmol/L dNTP,
0.1-0.3 μM of primer VicR-L, 0.1-0.3 μM of primer VicR-R, Taq enzyme 0.01-0.1U/ μ L, DNA profiling 10-100ng/ μ L.
PCR amplification programs are：92-95 DEG C of denaturation 3-6min, starts afterwards following circulation, and the program of each circulation is：
92-95 DEG C of degeneration 20-40s, 58-63 DEG C of annealing 20-40s, 68-74 DEG C of extension 20-40s；Common 30-35 circulation；Loop ends
Afterwards, 70-74 DEG C of extension 8-12min, is cooled to 4-15 DEG C, terminates.
Inventor Jing is tested and also found, the yield highest of the amplified production of 750bp or so, when electrophoretic band is most substantially clear
PCR reaction systems be：1 × PCR reaction buffers, 12.5mmol/L Mg2+, 0.25mmol/L dNTP, 0.2 μM of primer
VicR-L, 0.2 μM of primer VicR-R, Taq enzyme 0.04U/ μ L, DNA profiling 40ng/ μ L.
The yield highest of the amplified production of 750bp or so, PCR amplification programs when electrophoretic band is most substantially clear are：94
DEG C denaturation 5min, starts afterwards following circulation, and program of each circulation is：94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C are prolonged
Stretch 30s；Circulation totally 30；After loop ends, 72 DEG C of extension 10min are cooled to 12 DEG C, terminate.
Embodiment 2 detects the Evaluation on specificity of lactobacillus casei bacterial strain
1), the acquisition of strain gene group DNA template
Take the bacterium of 1 plant of lactobacillus casei reference culture (ATCC334), 18 plants of lactobacillus casei isolated strains and other kinds
14 plants (as shown in table 1) of strain, distinguishes collects thalline and presses CTAB methods and extract genomic DNA according to step as described in Example 1,
As the PCR reaction template of detection lactobacillus casei.
2), PCR is detected whether as lactobacillus casei bacterial strain
Take 2 μ L steps 1) obtained by every plant of bacterial strain DNA solution (DNA concentration 30ng/ μ L) as PCR reaction template add
Amplified reaction is carried out in PCR reaction systems.
PCR reaction systems are：16.1 μ L sterilized water, sequentially add 10 × PCR reaction buffers 2.5 μ L, 25mmol/L
Mg2+The μ L of 2.0 μ L, 2.5mmol/L dNTP 1.0,5 μM of μ L of primer VicR-L 1.0,5 μM of μ L of primer VicR-R 1.0,2.5U/ μ L
The μ L of Taq enzyme 0.4, finally add the μ L of template solution 2, and are negative control of the template as reaction using sterilized water.
PCR response procedures are：4 DEG C of denaturations 5min, start afterwards following circulation, and the program of each circulation is：94 DEG C of changes
Property 30s, 60 DEG C of annealing temperature, annealing time is 30s, 72 DEG C extension 30s, totally 35 circulation, after loop ends 72 DEG C extension
10min, is cooled to 12 DEG C, terminates.
Pcr amplification product is detected using agarose gel electrophoresiies, judges to whether there is single amplification bar in 750bp positions
Band, if there is the band of single amplification in 750bp positions, is labeled as "+", that is, be judged as the lactobacillus casei；Otherwise mark
It is designated as "-".1 is the results are shown in Table, electrophoresis result is shown in Fig. 1~3.
The Evaluation on specificity bacterial strain uses therefor of table 1. and result of the test
| Bacterial strain | Numbering | Bacterium number | As a result |
| Lactobacillus casei | ATCC334 | 1 | + |
| Lactobacillus casei | LC2W | 1 | + |
| Lactobacillus casei | BD-II | 1 | + |
| Lactobacillus casei | str-zhang | 1 | + |
| Lactobacillus casei | BD0059 | 1 | + |
| Lactobacillus casei | BD0090 | 1 | + |
| Lactobacillus casei | BD1649 | 1 | + |
| Lactobacillus casei | BD1803 | 1 | + |
| Lactobacillus casei | BD3552 | 1 | + |
| Lactobacillus casei | BD3553 | 1 | + |
| Lactobacillus casei | BD3554 | 1 | + |
| Lactobacillus casei | BD3555 | 1 | + |
| Lactobacillus casei | BDlb-01 | 1 | + |
| Lactobacillus casei | BDlb-02 | 1 | + |
| Lactobacillus paracasei | BDlb-03 | 1 | + |
| Lactobacillus paracasei | BD00095 | 1 | + |
| Lactobacillus paracasei | BD00116 | 1 | + |
| Lactobacillus paracasei | BD01950 | 1 | + |
| Lactobacillus paracasei | BD02004 | 1 | + |
| Lactobacillus reuteri | JCM112 | 1 | - |
| Lactobacillus rhamnosuss | LGG | 1 | - |
| Lactobacillus plantarum | ATCC14917 | 1 | - |
| Lactobacillus plantarum | ST-III | 1 | - |
| Streptococcus thermophiluss | BDST001 | 1 | - |
| Lactobacillus helveticuss | BDLH001 | 1 | - |
| Deshi Lactobacilluss | BDLD6240 | 1 | - |
| Bacillus acidophilus | BDLA6075 | 1 | - |
| Lactobacillus plantarum | BDLP6102 | 1 | - |
| Lactobacillus bulgaricus | L99 | 1 | - |
| Series bacillus | BD3526 | 1 | - |
| Leuconostoc mesenteroides, | BD1710 | 1 | - |
| Bifidobacterium longum | BDBL001 | 1 | - |
| Bifidobacterium bifidum | BDBB001 | 1 | - |
As can be known from the results of Table 1, in addition to the reference culture and separation strains of lactobacillus casei bacterial strain, remaining negative control bacterium
Strain does not have specific amplified band (750bp).Lactobacillus casei (Lactobacillus casei) has used 1 plant of standard in table 1
Bacterial strain (ATCC334), represents all kinds in the category of typical standard bacterial strain.Other bacterial strains in lactobacillus casei and Lactobacillus
Sibship recently, negative control of this test has used altogether 3 plants of (Lactobacillus reuteri of Lactobacillus reference culture
JCM112, lactobacillus rhamnosuss LGG and Lactobacillus plantarum ATCC14917), Lactobacillus isolated strains are 11 plants.Used
These bacterial strains and lactobacillus casei bacterial strain have nearer sibship.And these bacterial strains Jing the present invention primer VicR-L and draw
Thing VicR-R is expanded less than special fragment (750bp) after PCR experiment, then other and lactobacillus casei bacterial strain relationship
Relation bacterial strain farther out is just more difficult to amplification to the fragment, therefore through the checking of the nearer bacterial strain of these sibships, protects
The specificity of primer VicR-L and primer VicR-R described in embodiment 1 is demonstrate,proved, has also fully proved that the method can expand cheese breast bar
Any bacterial strain in strain, without expanding other any bacterial strains.
Additionally, 13 plants of lactobacillus casei isolated strains and 5 plants of Lactobacillus paracasei isolated strains are from food in table 1
The isolated strains of isolation identification, it carries out isolation identification all in accordance with GB GB 4789.35-2010, and by separate obtain can
Doubtful bacterial strain carries out first microscopy, then carries out following bio-chemical characteristics and is identified, the Physiology and biochemistry of all isolated strains
Test (table 2) consistent with the result of the reference culture of lactobacillus casei bacterial strain, illustrate 13 plants of lactobacillus casei isolated strains and 5 plants
Lactobacillus paracasei isolated strains are lactobacillus casei really.In table 2, lactobacillus casei can be under conditions of the specific carbon source
Normal growth is then labeled as "+"；Otherwise it is labeled as "-".
Table 2 identifies the carbon source reaction result of lactobacillus casei isolated strains
| Strain | Esculin | Cellobiose | Maltose | Mannitol | Salicin | Sorbitol | Sucrose | Cottonseed sugar |
| Lactobacillus casei | + | + | + | + | + | + | + | ‐ |
Also, this 13 plants of lactobacillus casei isolated strains and 5 plants of Lactobacillus paracasei isolated strains are individual differing：Root
According to the different 16S rRNA sequences and dnaK gene order (lists of references of these isolated strains：Huang Chien-Hsun,
Lee Fwu-ling,The dnaK gene as a molecular marker for the classification and
discrimination of the Lactobacillus casei group,Antonie van Leeuwenhoek,2011,
99:319-327), it becomes possible to be one by one distinguished these isolated strains.
In embodiment 1, bacterium is increased in MRS culture medium to be less than 19 hours, extracts genomic DNA about 3 by CTAB methods little
When, PCR detects whether about to spend 2 hours for lactobacillus casei bacterial strain, therefore the method for the detection lactobacillus casei needs altogether
24 hours or so.
Embodiment 3 detects the sensitivity evaluation of lactobacillus casei method
CTAB methods as described in embodiment 1 extract the method for genomic DNA and extract lactobacillus casei reference culture ATCC334
Genome DNA.The DNA of gained is dissolved in sterilized water, concentration is 1053ng/uL, then it is dilute with sterilized water 10 times of gradients of work
Release, 8 gradients are diluted altogether：105.3ng/PCR, 10.5ng/PCR, 1.05ng/PCR, 105pg/PCR, 10.5pg/PCR,
10.5pg/PCR, 105fg/PCR, each gradient takes respectively 5 μ L and adds PCR reaction systems as template.With as described in Example 1
VicR-L and primer VicR-R expanded according to PCR reaction systems as described in Example 2 and PCR response procedures, gel
Electrophoresis detection amplified production, observes Gel electrophoresis results, as shown in Figure 4 in gel imaging instrument.As shown in Figure 4, in the 6th article of swimming
Road can see clearly band (750bp), and corresponding DNA concentration is 10.5pg/PCR pg/PCR, and after the 7th article of swimming lane
Can't see amplified band.Therefore, PCR detection sensitivities are judged as 10.5pg/PCR, with higher sensitivity.
Whether contain lactobacillus casei in the detection food samples of embodiment 4
10 parts of food samples (including cheese and fermentation milk) are self-control.Respectively 25g samples are added to into the aseptic lifes of 225mL
Reason saline is diluted, and by National Standard Method GB 4789.35-2010 isolation identification lactobacillus casei is carried out, and by last qualification result
Compareed with PCR method.Meanwhile, take 1mL samples CTAB methods as described in Example 1 and extract genomic DNA, and by total base
Because a group DNA is diluted to 50ng/uL as pcr template, negative control, PCR reaction systems as described in Example 2 are made with sterilized water
Expanded with PCR response procedures, each experiment is repeated 3 times, and the results are shown in Table shown in 3, Fig. 5.In table 3, using agarose gel
The pcr amplification product of electrophoresis detection food samples, judges in 750bp positions with the presence or absence of single amplified band, if in 750bp positions
The band that there is single amplification is put, is then labeled as "+", that is, judge to contain lactobacillus casei in food samples；Otherwise it is labeled as
“-”.As shown in figure 5, have 5 parts of sample detection to special fragment (750bp), and 5 parts of sample Jing national standard methods are also separated to
Lactobacillus casei, it can be seen that the method that this experiment is set up has extreme high reliability.
The testing result of lactobacillus casei in the food samples of table 3.
| Sample ID | Numbering | Bacterium number | As a result |
| Cheese | 1 | 1 | + |
| Cheese | 2 | 1 | + |
| Cheese | 3 | 1 | + |
| Cheese | 4 | 1 | + |
| Fermentation milk | 5 | 1 | + |
| Cheese | 6 | 1 | - |
| Fermentation milk | 7 | 1 | - |
| Fermentation milk | 8 | 1 | - |
| Fermentation milk | 9 | 1 | - |
| Fermentation milk | 10 | 1 | - |
Comparative example 1
PCR response procedures are：4 DEG C of denaturations 5min, start afterwards following circulation, and the program of each circulation is：94 DEG C of changes
Property 30s, 50 DEG C of annealing temperature, annealing time is 30s, 72 DEG C extension 30s, totally 35 circulation, after loop ends 72 DEG C extension
10min, is cooled to 12 DEG C, terminates.
Remaining step and conditional parameter with embodiment 2, detected through gel electrophoresis amplified production, are as a result found in 750bp positions
Put be difficult to obtain it is any clearly, the band of single amplification, and other lactobacillus strains also have the expansion of non-specific band
Increase, illustrate that the PCR response procedures are unsuitable for detecting lactobacillus casei bacterial strain.
Comparative example 2
PCR response procedures are：4 DEG C of denaturations 5min, start afterwards following circulation, and the program of each circulation is：94 DEG C of changes
Property 30s, 68 DEG C of annealing temperature, annealing time is 30s, 72 DEG C extension 30s, totally 35 circulation, after loop ends 72 DEG C extension
10min, is cooled to 12 DEG C, terminates.
Remaining step and conditional parameter with embodiment 2, detected through gel electrophoresis amplified production, are as a result found in 750bp positions
Put be difficult to obtain it is any clearly, the band of single amplification, and some lactobacillus casei bacterial strains also cannot amplify it is expected
Band, illustrates that the PCR response procedures are unsuitable for detecting lactobacillus casei bacterial strain.
Presently preferred embodiments of the present invention is the foregoing is only, the restriction to right, ability is not constituted
Other replacements being substantially equal to that technical staff is contemplated that in domain, in the scope of the present invention.
Claims (13)
1. the primer pair of a species-specific amplification lactobacillus casei (Lactobacillus casei), it is characterised in that its nucleoside
Acid sequence is respectively as shown in SEQ ID NO.1 in sequence table and as shown in SEQ ID NO.2 in sequence table.
2. a kind of test kit for specific amplification lactobacillus casei, it is characterised in that it includes nucleotide sequence respectively such as
In sequence table shown in SEQ ID NO.1 and the primer pair as shown in SEQ ID NO.2 in sequence table.
3. test kit as claimed in claim 2, it is characterised in that also include in described test kit dNTP, PCR buffer,
Mg2+, Taq archaeal dna polymerases and ddH2One or more in O.
4. test kit as claimed in claim 3, it is characterised in that also try including genome DNA extraction in described test kit
Agent.
5. it is a kind of detection lactobacillus casei bacterial strain method, it is characterised in that it is comprised the following steps：
(1) genomic DNA of measuring samples is extracted as template, nucleotide sequence is respectively such as SEQ ID NO.1 in sequence table
Shown and primer pair as shown in SEQ ID NO.2 in sequence table enters performing PCR amplification as primer；
(2) whether there is single amplified production in 750bp positions in detecting step (1) pcr amplification product.
6. method as claimed in claim 5, it is characterised in that in step (1), the extracting method of the genomic DNA is
CTAB methods.
7. method as claimed in claim 5, it is characterised in that in step (1), the reaction system of the PCR amplifications is 1 ×
PCR reaction buffers, 10-15mmol/L Mg2+, 0.2-0.3mmol/L dNTP, the 0.1-0.3 μM of primer, Taq enzyme 0.01-
0.1U/ μ L, the template 10-100ng/ μ L；And/or, the response procedures of the PCR amplifications are 92-95 DEG C of denaturation 3-6min,
Start following circulation afterwards, the program of each circulation is：92-95 DEG C of degeneration 20-40s, 58-63 DEG C of annealing 20-40s, 68-74 DEG C
Extend 20-40s, circulation is common 30-35, after loop ends, 70-74 DEG C of extension 8-12min is cooled to 4-15 DEG C, terminates.
8. method as claimed in claim 7, it is characterised in that in step (1), the reaction system of the PCR amplifications is 1 ×
PCR reaction buffers, 12.5mmol/L Mg2+, 0.25mmol/L dNTP, 0.2 μM of primer, Taq enzyme 0.04U/ μ L are described
Template 40ng/ μ L；And/or, the response procedures of the PCR amplifications are 4 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s；
Circulation totally 30；After loop ends, 72 DEG C of extension 10min are cooled to 12 DEG C, terminate.
9. method as claimed in claim 5, it is characterised in that in step (2), described is detected as detected through gel electrophoresis.
10. method as claimed in claim 9, it is characterised in that described detected through gel electrophoresis are agarose gel electrophoresiies inspection
Survey or polyacrylamide gel electrophoresis detection.
11. methods as claimed in claim 10, it is characterised in that described agarose gel electrophoresiies are detected as 1.5% agar
Sugared detected through gel electrophoresis.
12. methods as claimed in claim 5, it is characterised in that in step (2), the determination methods of described detection are as follows：Such as
There is the single amplified production of 750bp positions in fruit, then illustrate to contain lactobacillus casei bacterial strain in measuring samples；If there is no
The single amplified production of 750bp positions, then do not contain lactobacillus casei bacterial strain in measuring samples.
13. methods as claimed in claim 5, it is characterised in that described measuring samples are cheese or Yoghourt.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410840051.5A CN104531873B (en) | 2014-12-24 | 2014-12-24 | Method for detecting lactobacillus casei strain and kit and primer pair used by method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410840051.5A CN104531873B (en) | 2014-12-24 | 2014-12-24 | Method for detecting lactobacillus casei strain and kit and primer pair used by method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104531873A CN104531873A (en) | 2015-04-22 |
| CN104531873B true CN104531873B (en) | 2017-05-17 |
Family
ID=52847492
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410840051.5A Active CN104531873B (en) | 2014-12-24 | 2014-12-24 | Method for detecting lactobacillus casei strain and kit and primer pair used by method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104531873B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349670B (en) * | 2015-11-30 | 2018-05-25 | 武汉轻工大学 | For fast qualitative, immue quantitative detection reagent box and the detection method of Lactobacillus casei added in feed and application |
| CN112029884B (en) * | 2020-09-29 | 2022-05-10 | 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 casei group |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101509034A (en) * | 2008-11-18 | 2009-08-19 | 东华大学 | Human body intestinal canal flora detection parting and quantitative reagent kit |
| 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 |
| CN103361439A (en) * | 2013-07-30 | 2013-10-23 | 青岛啤酒股份有限公司 | Method for integrated detection of nine beer-spoilage bacteria |
-
2014
- 2014-12-24 CN CN201410840051.5A patent/CN104531873B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101509034A (en) * | 2008-11-18 | 2009-08-19 | 东华大学 | Human body intestinal canal flora detection parting and quantitative reagent kit |
| 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 |
| CN103361439A (en) * | 2013-07-30 | 2013-10-23 | 青岛啤酒股份有限公司 | Method for integrated detection of nine beer-spoilage bacteria |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104531873A (en) | 2015-04-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mättö et al. | Genetic heterogeneity and functional properties of intestinal bifidobacteria | |
| Fujimoto et al. | Identification and quantification of Lactobacillus casei strain Shirota in human feces with strain-specific primers derived from randomly amplified polymorphic DNA | |
| Fasoli et al. | Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis | |
| Vera-Pingitore et al. | Screening and characterization of potential probiotic and starter bacteria for plant fermentations | |
| Cocolin et al. | The late blowing in cheese: a new molecular approach based on PCR and DGGE to study the microbial ecology of the alteration process | |
| Dal Bello et al. | Increased complexity of the species composition of lactic acid bacteria in human feces revealed by alternative incubation condition | |
| Amor et al. | Advanced Molecular Tools for the Identification of Lactic Acid Bacteria1 | |
| Vincent et al. | Characterization of bifidobacteria by random DNA amplification | |
| Fujimoto et al. | Quantitative detection of viable Bifidobacterium bifidum BF-1 cells in human feces by using propidium monoazide and strain-specific primers | |
| EP1883695B1 (en) | Probiotic bifidobacterial species | |
| CN103421898B (en) | The triple real-time fluorescent PCR testing primer of methicillin-resistant staphylococcus aureus, probe, detection kit and detection method | |
| Yu et al. | Phylogenetic study of Lactobacillus acidophilus group, L. casei group and L. plantarum group based on partial hsp 60, phe S and tuf gene sequences | |
| Ongol et al. | Main microorganisms involved in the fermentation of Ugandan ghee | |
| Maruo et al. | Monitoring the cell number of Lactococcus lactis subsp. cremoris FC in human feces by real-time PCR with strain-specific primers designed using the RAPD technique | |
| CN113564272B (en) | Rapid identification and detection method for cheese lactobacillus in fermented milk | |
| CN109439778B (en) | Mark primer pair of lactobacillus rhamnosus hsryfm1301 derived from Bama longevity village, identification method and application thereof | |
| CN104450941B (en) | Method for detecting lactobacillus casei strain and kit and primer pair thereof | |
| Kim et al. | Development of rapid and highly specific TaqMan probe-based real-time PCR assay for the identification and enumeration of Lactobacillus kefiri in kefir milk | |
| CN104531873B (en) | Method for detecting lactobacillus casei strain and kit and primer pair used by method | |
| DE URRAZA et al. | DNA fingerprinting of thermophilic lactic acid bacteria using repetitive sequence-based polymerase chain reaction | |
| Tafvizi et al. | Application of repetitive extragenic palindromic elements based on PCR in detection of genetic relationship of lactic acid bacteria species isolated from traditional fermented food products | |
| Ohud et al. | Detection of Salmonella strains in clinical samples from Saudi Arabia by invA and hilA polymerase chain reaction (PCR)-based assays | |
| RoushanZadeh et al. | Phenotypic and genotypic diversity of dominant lactic acid bacteria isolated from traditional yoghurts produced by tribes of Iran | |
| CN104531692B (en) | A kind of method and its kit and primer pair for detecting lactobacillus casei bacterial strain | |
| Shevtsov et al. | Identification of phenotypically and genotypically related Lactobacillus strains based on nucleotide sequence analysis of the groEL, rpoB, rplB, and 16S rRNA genes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |