CN101570781B - Detection kit and species-based detection method for lactobacilli - Google Patents
Detection kit and species-based detection method for lactobacilli Download PDFInfo
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- CN101570781B CN101570781B CN200910010802XA CN200910010802A CN101570781B CN 101570781 B CN101570781 B CN 101570781B CN 200910010802X A CN200910010802X A CN 200910010802XA CN 200910010802 A CN200910010802 A CN 200910010802A CN 101570781 B CN101570781 B CN 101570781B
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Abstract
The invention discloses a detection kit and a species-based detection method for lactobacilli, which are used for the detection and species-based identification of the bacteria of genus lactobacillus.The kit comprises Taq DNA polymerase with a concentration of 5U/mu L and a PCR reaction solution, wherein the PCR reaction solution contains 10 millimols of Tris.HCl, 50 millimols of KCl, 25 millimols of MgCl2, 2.5 millimols of dNTP and 0.2 millimol of general lactobacilli detection primer pair. The detection kit and the detection method of the invention can be used for synchronous detection of the bacteria of the genus lactobacillus and species-based detection of 8 species of bacteria of the genus lactobacillus. The kit has high specificity and sensitivity and can detect lactobacilli with aconcentration of 150 CFU/mL. The kit can realize high throughput detection of the bacteria of the genus lactobacillus and can solve the problem of synchronous and quick detection and identification of8 species of lactobacilli of the genus lactobacillus.
Description
Technical field
The present invention relates to lactobacillus genus and divide kind of a detection authentication method, relate in particular to and utilize universal primer PCR and sex change performance liquid chromatography (DHPLC) technology to detect the method for identifying 8 kinds of lactobacillus spps of lactobacillus genus simultaneously.Also relate to and detect employed compsn, i.e. test kit.
Background technology
Lactobacillus spp extensively is present in milk-product or the sticking Tetramune (like cheese, sour milk) of fermentable animal.Some lactobacillus spp of part such as Lactobacterium acidophilum, lactobicillus bulgaricus are usually used in fermentation industries such as beverage.Lactobacillus spp is usually used in bioassay, is widely used in various vitamins Bs and various amino acid whose calibrating like the cheese probiotic lactobacillus, and the Li Shi probiotic lactobacillus is used for the microbiology calibrating of cobalamin, and probiotic lactobacillus also can be in order to make fermented feed.Having some milk-acid bacterias, particularly probiotic lactobacillus in the beer that contains the hops material, to grow, is the predominantly bacteria that causes beer spoilage.The lactobacillus genus bacteria nutritional requirement is complicated, and cultural characters is more special, and the classic flat-plate incubation growth is slow, and separation and Culture needs 7～9 days qualification cycle approximately.Therefore it is imperative with the method for inspection of identifying lactobacillus spp to set up rapid detection.And because the metabolic characteristic and the technic characteristic of lactobacillus spp need be carried out safety and quality control to them in the lactobacillus ferment food production.The rapid and reliable authentication method that identifies milk-acid bacteria kind and bacterial strain level is significant for its fundamental research and industrial requirement.
At present, milk-acid bacteria is identified and still continues to use traditional method, mainly is morphological observation, gramstaining and sugar fermentating test.But also generally recognize some shortcomings of phenotype analytical; For example low, the similar phenotypic characteristic of repetition rate and identification capability is not equal to similar or genotype in close relations, therefore can not make clear and definite evaluation to lactobacillus strain based on the routine techniques of phenotypic assay.In general, identify milk-acid bacteria exactly, need 17 kinds of phenotype experiments at least, but the strain isolated of microecosystem needs authentication method more simply fast to the level of planting.Therefore, need carry out the molecular level research of phenotypic characteristic and the research of hereditary property, classify more reliably and discriminating to reach, and improve and detect the working efficiency of identifying.Along with the understanding to milk-acid bacteria genome structure and phylogenetic relationship, Protocols in Molecular Biology is used to classify in the evaluation work more and more.
All there are some problems in existing detection technique such as microbial culture and biochemical identification, immunological technique, round pcr etc.: conventional biochemical identification method complicated operation, and length consuming time, often the one-time detection experiment can only be identified a kind of or a few bacterium; Though the immunological technique susceptibility is high, be prone to pollute, the false positive phenomenon often appears; Round pcr generally will with the gel electrophoresis technology coupling, and electrophoresis result can not be as final conclusion, also need carry out other probe hybridizations experiments.Though it is low that the PCR-gel electrophoresis detection method detects cost, the reaction product electrophoretic process very easily pollutes; High specificity, highly sensitive real time fluorescent PCR method exists the detection cost higher, and the fluorescent probe shelf time is than problems such as weak points.Therefore, above-mentioned detection method can not satisfy the needs of rapid detection various bacteria simultaneously.Be badly in need of a kind of stable method fast for the discriminating of lactobacillus spp.
Many germs have the different gene type, they pathogenic widely different, but they other characteristic and gene are all closely similar, make the accurate somatotype of bacterium face very big technological challenge.In an epidemic situation outburst on a large scale, confirm that from the bacterial strain level pathogenic bacteria is vital, only know about pathogenic strains and could correctly select antibacterials, follow the trail of the source of germ etc.Therefore setting up a kind of high-throughout lactobacillus spp classifying method also is very important.
Summary of the invention
The object of the present invention is to provide a kind of sensitive rapid detection lactobacillus spp microorganism belonging to genus that is used for, and this is belonged to the quick detection kit based on mPCR-DHPLC that 8 kinds of lactobacillus spps branches are planted.
Test kit of the present invention comprises that concentration is Taq archaeal dna polymerase and the PCR reaction solution of 5U/ μ L; Wherein contain 10mM TrisHCl, 50mM KCl, 25mM MgCl2, each 2.5mM of dNTP and the general detection primer of lactobacillus genus in the PCR reaction solution to 0.2mM, described primer to sequence is:
Upstream primer: 5 '-GGGTTGTCTGCGAAAGCGAA-3 '
Downstream primer: 5 '-GTCTTCGTGCTGCGAGTTTG-3 '
This test kit-20 ℃ preservation.
The present invention also provides and has utilized the mentioned reagent box to carry out the method that lactobacillus spp detects and branch is planted, and this method comprises the steps:
1. get 2ul testing sample dna solution, add PCR damping fluid and 0.5 μ LTaq archaeal dna polymerase in the 12ul test kit, sterilization ultrapure water 35.5 μ L, TV 50 μ L; The centrifugal 10s of 5000r/min, carry out pcr amplification by following parameter then:
Sex change in advance: 93 ℃, 5min;
Get into circulation: 94 ℃ of sex change 30s, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, 35 circulations;
Stop extending: 72 ℃, 10min;
Preserve reaction product for 4 ℃;
2. analyze the PCR product under the non-sex change condition, to detect the lactobacillus spp microorganism belonging to genus, the DHPLC testing conditions is following:
Chromatographic column: PS-DVB & C18 DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 50 ℃;
Moving phase (volume ratio): 0min, 55.0% buffered soln A, 45.0% buffered soln B,
0.5min, 50.2% buffered soln A, 49.8% buffered soln B,
2.4min, 45.7% buffered soln A, 54.3% buffered soln B,
4.3min, 42.9% buffered soln A, 57.1% buffered soln B,
6.1min, 40.9% buffered soln A, 59.1% buffered soln B,
8.0min, 39.4% buffered soln A, 60.6% buffered soln B,
Wherein, buffered soln A is that 50ml TEAA and 250 μ l acetonitriles mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution; Buffered soln B is that 50ml TEAA and 250ml acetonitrile mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluoroscopic examination, light source: 150W Xenon lamp; PLE bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR product 5 μ L;
3. analyze the PCR product under the partially denaturing condition, to the branch kind detection of 8 kinds of lactobacillus spps, the DHPLC testing conditions is following:
Chromatographic column: PS-DVB & C18 DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 59 ℃;
Moving phase (volume ratio): 0.0min, 50.3% buffered soln A, 49.7% buffered soln B,
0.5min, 45.3% buffered soln A, 54.7% buffered soln B,
5.0min, 36.3% buffered soln A, 63.7% buffered soln B,
Wherein, buffered soln A is that 50ml TEAA and 250 μ l acetonitriles mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution; Buffered soln B is that 50ml TEAA and 250ml acetonitrile mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluoroscopic examination, light source: 150W Xenon lamp; PLE bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR product 5 μ L.
Use test kit of the present invention and detection method, can detect sensitivity, special, detect 8 kinds of lactobacillus spps and it is carried out the branch kind apace, swift to operate, easy, and it is low to detect cost.
Description of drawings
Accompanying drawing 10 width of cloth of the present invention, wherein:
Fig. 1 is that PCR-DHPLC detects collection of illustrative plates under 50 ℃ of non-sex change conditions of lactobacillus spp, and wherein 1～6 is respectively lactobicillus bulgaricus, lactobacillus lactis bacteria, plant lactobacillus, lactobacterium casei, lactobacillus fermentum and form probiotic lactobacillus, the 7th, and intestinal bacteria;
Fig. 2 is that PCR-DHPLC detects collection of illustrative plates under 59 ℃ of partially denaturing conditions of lactobacillus delbruockii subspecies bulgaricus;
Fig. 3 is that PCR-DHPLC detects collection of illustrative plates under 59 ℃ of partially denaturing conditions of lactobacterium casei;
Fig. 4 is that PCR-DHPLC detects collection of illustrative plates under 59 ℃ of partially denaturing conditions of lactobacillus lactis bacteria;
Fig. 5 is that PCR-DHPLC detects collection of illustrative plates under 59 ℃ of partially denaturing conditions of lactobacillus fermentum;
Fig. 6 is that PCR-DHPLC detects collection of illustrative plates under 59 ℃ of partially denaturing conditions of plant lactobacillus;
Fig. 7 is that PCR-DHPLC detects collection of illustrative plates under 59 ℃ of partially denaturing conditions of form probiotic lactobacillus;
Fig. 8 is that PCR-DHPLC detects collection of illustrative plates under 59 ℃ of partially denaturing conditions of short lactobacillus;
Fig. 9 is that PCR-DHPLC detects collection of illustrative plates under 59 ℃ of partially denaturing conditions of cheese probiotic lactobacillus;
Accompanying drawing 10 is that sensitivity test detects spectrogram, and wherein: 1. template is 2 * 10
3The lactobacillus spp of CFU/mL; 2. template is 1.5 * 10
2The lactobacillus spp of CFU/mL; 3. template is 1 * 10
1The lactobacillus spp of CFU/mL; 4. intestinal bacteria make negative control;
In the above-mentioned accompanying drawing, X-coordinate is RT, and (unit: minute min), ordinate zou is represented absorption peak strength of signal (unit: millivolt mV).
Embodiment
Be specific embodiment of the present invention below, its foundation and application thereof to present method is further described, but does not limit content of the present invention in any form.
If no specified otherwise, the employed main agents in this part, instrument and merchandise resources thereof are: reagent such as bacterial genomes DNA a small amount of purification kit (TakaRa MiniBEST Bacterial Genomic DNA Extractionkit), Taq enzyme and PCR damping fluid are all available from precious biotechnology (Dalian) ltd; Triethylamine acetyl salt (TEAA, chromatographically pure) is available from Transgenomic company; Acetonitrile (chromatographically pure) is available from Fisher company; Regular-PCR appearance PE24000 (PerkinElmer company, the U.S.); Sex change high performance liquid chromatograph NAV-99-4500 (Transgenomic company, the U.S.); Supercentrifuge centrifuge 5804 (Eppendorf company, Germany).
This patent institute with reference culture all available from U.S. typical case DSMZ (ATCC) and Chinese medicine microbial strains preservation administrative center (CMCC).Each bacterial strain uses bacterial classification to preserve-80 ℃ of preservations of pipe, and activation culture etc. are all carried out according to relevant national standard (GB), inspection and quarantine industry standard (SN) or internal authority standard method.
Table 1. test strain and numbering thereof
The foundation of embodiment 1, lactobacillus spp detection kit and branch kind of detection method
(1) foundation of lactobacillus spp detection kit:
The primer that the present embodiment independent design is used to detect is:
Upstream primer: 5 '-GGGTTGTCTGCGAAAGCGAA-3 '
Downstream primer: 5 '-GTCTTCGTGCTGCGAGTTTG-3 '
Expection amplified fragments 250bp
On this basis, set up the lactobacillus spp detection kit that is used for pcr amplification, this test kit contains Taq archaeal dna polymerase and the PCR reaction solution that concentration is 5U/ μ L; Wherein contain 10mMTrisHCl, 50mM KCl, 25mM MgCl in the PCR reaction solution
2, dNTP (dATP, dGTP, dCTP and dTTP) each 2.5mM and the general detection primer of above-mentioned lactobacillus genus be to 0.2mM.
(2) foundation of the detection of lactobacillus spp and branch kind of detection method comprises the steps:
1. the preparation of sample to be checked: adopt the test kit extraction method to prepare testing sample DNA genome:
Test sample adding lactobacillus broth substratum under aerobic and anaerobic condition, in 37 ℃ of constant temperature culture 48 ± 3h, extracts template DNA with broth culture respectively.Use bacterial genomes DNA a small amount of purification kit (TakaRa MiniBEST Bacterial Genomic DNA Extraction kit) to extract its genomic dna, produce pcr template.Mark is directly as pcr template or-20 ℃ of preservations.
2. PCR reaction:
Get 2ul testing sample dna solution, add PCR damping fluid and 0.5 μ L TaqDNA polysaccharase in the 12ul test kit, sterilization ultrapure water 35.5 μ L, TV 50 μ L; The centrifugal 10s of 5000r/min, carry out pcr amplification by following parameter then:
Sex change in advance: 93 ℃, 5min;
Get into circulation: 94 ℃ of sex change 30s, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, 35 circulations;
Stop extending: 72 ℃, 10min;
Preserve reaction product for 4 ℃;
3. analyze the PCR product under the non-sex change condition, to detect the lactobacillus spp microorganism belonging to genus, the DHPLC testing conditions is following:
Chromatographic column: PS-DVB & C18 DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 50 ℃;
Moving phase (volume ratio): 0min, 55.0% buffered soln A, 45.0% buffered soln B,
0.5min, 50.2% buffered soln A, 49.8% buffered soln B,
2.4min, 45.7% buffered soln A, 54.3% buffered soln B,
4.3min, 42.9% buffered soln A, 57.1% buffered soln B,
6.1min, 40.9% buffered soln A, 59.1% buffered soln B,
8.0min, 39.4% buffered soln A, 60.6% buffered soln B,
Wherein, buffered soln A is that 50ml TEAA and 250 μ l acetonitriles mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution; Buffered soln B is that 50ml TEAA and 250ml acetonitrile mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluoroscopic examination, light source: 150W Xenon lamp; PLE bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR product 5 μ L;
4. analyze the PCR product under the partially denaturing condition, to the branch kind detection of 8 kinds of lactobacillus spps, the DHPLC testing conditions is following:
Chromatographic column: PS-DVB & C18 DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 59 ℃;
Moving phase (volume ratio): 0.0min, 50.3% buffered soln A, 49.7% buffered soln B,
0.5min, 45.3% buffered soln A, 54.7% buffered soln B,
5.0min, 36.3% buffered soln A, 63.7% buffered soln B,
Wherein, buffered soln A is that 50ml TEAA and 250 μ l acetonitriles mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution; Buffered soln B is that 50ml TEAA and 250ml acetonitrile mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluoroscopic examination, light source: 150W Xenon lamp; PLE bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR product 5 μ L.
Use the test kit and the method for above-mentioned foundation, simultaneously 8 kinds of lactobacillus spps such as lactobicillus bulgaricus, lactobacillus lactis bacteria, plant lactobacillus, lactobacterium casei, lactobacillus fermentum, form probiotic lactobacillus, short lactobacillus and cheese probiotic lactobacillus of augmentation detection lactobacillus genus.PCR product applying step 50 ℃ of non-sex change conditions are 3. analyzed, and detect collection of illustrative plates shown in accompanying drawing 1.A single elution peak all appears in 8 kinds of lactobacillus spps at the title product place, baseline is smooth, and the residence time is consistent, has height identity.
Use the test kit and the method for above-mentioned foundation, simultaneously 8 kinds of lactobacillus spps such as the lactobacillus plantarum of augmentation detection lactobacillus genus, lactobacillus johnsonii, lactobacillus delbruockii subspecies bulgaricus, lactobacillus lactis bacteria, lactobacillus fermentum, form probiotic lactobacillus, short lactobacillus, cheese probiotic lactobacillus.PCR product applying step 59 ℃ of partially denaturing conditions are 4. analyzed, and detect collection of illustrative plates shown in accompanying drawing 2～9.8 kinds of bacterium are under the partially denaturing condition; Unwind to some extent from differential gene site separately; Along with the segmental territory difference of unwinding; Three ethyl and the alkyl generation hydrophobic interaction power on stationary phase C18 surface of nucleotide fragments in the TEAA molecule attracts each other separately; Gradient elution through the acetonitrile in the moving phase reaches the separation with 8 kinds of lactobacillus spp nucleotide fragments molecules of different nature, thereby has occurred specific somatotype collection of illustrative plates separately, can clearly from detecting on the collection of illustrative plates 8 kinds of lactobacillus spps be made a distinction.
Get reference strain dna profiling storehouse listed in the table 1, test kit and the method for using embodiment 1 to be set up detect somatotype.Test-results shows to have only lactobacillus spp positive absorption peak to occur, and other kinds microorganism belonging to genus does not all have positive absorption peak and occurs.
The above results shows that test kit that embodiment 1 is set up and analytical procedure identify that to lactobacillus spp very high specificity is arranged, and avoids the disturbance reponse of other kind bacterium.
For the fragment that confirms that this positive absorption peak is the lactobacillus spp specific gene; Clone after the present invention reclaims the positive absorption peak product of DHPLC and check order; The dna sequence dna comparison of result and lactobacillus spp has proved that the positive absorption peak of DHPLC is a lactobacillus spp specific gene fragment.
(1) 10 times of ratio of two term dilutions of lactobacillus spp reference culture pure growth, corresponding gradient culture extracts template DNA according to the method that embodiment 1 is set up respectively.The method of using embodiment 1 to be set up is carried out sensitivity test, and test-results is seen shown in the accompanying drawing 10.DHPLC peak type is followed successively by to low from height: 2 * 10
3The lactobacillus spp of CFU/mL; 1.5 * 10
2The lactobacillus spp of CFU/mL; 1 * 10
1The lactobacillus spp of CFU/mL.Test-results shows that the method detectability can reach about 0.85pg/ul lactobacillus spp DNA, detects lower limit and approximately can detect 150 lactobacillus spps.
(2) with the comparison of additive method
The present invention adopts PCR-gel electrophoresis, real-time fluorescence PCR method, milk-acid bacteria detection method to carry out the comparison that sensitivity detects to the lactobacillus spp enrichment liquid of different gradients simultaneously, and several kinds of detection method results relatively are shown in following table (table 2).
Table 2, three kinds of different methods detection sensitivity results' comparison
Visible from above-mentioned comparative result: PCR-DHPLC method of the present invention detects the highly sensitive of lactobacillus spp.
With the positive strain isolated of the lactobacillus spp that isolation identification also kept from the actual detected sample in the past, adopt test kit and the method for embodiment 1 to verify, with the accuracy of comparison method.Get the DNA of the 7 strain lactobacillus spp strain isolateds of from sample, isolating and retaining; Carry out the proof test of PCR-DHPLC; This 7 strain lactobacillus spp strain isolated has all detected the amplification absorption peak through DHPLC as a result; And appearance time and peak type circulation ratio are better, and be then negative with the detected result of the negative contrast of intestinal bacteria.
The result shows: test kit of the present invention and PCR-DHPLC detect the method for lactobacillus spp, have good accordance and circulation ratio as a result with the cultivation biochemical identification method of classics.
Test kit and the method for embodiment 1 are used for actual survey work; And with existing other detection methods-" milk-acid bacteria method of inspection part 1 in the import and export food: separate and method of counting " (SN/T1941.1-2007); " the milk-acid bacteria method of inspection the 3rd part in the import and export food: the PCR method of lactobacillus spp " (SN/T 1941.3-2007); Compare checking practicality and safety.During 7 months of year June in December, 2007 to 2008, adopt test kit and the PCR-DHPLC method rapid screening of embodiment 1, adopt the culture identification method of inspection and quarantine industry standard and PCR method to verify comparison simultaneously.Detect actual sample, detect 5 big types of 1321 kinds of samples altogether, the goods such as beer, sour milk of re-detection.The result shows, with 34 parts of positive sample that the screening of PCR-DHPLC method detects, adopts classical biochemical method to verify that it is positive to confirm that all the PCR-DHPLC method detects.The result is as shown in table 3:
Table 3, three kinds of comparative results that method validation detects
SEQUENCE?LISTING
< 110>Zheng, Qiu Yue
< 120>a lactobacillus spp detection kit and branch kind of a detection method
<130>N/A
<160>2
<170>PatentIn?version?3.3
<210>1
<211>20
<212>DNA
< 213>artificial sequence
<400>1
gggttgtctg?cgaaagcgaa 20
<210>2
<211>20
<212>DNA
< 213>artificial sequence
<400>2
gtcttcgtgc?tgcgagtttg 20
Claims (2)
1. lactobacillus spp detection kit, comprising: concentration is Taq archaeal dna polymerase and the PCR reaction solution of 5U/ μ L; Wherein contain 10mM TrisHCl, 50mM KCl, 25mM MgCl in the PCR reaction solution
2, each 2.5mM of dNTP and the general detection primer of lactobacillus genus be to 0.2mM, described primer to sequence is:
Upstream primer SEQ ID NO.1:5 '-GGGTTGTCTGCGAAAGCGAA-3 '
Downstream primer SEQ ID NO.2:5 '-GTCTTCGTGCTGCGAGTTTG-3 ' this test kit-20 ℃ preservation.
2. the branch kind detection method of the lactobacillus spp of non-medical purpose is characterized in that using the described test kit of claim 1, comprises the steps:
1. get 2ul testing sample dna solution, add PCR damping fluid and 0.5 μ LTaq archaeal dna polymerase in the 12ul test kit, sterilization ultrapure water 35.5 μ L, TV 50 μ L; The centrifugal 10s of 5000r/min, carry out pcr amplification by following parameter then:
Sex change in advance: 93 ℃, 5min;
Get into circulation: 94 ℃ of sex change 30s, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, 35 circulations;
Stop extending: 72 ℃, 10min;
Preserve reaction product for 4 ℃;
2. analyze the PCR product under the non-sex change condition, to detect the lactobacillus spp microorganism belonging to genus, the DHPLC testing conditions is following:
Chromatographic column: PS-DVB&C18DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 50 ℃;
Moving phase, in volume ratio: 0min, 55.0% buffered soln A, 45.0% buffered soln B,
0.5min, 50.2% buffered soln A, 49.8% buffered soln B,
2.4min, 45.7% buffered soln A, 54.3% buffered soln B,
4.3min, 42.9% buffered soln A, 57.1% buffered soln B,
6.1min, 40.9% buffered soln A, 59.1% buffered soln B,
8.0min, 39.4% buffered soln A, 60.6% buffered soln B,
Wherein, buffered soln A is that 50ml TEAA and 250 μ l acetonitriles mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution; Buffered soln B is that 50ml TEAA and 250ml acetonitrile mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluoroscopic examination, light source: 150WXenon lamp; PLE bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR product 5 μ L;
3. analyze the PCR product under the partially denaturing condition, to the branch kind detection of 8 kinds of lactobacillus spps, the DHPLC testing conditions is following:
Chromatographic column: PS-DVB&C18DNASep chromatographic column, 4.6mm * 50mm, granularity 3 μ m;
Column temperature: 59 ℃;
Moving phase, in volume ratio: 0.0min, 50.3% buffered soln A, 49.7% buffered soln B,
0.5min, 45.3% buffered soln A, 54.7% buffered soln B,
5.0min, 36.3% buffered soln A, 63.7% buffered soln B,
Wherein, buffered soln A is that 50ml TEAA and 250 μ l acetonitriles mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution; Buffered soln B is that 50ml TEAA and 250ml acetonitrile mix, and adds the sterilization ultrapure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluoroscopic examination, light source: 150W Xenon lamp; PLE bandwidth: 15nm; Emission spectrum bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR product 5 μ L.
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| US12084725B2 (en) * | 2021-01-13 | 2024-09-10 | Bened Biomedical Co., Ltd. | Oligonucleotides for detecting Lactobacillus and method for detecting Lactobacillus by using same |
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