CN102994644B - Lactobacillus plantarum quantitative detection method and detection kit and application thereof - Google Patents
Lactobacillus plantarum quantitative detection method and detection kit and application thereof Download PDFInfo
- Publication number
- CN102994644B CN102994644B CN201210594264.5A CN201210594264A CN102994644B CN 102994644 B CN102994644 B CN 102994644B CN 201210594264 A CN201210594264 A CN 201210594264A CN 102994644 B CN102994644 B CN 102994644B
- Authority
- CN
- China
- Prior art keywords
- plant lactobacillus
- lactobacillus
- quantitative
- primer
- lactobacillus plantarum
- 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
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a lactobacillus plantarum quantitative detection method and a detection kit and application thereof. The lactobacillus plantarum quantitative detection method includes: (1) total ribonucleic acid (RNA) of a sample to be detected is extracted; (2) the extracted total RNA is reversely transcribed into complementary deoxyribonucleic acid (cDNA); (3) primer pairs of specific amplification lactobacillus plantarum are adopted, the obtained cDNA is used as a template to perform fluorescence quantitative polymerase chain reaction (PCR) amplification, an upstream primer sequence is shown as SEQ ID No:1, and a downstream primer sequence is shown as SEQ ID No:2; and (4) a Ct value of a sample to be detected and a standard curve of quantitative outer standard substance are compared to measure quantity of the lactobacillus plantarum. The lactobacillus plantarum quantitative detection method can effectively remove interference of other bacteria, especially closely related bacteria, and quantity of the live lactobacillus plantarum in the sample is detected. The lactobacillus plantarum quantitative detection kit is simple and convenient to use and high in detection efficiency.
Description
Technical field
The invention belongs to bioengineering field, particularly relate to a kind of plant lactobacillus quantitative detecting method, described plant lactobacillus quantitative detecting method is quantitative fluorescence PCR method, a kind of plant lactobacillus immue quantitative detection reagent box and application thereof.
Background technology
Plant lactobacillus (Lactobacillus plantarum) is a kind of in lactobacillus, in close relations with the mankind's life, is often present in the vegetables and fruit juice of fermentation.Plant lactobacillus, as the beneficial flora of human gastrointestinal tract, has colony balance in the enteron aisle of maintaining, improves immunity of organisms and promote the several functions such as absorption of nutrient ingredients, thereby as probiotic bacterium, added in functional foodstuff and healthcare products widely.Large quantity research shows to only have the viable count of probiotic bacterium in product to reach 1 * 10
6individual/ML can bring into play its due effect, thereby probiotics viable bacteria quantity is to evaluate the important indicator of probiotic composition quality.Yet at present to the qualitative and quantitative analysis aspect of the plant lactobacillus in many bacterium composite prod lack science, rationally, method fast.Tradition Physiology and biochemistry method is subject to select the factor impacts such as substratum, culture condition, strain properties, and detection efficiency is limited, and the time consuming time is long, generally takes 2-3 days; The 16S rDNA sequence homology analysis of usining has been widely used in the classification evaluation of milk-acid bacteria as BACTERIAL PHYLOGENY and sibship research, but because plant lactobacillus and Lactobacillus pentosus, secondary plant lactobacillus etc. have 99% similarity, utilize the method to identify and belong to and cannot be accurate to kind.
Be accompanied by the fast development of high throughput sequencing technologies, the Genome Atlas of a large amount of lactic bacterium strains is resolved.These genomic datas can make us understand genetic construction and the feature of different probiotic bacteriums, find out the proprietary feature of different strains, thereby for differentiation and the evaluation of milk-acid bacteria of the same race are not laid a good foundation.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is subject to the factor impacts such as substratum, culture condition, strain properties for the fungal counting method of existing plant lactobacillus (Lactobacillus plantarum) exactly, and it is long to detect the time consuming time, the defect that detection efficiency is lower, provides a kind of plant lactobacillus quantitative detecting method and detection kit and application.Plant lactobacillus quantitative detecting method of the present invention and detection kit thereof are highly sensitive, high specificity, and good stability, simple to operate, fast, can greatly improve the detector efficiency of Bacterium lacticum quantity in milk-product.
For solving the problems of the technologies described above, one of technical scheme that the present invention takes is: a kind of plant lactobacillus (Lactobacillus plantarum) quantitative detecting method, and wherein said method comprises the following steps:
(1) extract total RNA of sample to be checked;
(2) total RNA reverse transcription of step (1) being extracted becomes cDNA;
(3) adopt the primer pair of specific amplification plant lactobacillus, step (2) the gained cDNA of take is template, carry out fluorescent quantitative PCR detection, the primer pair of wherein said specific amplification plant lactobacillus is: upstream detection primer, and its sequence is as shown in SEQ ID No:1 in sequence table; Detected downstream primer, its sequence is as shown in SEQ ID No:2 in sequence table;
(4) by the quantity of plant lactobacillus in the Ct value of sample more to be checked and the standard curve determination sample of quantitative outer standard substance.
Plant lactobacillus of the present invention (Lactobacillus plantarum) also can be abbreviated as plant lactobacillus (L.plantarum), and all the other Bacterium lacticum naming rules are identical with it.
Wherein the method for the total RNA of the described extraction testing sample of step (1) is the method for extracting total RNA of this area routine.The extraction of described total RNA is preferably carried out for the RNA by the various commercializations in this area extracts test kit.
Wherein to become the method for cDNA be the reverse transcription method of this area routine to the described RNA reverse transcription of step (2).Described reverse transcription is preferably for utilizing various commercial reverse transcription test kits to carry out.
Wherein the primer pair of the described specific amplification plant lactobacillus of step (3) comprises upstream sequencing primer and downstream sequencing primer, wherein:
Upstream sequencing primer: 5 '-GGAGCCGCTATTAGTATTTTCAT-3 ', its sequence is as shown in SEQ ID No:1 in sequence table;
Detected downstream primer: 5 '-AATACAAGCAAGTCTTGGACCAG-3 ', its sequence is as shown in SEQ ID No:2 in sequence table.The preparation method of described primer is the conventional preparation method in this area, preferably for by synthetic approach and get final product, can Delegation Server business carries out complete sequence and synthesizes and get final product.
Wherein the described fluorescent quantitative PCR of step (3) is the fluorescent quantitative PCR technology of this area routine.Described quantitative fluorescent PCR program is preferably: (1) 94 ℃～95 ℃, and 25～30 seconds; (2) 94 ℃～95 ℃, 5～15 seconds, (3) 60 ℃, 20～30 seconds, wherein 30～45 circulations were repeated in step (2)～(3), are more preferably: (1) 95 ℃ of 30S; (2) 95 ℃ of 5S, (3) 60 ℃ of 25S, 40 circulations are repeated in step (2)～(3); 4 ℃ of preservations.
Wherein the described plant lactobacillus of step (3) (Lactobacillus plantarum) is the plant lactobacillus of this area routine.Plant lactobacillus of the present invention is preferably selected from one or more in plant lactobacillus (L.plantarum) ST-III, plant lactobacillus (L.plantarum) WCFS1, plant lactobacillus (L.plantarum) P8 and plant lactobacillus (L.plantarum) CCTCC No.M206033.
Total cDNA of gained after total RNA reverse transcription that wherein the described quantitative outer standard substance of step (4) are preferably plant lactobacillus (L.plantarum) ATCC14917, wherein said typical curve is preferably outer standard substance is adopted to 10 times of gradient water dilutions, the outer standard substance of usining after dilution carry out quantitative PCR reaction as substrate and obtain Ct value, according to the copy number of standard substance after dilution, draw and obtain with its corresponding Ct value.
The fluorescent signal that fluorescence dye produces can be detected by the fluorescent signal acquisition system in quantitative real time PCR Instrument, the accumulation volume relation in direct ratio of the increase of fluorescence volume and PCR product.To plant lactobacillus quantitatively can by the cycle threshold with plasmid standards for quantitation (Ct, Threshold Cycle), comparing draws.Ct value is in PCR process, and the accumulation of fluorescence volume surpasses the cycle number of substrate fluorescence volume.Ct value and starting template are counted proportion relation, and Ct value is less, and starting template number is more; On the contrary, Ct value is larger, and starting template number is fewer.Therefore, utilize the Ct value of gradient dilution standard substance to make typical curve, then according to the Ct value of testing sample, can accurately measure the initial copy number of this sample.
For solving the problems of the technologies described above, two of the technical scheme that the present invention takes is: a kind of primer pair of specific amplification plant lactobacillus, and the sequence of the upstream detection primer in described primer pair is as shown in SEQ ID No:1 in sequence table; Wherein the sequence of detected downstream primer is as shown in SEQ ID No:2 in sequence table.
The sequence of the primer pair of wherein said specific amplification plant lactobacillus is respectively:
Upstream sequencing primer: 5 '-GGAGCCGCTATTAGTATTTTCAT-3 ', its sequence is as shown in SEQ ID No:1 in sequence table;
Detected downstream primer: 5 '-AATACAAGCAAGTCTTGGACCAG-3 ', its sequence is as shown in SEQ ID No:2 in sequence table.The preparation method of described primer is the conventional preparation method in this area, preferably for by synthetic approach and get final product, can Delegation Server business carries out complete sequence and synthesizes and get final product.
For solving the problems of the technologies described above, three of the technical scheme that the present invention takes is: a kind of plant lactobacillus immue quantitative detection reagent box, described immue quantitative detection reagent box comprises: SYBR Green I fluorescence dye, PCR damping fluid, dNTPs solution, archaeal dna polymerase, aseptic double-distilled water, upstream detection primer and detected downstream primer, the sequence of wherein said upstream detection primer is as shown in SEQ ID No:1 in sequence table, and the sequence of described detected downstream primer is as shown in SEQ ID No:2 in sequence table.
Wherein said SYBR Green I fluorescence dye, PCR damping fluid, dNTPs solution, archaeal dna polymerase, aseptic double-distilled water are this area normal experiment reagent, all commercially available obtaining.
Wherein said plant lactobacillus immue quantitative detection reagent box, preferably also comprises quantitatively outer standard substance.Described quantitatively outer standard substance are outer standard substance of this area routine, are preferably the total cDNA of gained after total RNA reverse transcription of plant lactobacillus ATCC14917.
Wherein said plant lactobacillus immue quantitative detection reagent box, preferably also comprises that RNA extracts reagent and/or reverse transcription reagent.Described RNA extracts reagent: Trizol, RNA enzyme inhibitors.Wherein said reverse transcription reagent is preferably: reversed transcriptive enzyme (AMV), AMV damping fluid.Wherein said AMV damping fluid preferably comprises: dNTPs, random primer, RNA enzyme inhibitors and aseptic double-distilled water.Wherein said RNA extraction reagent and/or reverse transcription reagent is commercially available obtaining all.
For solving the problems of the technologies described above, four of the technical scheme that the present invention takes is: described plant lactobacillus immue quantitative detection reagent box is the application in plant lactobacillus quantity in detecting sample.
Application of the present invention is preferably: utilize described plant lactobacillus immue quantitative detection reagent box to extract total RNA of plant lactobacillus (L.plantarum) ATCC14917, ultraviolet spectrophotometer is measured after absorbance (260nm), reverse transcription obtains cDNA, according to Genome Size, calculate copy number, dilute, the multiple of described dilution is preferably 10 times of gradient dilutions, usings to dilute rear gained cDNA and carry out real-time fluorescence quantitative PCR reaction as template.The logarithm of positive template quantity of different copy numbers of take is X-coordinate, the initial cycle number (Ct value) that arrives fluorescence threshold in real-time fluorescence quantitative PCR reaction process of take obtains plant lactobacillus (L.plantarum) ATCC14917RT-PCR typical curve as ordinate zou, and obtains regression equation.The total cDNA of lactobacillus in testing sample of take is template, utilize upstream detection primer and detected downstream primer in described test kit, with identical system, carry out the fluorescent quantitative PCR of plant lactobacillus characteristic fragment, record Ct value, bring the Ct value of testing sample into aforesaid standards curve, draw the quantity of plant lactobacillus in sample.
The raw material that the present invention is used or reagent except special instruction, equal commercially available obtaining.
Than prior art, beneficial effect of the present invention is as follows:
1, fast, the high specificity of detection speed
Common biochemical identification for microorganism generally needs 2～3 days, and plant lactobacillus quantitative detecting method of the present invention evaluation only needs 2～3 hours; And can distinguish the nearly edge lactobacillus that common biochemical identification method cannot be distinguished.PCR primer pair of the present invention is according to the specificity marker fragment design of the CAAX family protein enzyme gene of coded plant lactobacterium strain, this primer pair can amplify corresponding DNA fragmentation in plant lactobacillus, and specific amplification plant lactobacillus gene only, and can in other bacterium, not amplify respective segments, comprise the nearly edge bacterium of plant lactobacillus Lactobacillus pentosus, secondary plant lactobacillus etc.Therefore, take the PCR reactive system that this gene fragment is basic design has good microorganism kind specificity.
2, detection sensitivity is high, cost is low
Plant lactobacillus quantitative detecting method of the present invention is highly sensitive, can be by the template amplification initial to be measured of pieck stage to microgram level: even if only contain 3 target bacteria in testing sample, also can detect.Common biochemical identification method detection reagent used is of a great variety, and step is complicated, and the indentifying substance kind that plant lactobacillus quantitative detecting method of the present invention is used is few, simple to operate, and cost is lower.In addition, compare with the authentication method by genome sequencing, described plant lactobacillus quantitative detecting method expense is lower, also shorten dramatically experimental period, conveniently drops into practical application.
In sum, advantage of the present invention is, the detection primer that described plant lactobacillus quantitative detecting method and detection kit thereof adopt only increases to the characteristic fragment of plant lactobacillus, thereby has effectively got rid of the particularly interference of nearly edge bacterium to detection of other bacteriums; The plant lactobacillus that the RNA of take can effectively get rid of dead in sample as basic fluorescence quantifying PCR method simultaneously pollutes, and only detects the plant lactobacillus quantity of surviving in sample.
Accompanying drawing explanation
Fig. 1 is the primer specificity the result figure of plant lactobacillus.Wherein 1. plant lactobacilluss (L.plantarum) ATCC14917 is as positive control; 2.H
2o makes negative control; 3. plant lactobacillus (L.plantarum) ST-III; 4. plant lactobacillus (L.plantarum) WCFS1; 5. plant lactobacillus (L.plantarum) P8; 6. plant lactobacillus (L.plantarum) CCTCC No.M206033; 7. Lactobacillus pentosus (Lactobacillus pentosus) ATCC8041; 8. secondary plant lactobacillus (L.paraplantarum) ATCC700211; 9. lactobacterium casei (L.casei) ATCC334; 10. Lactobacterium acidophilum (L.acidophilus) ATCC4356; 11. lactobacillus fermentums (L.fermentum) 1.1880; M is 500bp Marker.
Fig. 2 is plant lactobacillus (L.plantarum) ATCC14917RT-PCR typical curve.
Fig. 3 is the dependency schematic diagram that real time fluorescence quantifying PCR method and dull and stereotyped number scale are measured plant lactobacillus (L.plantarum) ATCC14917 quantity.
Embodiment
Plant lactobacillus (Lactobacillus plantarum) ATCC14917 is purchased from U.S. ATCC;
Lactobacillus pentosus (Lactobacillus pentosus) ATCC8041, purchased from U.S. ATCC;
Secondary plant lactobacillus (L.paraplantarum) ATCC700211, purchased from U.S. ATCC;
Lactobacterium casei (L.casei) ATCC334, purchased from U.S. ATCC;
Lactobacterium acidophilum (L.acidophilus) ATCC4356, purchased from U.S. ATCC;
Lactobacillus fermentum (L.fermentum) CGMCC1.1880, purchased from Chinese common micro-organisms preservation administrative center;
Plant lactobacillus (L.plantarum) ST-III, bright milk industry, patent publication No.: CN1467290;
Plant lactobacillus (L.plantarum) WCFS1, Wageningen, Netherlands NIZO Food Research Inst. (refers to: Complete genome sequence of Lactobacillus plantarum WCFS1.Kleerebezem M, Boekhorst J, van Kranenburg R, et.al Proc Natl Acad Sci U S A.2003,18:1990-1995);
Plant lactobacillus (L.plantarum) P8， Agricultural University of the Inner Mongol, number of patent application: CN201210062159.7;
Plant lactobacillus (L.plantarum) CCTCC No.M206033, Southern Yangtze University, patent publication No.: CN1928071.
The design of embodiment 1 Auele Specific Primer
Gained plant lactobacillus specific detection primer of the present invention is as follows:
Upstream detection primer: 5 '-GGAGCCGCTATTAGTATTTTCAT-3 ';
Detected downstream primer: 5 '-AATACAAGCAAGTCTTGGACCAG-3 ';
Wherein upstream detection primer sequence is as shown in SEQ ID No:1 in sequence table, and detected downstream primer sequence is as shown in SEQ ID No:2 in sequence table, and described primer is synthetic by Shanghai Sheng Gong biotechnology company limited.
Use the cDNA of different strains as template, utilize above-mentioned primer to carry out pcr amplification, select 30 lactobacillus plantarum strains and 50 strain non-plant lactobacillus strainss (being mainly its nearly edge bacterium) to carry out primer specificity checking, part of test results as shown in Figure 1.
Experimental result shows that primer provided by the present invention only can amplify plant lactobacillus, and to other nearly edge bacterial strains particularly Lactobacillus pentosus and secondary plant lactobacillus etc. all without corresponding amplification.
The Quantitative detection of plant lactobacillus in embodiment 2 self-control milk-product
A. make lactobacillus yogurt product by oneself
Plant lactobacillus ATCC14917 bacterial strain is activated in test tube, and it is that 12% fat cow's milk (adding 1% peptone) is cultivated that 2% inoculum size is inoculated in 100mL massfraction.Cultivate 6～8 hours to curdled milk for 37 ℃, finish fermentation, put refrigerator standby.
B. total RNA preparation of plant lactobacillus milk-product
Adopt Trizol method to extract: the sample of getting respectively seven different times of above-mentioned Yoghourt (being respectively 1,3,5,7,9,11,13 day apart from the fermentation ends date), being placed in the mortar that fills liquid nitrogen grinds, then add rapidly 1mL Trizol reagent, press Trizol test kit specification sheets (
plus RNA Purification System, U.S. invitrogen company, article No. 12183-555) extract sample RNA, then with DNaseI, process twice, the DNA that guarantees to eliminate completely in RNA pollutes, obtain the RNA sample of purifying, after detectable level, above-mentioned RNA sample integrity detects by 1% agarose gel electrophoresis.
C. post transcription cloning
According to reverse transcription test kit (M-MLV RTase cDNA Synthesis Kit, Dalian precious biotechnology company limited, article No. D6130) specification sheets, carry out above-mentioned RNA sample post transcription cloning, obtain cDNA template.
D. fluorescence quantifying PCR method
Get embodiment 1 and prepare gained plant lactobacillus species specificity detection primer, its sequence is as described below:
Upstream primer 5 '-GGAGCCGCTATTAGTATTTTCAT-3 ' (as shown in SEQ ID No:1 in sequence table);
Downstream primer 5 '-AATACAAGCAAGTCTTGGACCAG-3 ' (as shown in SEQ ID No:2 in sequence table), described primer amplification fragment length is 107bp.
Quantitative fluorescent PCR according to
primeScript
tMrT-PCR Kit(Dalian precious biotechnology company limited, article No. DRR063A) specification sheets carries out.Reaction system is 25.0 μ L, and each reactant final concentration is: 1 *
premix Ex Taq
tM, 0.2 μ mol/L upstream and downstream primer (SEQ ID No:1, SEQ ID No:2), the cDNA template of 1.0ng/ μ L, uses ddH
2o supplies 25.0 μ L.
Quantitative fluorescent PCR response procedures is: (1) 95 ℃ of 30S; (2) 95 ℃ of 5S, (3) 60 ℃ of 25S, 40 circulations are repeated in step (2)-(3); 4 ℃ of preservations.
E. the quantitatively preparation of outer standard substance and the drafting of typical curve
According to method described in step b, extract total RNA of plant lactobacillus ATCC14917, through ultraviolet spectrophotometer, measure absorbance (260nm), measuring total rna concentration is 920ng/ μ L, and according to the Genome Size of plant lactobacillus ATCC14917, calculating copy number is 2.26 * 10
8cFU/mL.By method reverse transcription described in step c, obtain cDNA, do 10 times of serial dilutions, the cDNA masterplate after diluting with this carries out quantitative fluorescent PCR reaction described in steps d.The logarithm of positive template of different copy numbers of take is X-coordinate, the initial cycle number (Ct value) that arrives fluorescence threshold in quantitative fluorescent PCR reaction process of take obtains the RT-PCR typical curve of plant lactobacillus ATCC14917 as ordinate zou, its regression equation is Y=-3.36lgX+40.67, R
2=0.996, as shown in Figure 2.
Wherein the minimum concentration of plant lactobacillus ATCC14917 dilution is 2.26 * 10
2cFU/mL, lower than this concentration, fluorescent signal is mixed and disorderly, therefore under the dilution of plant lactobacillus quantitative detecting method of the present invention, is limited to 2.26 * 10
2cFU/mL.
F. plant lactobacillus quantity detected result
Utilize method described in step c to prepare the cDNA template of testing sample, utilize specific detection primer described in steps d, with identical method, carry out the fluorescent quantitative PCR of plant lactobacillus characteristic fragment, detect the Ct value of testing sample.According to the Ct value of regression equation: Y=-3.36lgX+40.67 and testing sample, can calculate the quantity of plant lactobacillus ATCC14917 in sample, detected result is as shown in table 1:
The quantitative result of plant lactobacillus ATCC14917 content in table 1 product
Above-mentioned sample is detected to the quantity of plant lactobacillus ATCC14917 by colony counting method, its result is as shown in table 1, and (described plate count method please refer to: Zhao Jianxin, field Fenghua, Chen Wei, Deng. the research [J] of Lactobacterium acidophilum and plant lactobacillus detection method in Yoghourt. China Dairy Industry, 2005,33 (2): 54-57.).
The plant lactobacillus quantity that the plant lactobacillus quantity that fluorescence quantitative PCR method is recorded is measured with colony counting method is analyzed known: two kinds of method detected results close (± 10%), within the scope of the nominal error of plate count, dependency demonstration is obvious positive correlation (R
2=0.994, regression equation is: Y=0.95X+0.45), its result as shown in Figure 3.Two kinds of bacterial count detection methods are compared, and adopting conventional plate count method consuming time is 2～3 days, and adopts quantitative detecting method of the present invention only to need time half a day, thereby has greatly saved time cost.
The quantitative detecting method of plant lactobacillus ST-III in the commercially available probiotic bacteria milk product of embodiment 3
A. the preparation of the total RNA of sample
Using what buy on market, contain plant lactobacillus ST-III leben (bright smooth excellent plant lactobacillus drink) as implementing sample, be respectively apart from the date manufactured be 10 days, three samples of 15 days and 20 days, being placed in the mortar that fills liquid nitrogen grinds, (health is century Liquid Sample RNA Reagent according to RNA, to extract test kit, article No. CW0534) extract sample RNA, then with DNaseI, process twice, the DNA that guarantees to eliminate completely in RNA pollutes, obtain the RNA sample of purifying, survey concentration, RNA integrity detects by 1% agarose gel electrophoresis.
B. post transcription cloning
According to reverse transcription test kit (HiFi-MMLV cDNA Kit, Kang Wei century bio tech ltd, Beijing, article No. CW0744) specification sheets, carry out above-mentioned RNA sample post transcription cloning, obtain cDNA template.
C. real time fluorescence quantifying PCR method
Get embodiment 1 gained plant lactobacillus species specificity and detect primer, its sequence is as described below:
Upstream primer 5 '-GGAGCCGCTATTAGTATTTTCAT-3 ' (as shown in SEQ ID No:1 in sequence table);
Downstream primer 5 '-AATACAAGCAAGTCTTGGACCAG-3 ' (as shown in SEQ ID No:2 in sequence table), described primer amplification fragment length is 107bp.
Quantitative fluorescent PCR is according to Power
green PCR Master Mix(U.S. invitrogen company, article No. 4367659) specification sheets carries out.Reaction system is 20.0 μ L, and each reactant final concentration is: 1 *
pCR Master mix, 0.25 μ mol/L upstream and downstream primer (SEQ ID No:1, SEQ ID No:2), the cDNA template of 2.0ng/ μ L, uses ddH
2o supplies 20.0 μ L.
Quantitative fluorescent PCR response procedures is: (1) 94 ℃ of 30S; (2) 94 ℃ of 5S, (3) 60 ℃ of 20S, 30 circulations are repeated in step (2)～(3); 4 ℃ of preservations.
D. plant lactobacillus quantity detected result
According to the method for embodiment 2, carry out the quantitatively preparation of outer standard substance and the drafting of typical curve, the cDNA of testing sample and standard substance of take is template, with the species-specific primer of plant lactobacillus, with identical system, carry out the fluorescent quantitative PCR of plant lactobacillus characteristic fragment gene fragment simultaneously.According to the Ct value of regression equation: Y=-3.33lgX+39.56 and testing sample, can calculate the quantity of plant lactobacillus ST-III in sample, detected result is as shown in table 2:
The detected result of plant lactobacillus ST-III content in table 2 sample
| Apart from the date manufactured (my god) | Ct value | Count results (CFU/mL) |
| 10 | 16.37 | 9.20×10 6 |
| 15 | 17.81 | 3.40×10 6 |
| 20 | 19.9 | 8.02×10 5 |
The detection by quantitative of plant lactobacillus in embodiment 4 biased samples
In order to verify whether the method is subject to the interference of other nearly edge bacterium, the present embodiment is measured the wherein bacterium number of plant lactobacillus after single bacterium fermented-milk of three kinds of known cell concentrations is mixed.Three kinds of bacterial strains are respectively: plant lactobacillus CCTCC No.M206033, secondary plant lactobacillus ATCC700211 and Lactobacillus pentosus ATCC8041.Three kinds of single bacterium fermented-milks mix in the ratio of different concns, as shown in table 3:
Each bacterial classification blending ratio of table 3
According to preceding method, measure the wherein quantity of plant lactobacillus, reaction system is 30.0 μ L, and each reactant final concentration is: 1 *
pCR Master mix, 0.3 μ mol/L upstream and downstream primer (as shown in SEQ ID No:1 in sequence table and SEQ ID No:2), the DNA profiling of 2.0ng/ μ L, uses ddH
2o supplies 30.0 μ L.
Quantitative fluorescent PCR response procedures is: (1) 95 ℃, and 25S, (2) 95 ℃, 15S, (3) 60 ℃, 30S, 45 circulations are repeated in step (2)～(3); 4 ℃ of preservations.Fluorescent quantitative PCR result is as shown in table 4:
Table 4 plant lactobacillus recall rate table
| ? | Variable (CFU/mL) | Measured value (CFU/mL) | Recall rate (%) |
| Ratio 1 (%) | 5.31×10 7 | 5.26×10 7 | 0.99 |
| Ratio 2 (%) | 1.45×10 8 | 1.56×10 8 | 1.08 |
| Ratio 3 (%) | 8.05×10 7 | 8.75×10 7 | 1.09 |
| Ratio 4 (%) | 8.05×10 6 | 8.64×10 6 | 1.07 |
As can be seen from Table 4: plant bacillus quantity detects error all in 10%, show that Bacterium lacticum quantitative detecting method of the present invention can get rid of the interference of nearly edge bacterium, effective and feasible.
Should be understood that, after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (9)
1. plant lactobacillus (Lactobacillus plantarum) quantitative detecting method, is characterized in that, described plant lactobacillus quantitative detecting method comprises the following steps:
(1) extract total RNA of sample to be checked;
(2) total RNA reverse transcription of step (1) being extracted becomes cDNA;
(3) adopt the primer pair of specific amplification plant lactobacillus, step (2) the gained cDNA of take is template, carry out fluorescent quantitative PCR detection, the primer pair of wherein said specific amplification plant lactobacillus is: upstream detection primer, and its sequence is as shown in SEQ ID No:1 in sequence table; Detected downstream primer, its sequence is as shown in SEQ ID No:2 in sequence table;
(4) by the quantity of plant lactobacillus in the Ct value of sample more to be checked and the standard curve determination sample of quantitative outer standard substance, wherein said plant lactobacillus is selected from one or more in plant lactobacillus (Lactobacillus plantarum) ST-III, plant lactobacillus (Lactobacillus plantarum) WCFS1, plant lactobacillus (Lactobacillus plantarum) P8 and plant lactobacillus (Lactobacillus plantarum) CCTCC No.M206033.
2. plant lactobacillus quantitative detecting method as claimed in claim 1, is characterized in that, the described fluorescent quantitative PCR program of step (3) is: (1) 94 ℃～95 ℃, and 25～30 seconds; (2) 94 ℃～95 ℃, 5～15 seconds, (3) 60 ℃, 20～30 seconds, wherein 30～45 circulations were repeated in step (2)～(3).
3. plant lactobacillus quantitative detecting method as claimed in claim 1, it is characterized in that, total cDNA of gained after total RNA reverse transcription that the described quantitative outer standard substance of step (4) are plant lactobacillus (Lactobacillus plantarum) ATCC14917, wherein said typical curve is that outer standard substance are adopted to 10 times of gradient dilutions, the outer standard substance of usining after dilution carry out quantitative fluorescent PCR reaction as substrate and obtain Ct value, according to the copy number of standard substance after dilution, draw and obtain with its corresponding Ct value.
4. a primer pair for specific amplification plant lactobacillus, is characterized in that, the sequence of the upstream detection primer in described primer pair is as shown in SEQ ID No:1 in sequence table; Wherein the sequence of detected downstream primer is as shown in SEQ ID No:2 in sequence table.
5. a plant lactobacillus immue quantitative detection reagent box, it is characterized in that, described immue quantitative detection reagent box comprises: SYBR Green I fluorescence dye, PCR damping fluid, dNTPs solution, archaeal dna polymerase, aseptic double-distilled water, upstream detection primer and detected downstream primer, the sequence of wherein said upstream detection primer is as shown in SEQ ID No:1 in sequence table, and the sequence of described detected downstream primer is as shown in SEQ ID No:2 in sequence table.
6. plant lactobacillus immue quantitative detection reagent box as claimed in claim 5, is characterized in that, described plant lactobacillus immue quantitative detection reagent box comprises quantitatively outer standard substance.
7. plant lactobacillus immue quantitative detection reagent box as claimed in claim 6, is characterized in that, the cDNA of gained after total RNA reverse transcription that described quantitatively outer standard substance are plant lactobacillus (Lactobacillus plantarum) ATCC14917.
8. plant lactobacillus immue quantitative detection reagent box as claimed in claim 7, is characterized in that, described plant lactobacillus immue quantitative detection reagent box also comprises that RNA extracts reagent and reverse transcription reagent.
9. the application of the plant lactobacillus immue quantitative detection reagent box as described in claim 5～8 any one in detecting plant lactobacillus quantity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210594264.5A CN102994644B (en) | 2012-12-31 | 2012-12-31 | Lactobacillus plantarum quantitative detection method and detection kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210594264.5A CN102994644B (en) | 2012-12-31 | 2012-12-31 | Lactobacillus plantarum quantitative detection method and detection kit and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102994644A CN102994644A (en) | 2013-03-27 |
| CN102994644B true CN102994644B (en) | 2014-01-22 |
Family
ID=47923749
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210594264.5A Active CN102994644B (en) | 2012-12-31 | 2012-12-31 | Lactobacillus plantarum quantitative detection method and detection kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102994644B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525914B (en) * | 2013-09-23 | 2015-06-17 | 光明乳业股份有限公司 | Method, primer and kit for counting number of live bacteria of lactobacillus plantarum |
| CN105331723B (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 lactobacillus plantarum for being added in feed and application |
| CN110564874B (en) * | 2019-08-08 | 2022-10-14 | 河北省食品检验研究院（国家果类及农副加工产品质量监督检验中心、河北省食品安全实验室） | High-precision specific digital PCR detection method for series of mixed bacteria pollution mainly caused by glucose bacillus in fermented milk |
| CN113957127A (en) * | 2020-07-20 | 2022-01-21 | 中国科学院微生物研究所 | Method for quantitatively detecting plant tissue endophytic bacteria by non-culture method |
| CN112553353B (en) * | 2020-12-11 | 2023-08-25 | 江南大学 | Probes, methods and kits for absolute quantification of lactobacillus and application of probes, methods and kits |
| CN113005182A (en) * | 2021-05-08 | 2021-06-22 | 上海应用技术大学 | Quantitative detection method and quantitative detection kit for radiation-resistant acinetobacter and application of quantitative detection kit |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102453767A (en) * | 2011-12-23 | 2012-05-16 | 光明乳业股份有限公司 | Method and kit for quickly and quantificationally detecting lactobacillus plantarum ST-III |
| CN102703600A (en) * | 2012-07-10 | 2012-10-03 | 光明乳业股份有限公司 | Qualitative and quantitative determination method of lactobacillus plantarum intestinal tract colonization |
-
2012
- 2012-12-31 CN CN201210594264.5A patent/CN102994644B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102453767A (en) * | 2011-12-23 | 2012-05-16 | 光明乳业股份有限公司 | Method and kit for quickly and quantificationally detecting lactobacillus plantarum ST-III |
| CN102703600A (en) * | 2012-07-10 | 2012-10-03 | 光明乳业股份有限公司 | Qualitative and quantitative determination method of lactobacillus plantarum intestinal tract colonization |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102994644A (en) | 2013-03-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102994644B (en) | Lactobacillus plantarum quantitative detection method and detection kit and application thereof | |
| Ben Omar et al. | Microbial community dynamics during production of the Mexican fermented maize dough pozol | |
| Yu et al. | Diversity of lactic acid bacteria associated with traditional fermented dairy products in Mongolia | |
| CN102453767B (en) | Method and kit for quickly and quantificationally detecting lactobacillus plantarum ST-III | |
| CN101613745B (en) | Staphylococcus aureus multiple PCR-MIX rapid detection kit and detection method thereof | |
| CN101712989B (en) | Method for quickly, qualitatively and quantitatively measuring Lactobacillus casei in probiotic dairy products | |
| da Silva Duarte et al. | Comparative evaluation of cheese whey microbial composition from four Italian cheese factories by viable counts and 16S rRNA gene amplicon sequencing | |
| Kim et al. | Microbial diversities and potential hazards of Korean turbid rice wines (makgeolli): Multivariate analyses | |
| Kahve et al. | Identification and technological characterization of endogenous yeast isolated from fermented black carrot juice, shalgam | |
| Yoon et al. | Populations and potential association of Saccharomyces cerevisiae with lactic acid bacteria in naturally fermented Korean rice wine | |
| McEniry et al. | Bacterial community dynamics during the ensilage of wilted grass | |
| CN108048582A (en) | The Pseudomonas fluorescens method of thermophilic protease is produced in a kind of quantitative quick detection raw milk using LAMP primer group | |
| CN101712986A (en) | Method for quickly, qualitatively and quantitatively measuring Lactobacillus acidophilus in probiotic dairy products | |
| Wang et al. | Study on bacterial diversity in traditional sour whey of Yunnan province | |
| Kačániová et al. | Determination of wine microbiota using classical method, polymerase chain method and Step One Real-Time PCR during fermentation process | |
| CN101974641A (en) | Multiple fluorescence quantitative PCR method for simultaneous and fast detecting three types of pathogenic bacteria in food | |
| Mishra et al. | Identification and characterization of yeast strains associated with the fermented rice beverages of Garo Hills, Meghalaya, India | |
| CN101649352B (en) | Quick qualitative and quantitative measuring method of fermented lactobacillus in probiotic milk products | |
| Kim et al. | Development of a rapid and reliable TaqMan probe-based real-time PCR assay for the detection and enumeration of the multifaceted yeast Kluyveromyces marxianus in dairy products | |
| CN101712988B (en) | Method for quickly, qualitatively and quantitatively measuring Bifidobacteria in probiotic dairy products | |
| Ren et al. | Diversity analysis and quantification of lactic acid bacteria in traditionally fermented yaks’ milk products from Tibet | |
| CN116121422A (en) | Primer probe, kit and method for identifying lactobacillus paracasei | |
| CN101712990A (en) | Method for quickly, qualitatively and quantitatively measuring Lactobacillus rhamnosus in probiotic dairy products | |
| CN103981266A (en) | Method for detecting coliforms in dairy products and primer used thereby | |
| CN102433385B (en) | Primers for identifying leuconostoc mesenteroides and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |