CN101812503A - Reagent kit for detecting live bifidobacteriums in dairy products - Google Patents
Reagent kit for detecting live bifidobacteriums in dairy products Download PDFInfo
- Publication number
- CN101812503A CN101812503A CN200910072460A CN200910072460A CN101812503A CN 101812503 A CN101812503 A CN 101812503A CN 200910072460 A CN200910072460 A CN 200910072460A CN 200910072460 A CN200910072460 A CN 200910072460A CN 101812503 A CN101812503 A CN 101812503A
- Authority
- CN
- China
- Prior art keywords
- ema
- sample
- milk
- pcr
- product
- 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.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a reagent kit for detecting live bifidobacteriums in dairy products. The traditional method for detecting live bifidobacteriums in dairy products requires special equipment and has the defects of complex process and low specificity. The reagent kit comprises the following components by volume ratio: 3900 ul of PCR reaction liquid of added specificity primer and molecular beacon probe, 100 ul of Taq DNA polymerase, 300 ul of EMA solution and 300 ul of ROX reference dye. The detection process comprises the following steps: sampling, carrying out EMA treatment on the sample with the EMA solution in the reagent kit, extracting the DNA of the sample, carrying out real-time PCR detection with the reaction liquid in the reagent kit, and calculating the content of bifidobacterium. The invention is used for detecting the content of the live bifidobacterium in the dairy products.
Description
Technical field:
The present invention relates to reagent kit for detecting live bifidobacteriums in a kind of milk-product.
Background technology:
Bifidus bacillus is microorganism useful to body in the mankind and the animal intestinal, and is closely related with the body health situation, thereby is applied to aspects such as medical treatment, health care, food by extensive exploitation.But have only when the bifidobacteria viable bacteria number and reach 10
6When CFU/mL is above, the competence exertion normal physiological function, so the number of viable of bifidus bacillus determines or influences the quality of product.At present, the technology that detects live bifidobacterium bacteria bacterium number mainly is divided into two big classes, and a class is the detection technique based on the classic flat-plate enumeration, and another kind of is detection technique based on molecular biology.Because the bifidus bacillus strictly anaerobic, therefore traditional flat-plate bacterial colony technological method needs special equipment, and process is more loaded down with trivial details; And adopt enzyme linked immunosorbent assay (ELISA), and the F6PPK enzymatic detection techniques, immunoblotting (immuno-blotting), the specificity that methods such as round pcr detect bifidus bacillus is lower.
Summary of the invention:
The purpose of this invention is to provide reagent kit for detecting live bifidobacteriums in a kind of milk-product, can effectively remedy detect in the past bifidobacterium assays process complexity, time than length can't distinguish extremely, the defective of viable bacteria, have quick, sensitive, easy, accurate quantitative characteristics.
Above-mentioned purpose realizes by following technical scheme:
Reagent kit for detecting live bifidobacteriums in the milk-product, its composition comprises: add PCR reaction solution, Taq archaeal dna polymerase, EMA solution, the ROX reference dyestuff of Auele Specific Primer and molecular beacon probe, its volume ratio is: PCR reaction solution 3900ul, the Taq archaeal dna polymerase 100ul, EMA solution 300ul, the ROX reference dyestuff 300ul that add Auele Specific Primer and molecular beacon probe.
Reagent kit for detecting live bifidobacteriums in the described milk-product, described Auele Specific Primer comprises forward primer and reverse primer, described forward primer is Bif-F, length is 19bp, sequence is 5 '-TCTGGCTCAGGATGAACGC-3 ', binding site drops on 20～38bp position of bifidus longum bb 16S rDNA sequence; Described reverse primer is Bif-R, and length is 20bp, sequence is 5 '-CACCGTTACACCGGGAATTC-3 ', binding site drops on 655～674bp position of bifidus longum bb 16S rDNA sequence.
Reagent kit for detecting live bifidobacteriums in the described milk-product, described molecular beacon probe sequence is: 5 '-FAM-CCAGGCATCCGGCATTACC ACCCG TCCTGG-3 '-DABCYL, wherein 5 ' end mark fluorescent group FAM, 6-hydroxyl fluorescein, the fluorescent emission peak value is at the 518nm place, 3 ' end mark quenching group DABCYL, 4-4 '-oxane amino-benzene azobenzoic acid, the fluorescent emission peak value is at the 491nm place.
Reagent kit for detecting live bifidobacteriums in the described milk-product, described PCR reaction solution are to be that 10 μ mol/L, downstream primer concentration are 10 μ mol/L according to upstream primer concentration, and molecular beacon probe concentration is 5 μ mol/L; With upstream primer, downstream primer each 200 μ L, molecular beacon probe 200 μ L with do not contain Mg
2+10 * PCR Buffer250 μ L, Mg
2+250 μ L, dNTP mixture 200 μ L, it is even to join in the centrifuge tube of aseptic brown 1.5mL thorough mixing under aseptic condition, described Taq archaeal dna polymerase concentration is 2.5U/ μ L, described EMA solution, be under the condition of lucifuge, the EMA powder to be dissolved in the DMF solution, final concentration is 1 μ g/ μ L, and described ROX reference dye strength is 0.02mol/L.
The method that live bifidobacterium bacteria detects in a kind of milk-product, at first sampling is after the sampling, the EMA solution of using in the described test kit carries out the EMA processing to sample, extract the DNA of sample, the reaction solution of using again in the test kit carries out the PCR in real time detection, calculates the content of bifidus bacillus.
The method that live bifidobacterium bacteria detects in the described milk-product, described sampling is in aseptic technique the sample that contains the bifidus bacillus goods of abundant mixing to be put into the even diluent that the sterilization Erlenmeyer flask that contains the PBS damping fluid was made 1: 10, and room temperature leaves standstill hydration 30min.
The method that live bifidobacterium bacteria detects in the described milk-product, it is to get the sample diluting liquid that contains the bifidus bacillus goods that mixes that described EMA handles sample, adding concentration is 1 μ g/ μ L EMA solution, abundant mixing, lucifuge reaction 5min adopts the halogen lamp of 650W to shine 2min, and sample is apart from light source 20cm, operate on ice and carry out, each sample do two parallel.
The method that live bifidobacterium bacteria detects in the described milk-product, described DNA extraction process is: get the sample of 1mL after EMA handles, the Trisodium Citrate and 100 μ L 4% (w/v) sodium hydroxide that add 150 μ L 18% (w/v), the centrifugal 2min of 10000r/min, collect somatic cells, add 1mL PBS damping fluid piping and druming precipitation, in the centrifugal 2min of 11500g, abandon supernatant, add 1mL deionized water piping and druming precipitation again, in the centrifugal 2min of 11500g, abandon supernatant, extract test kit according to bacterial genomes again and operate.
The method that live bifidobacterium bacteria detects in the described milk-product, the method that described PCR in real time detects is: get PCR reaction solution 26 μ L, ROX reference dyestuff 2 μ L, Taq archaeal dna polymerase 0.5 μ L, template DNA 5 μ L, adding deionized water mends to 50 μ L, mix, carrying out PCR in real time detects, the annealing temperature of fluorescent signal collection is 40 ℃, and loop parameter is: 94 ℃ * 3min → 94 ℃ * 30s → 40 ℃ * 35s → 59 ℃ * 30s → 72 ℃ * 1min, cycle number is 40.
Beneficial effect of the present invention:
1, the detection kit of EMA-PCR in real time is with primer, molecular beacon probe, Buffer, dNTP, Mg
2+Deng being pre-mixed, can prepare reaction solution when testing fast, detection by quantitative goes out the quantity of live bifidobacterium bacteria then, increases the sensitivity that detects, and shortens detection time.EMA-PCR in real time test kit is 10 to the minimum detectability that contains live bifidobacterium bacteria in the bifidus bacillus goods
4CFU/mL (g); Be 3.5h detection time; Non-bifidus bacillus microorganism belonging to genus does not have influence to the detected result of present method.This test kit can be used for containing the fast quantification detection of bifidus bacillus in the bifidus bacillus goods.
Contain ethidium bromide trinitride (Ethidium bromide monoazide in the detection kit of the bifidus bacillus EMA-PCR in real time that 2, the present invention developed, EMA), it is a kind of fluorescent core acid dye, can combine with the DNA of dead thalline and form covalent compound, the DNA that can suppress dead thalline carries out pcr amplification, thereby this test kit can only detect viable count.
The detection kit good reproducibility of the bifidus bacillus EMA-PCR in real time that 3, the present invention developed, batch in and interassay coefficient of variation all less than 5%; High specificity, amplification curve present tangible S type, no non-specific amplification; Highly sensitive, detect and be limited to 10
4CFU/ml; Linearity range is wide, and the starting template number is 10
8CFU/ml～10
4Have good linear relationship between the CFU/ml, relation conefficient is greater than 98%.
4, compare with traditional enumeration detection method, the detection kit of the EMA-PCR in real time that the present invention developed detects the feasibility of bifidus bacillus.Confirmatory experiment is the result show, the detection kit of the bifidus bacillus EMA-PCR in real time of being set up has sensitivity, special, easy and characteristics fast, can be used for the detection by quantitative to the bifidobacteria viable bacteria number.
5, real time pcr is compared with traditional round pcr, whole process adopts complete stopped pipe to detect, therefore the amplified production contamination of heavy is very low, can be qualitative, quantitative in same instrument, sudden change and multiple assay, and quantitatively scope exceeds 2～3 orders of magnitude than conventional P CR method.
6, owing to saved operation stepss such as traditional electrophoresis, photograph, bromination second pyridine etc. is polluted and harm when having avoided radioactive substance or gel electrophoresis dyeing, and is therefore safe and reliable, and required time is shorter; Detection sensitivity exceeds 10～100 times than conventional P CR method.
7, because real time pcr is to have increased a fluorescent probe on the regular-PCR basis, the variation by special instrument fluorescence intensity in amplification has improved detection sensitivity greatly.Fluorescent probe has played the effect of DNA hybridization probe again simultaneously, is equivalent to hybridize both by PCR and DNA and reacts the specificity that checking detects.And, the entire operation process only needs to uncap once when application of sample, thereafter process is the stopped pipe operation fully, and do not need the PCR product is carried out the dyeing of gel electrophoresis, staining agent, operates after PCR such as observing under the UV-light, significantly reduced the chance of polluting, also avoided the toxic action of staining agent simultaneously human body.
8, EMA-PCR in real time detection by quantitative test kit does not need loaded down with trivial details cultivation enrichment, and operating process is simple, and instrument provides the result automatically, and detection time is short, only needs 3.5h (comprising time for sample pretreatment).In addition, the detection kit of bifidus bacillus EMA-PCR in real time can detect the viable count of bifidus bacillus in the milk-product.
Embodiment:
Embodiment 1:
Reagent kit for detecting live bifidobacteriums in the milk-product, its composition comprises: add PCR reaction solution, Taq archaeal dna polymerase, EMA solution, the ROX reference dyestuff of Auele Specific Primer and molecular beacon probe, its volume ratio is for adding PCR reaction solution 3900ul, Taq archaeal dna polymerase 100ul, EMA solution 300ul, the ROX reference dyestuff 300ul of Auele Specific Primer and molecular beacon probe.
Reagent kit for detecting live bifidobacteriums in the described milk-product, described Auele Specific Primer comprises forward primer and reverse primer, described forward primer is Bif-F, length is 19bp, sequence is 5 '-TCTGGCTCAGGATGAACGC-3 ', binding site drops on 20～38bp position of bifidus longum bb 16S rDNA sequence; Described reverse primer is Bif-R, and length is 20bp, sequence is 5 '-CACCGTTACACCGGGAATTC-3 ', binding site drops on 655～674bp position of bifidus longum bb 16S rDNA sequence.
Reagent kit for detecting live bifidobacteriums in the described milk-product, described molecular beacon probe sequence is: 5 '-FAM-CCAGGCATCCGGCATTACCACCCGTCCTGG-3 '-DABCYL, wherein 5 ' end mark fluorescent group FAM, 6-hydroxyl fluorescein, the fluorescent emission peak value is at the 518nm place, 3 ' end mark quenching group DABCYL, 4-4 '-(oxane amino-benzene azo) phenylformic acid, the fluorescent emission peak value is at the 491nm place.
Reagent kit for detecting live bifidobacteriums in the described milk-product, described PCR reaction solution is to be 10 μ mol/L according to upstream primer concentration for, downstream primer concentration, molecular beacon probe concentration is 5 μ mol/L, and upstream and downstream primer each 200 μ L, molecular beacon probe 200 μ L and 10 * PCR Buffer (are not contained Mg
2+) 250 μ L, Mg
2+250 μ L, dNTP mixture 200 μ L, it is even to join in the centrifuge tube of aseptic brown 1.5mL thorough mixing under aseptic condition, described Taq archaeal dna polymerase concentration is 2.5U/ μ L, described EMA solution, be under the condition of lucifuge, the EMA powder to be dissolved in the DMF solution, final concentration is 1 μ g/ μ L, and described ROX reference dye strength is 0.02mol/L.
Embodiment 2:
The method that live bifidobacterium bacteria detects in the milk-product, its technological process is: after the sampling, the EMA solution of using in the described test kit carries out the EMA processing to sample, extracts the DNA of sample, the reaction solution of using again in the test kit carries out the PCR in real time detection, calculates the content of bifidus bacillus.
The method that live bifidobacterium bacteria detects in the described milk-product, described sampling is in aseptic technique the sample that contains the bifidus bacillus goods of abundant mixing to be put into the even diluent that the sterilization Erlenmeyer flask that contains the PBS damping fluid was made 1: 10, and room temperature leaves standstill hydration 30min.
The method that live bifidobacterium bacteria detects in the described milk-product, it is to get the sample diluting liquid that contains the bifidus bacillus goods that mixes that described EMA handles sample, adding concentration is 1 μ g/ μ L EMA storage liquid, abundant mixing, lucifuge reaction 5min adopts the halogen lamp of 650W to shine 2min, and sample is apart from light source 20cm, operate on ice and carry out, each sample do two parallel.
The method that live bifidobacterium bacteria detects in the described milk-product, described DNA extraction process is: get the sample of 1mL after EMA handles, the Trisodium Citrate and 100 μ L 4% (w/v) sodium hydroxide that add 150 μ L 18% (w/v), the centrifugal 2min of 10000r/min, collect somatic cells, add 1mL PBS damping fluid piping and druming precipitation, in the centrifugal 2min of 11500g, abandon supernatant, add 1mL deionized water piping and druming precipitation again, in the centrifugal 2min of 11500g, abandon supernatant, extract test kit (TIANGEN) according to bacterial genomes again and operate.
The method that live bifidobacterium bacteria detects in the described milk-product, the method that described PCR in real time detects is: get PCR reaction solution 26 μ L, ROX reference dyestuff 2 μ L, Taq archaeal dna polymerase 0.5 μ L, template DNA 5 μ L, adding deionized water mends to 50 μ L, mix, carrying out PCR in real time detects, the annealing temperature of fluorescent signal collection is 40 ℃, and loop parameter is: 94 ℃ * 3min → 94 ℃ * 30s → 40 ℃ * 35s → 59 ℃ * 30s → 72 ℃ * 1min, cycle number is 40.
The principle that real time pcr detects is, added a fluorescently-labeled probe on the basis of regular-PCR technology, and 5 of probe ' one of end mark reports that fluorophor is reporter, R; Near cancellation fluorophor of 3 ' end mark is quencher, Q; During about between the two 7～10nm just FRET can take place.When probe was kept perfectly, report fluorophor fluorescent signal emitted was absorbed by the cancellation fluorophor, and the cancellation fluorophor has suppressed the fluorescent signal of report fluorophor and not luminous, and detection system can not detect the fluorescent signal of report fluorophor.When both distances were far away, restraining effect disappeared, and fluorescent signal strengthens.Fluorescent signal and PCR product increase by on a year-on-year basis, increase with the PCR product strengthens, the ethidium bromide trinitride is a kind of fluorescent core acid dye, can combine with the DNA of dead thalline and form covalent compound, and then the DNA that can suppress dead thalline carries out pcr amplification, when EMA joins in the sample that contains the life or death bacterium, the cytolemma of viable bacteria is complete, after adding EMA, only there is the surface of bacterium in EMA, can not be penetrated in the cell of viable bacteria, shine by halogen light source, after exposure for some time, viable bacteria surface free EMA decomposes or the formation hydroxylamine compound, because the cytolemma of dead thalline destroys, EMA enters into the cell of bacterium, in the process of DNA extraction, the free EMA exposure of viable bacteria surface is decomposed or the formation hydroxylamine compound, not binding of DNA to viable bacteria, and be present in EMA in the dead somatic cells, in leaching process, combine the formation covalent compound with the DNA of dead thalline, the DNA of viable bacteria can carry out pcr amplification, but form covalent compound because EMA combines with the DNA of dead thalline, the DNA of therefore dead thalline can not carry out pcr amplification, drawing standard curve, EMA combined with the method for PCR in real time not only can well distinguish anyway bacterium, and can carry out detection by quantitative.
The foundation of typical curve:
Count logarithmic value (Log with different bacterium
10CFU/mL) be X-coordinate, with the initial cycle number (Ct) that occurs fluorescent signal in the quantitative fluorescent PCR reaction process is ordinate zou, the drawing standard curve, the result shows: the typical curve equation that EMAReal-time PCR detects live bifidobacterium bacteria is: y=-3.96X+47.66, coefficient R
2Value is greater than 0.98, and the concentration of bifidus bacillus nutrient solution is positioned at 10
4CFU/mL～10
8Has good linear relationship between the CFU/mL.
The cubage of the bifidus bacillus in the milk-product sample of examining:
Method of calculation are as follows as a result: according to typical curve y=-3.96X+47.66, y represents the Ct value of PCR in real time detected result.X representative is to be measured to contain the logarithmic value (LogCFU/mL) of the bifidobacteria viable bacteria number in the sample of bifidus bacillus goods.
Claims (9)
1. reagent kit for detecting live bifidobacteriums in the milk-product, its composition comprises: add PCR reaction solution, Taq archaeal dna polymerase, EMA solution, the ROX reference dyestuff of Auele Specific Primer and molecular beacon probe, it is characterized in that: its volume ratio is: PCR reaction solution 3900ul, the Taq archaeal dna polymerase 100ul, EMA solution 300ul, the ROX reference dyestuff 300ul that add Auele Specific Primer and molecular beacon probe.
2. reagent kit for detecting live bifidobacteriums in the milk-product according to claim 1, it is characterized in that: described Auele Specific Primer comprises forward primer and reverse primer, described forward primer is Bif-F, length is 19bp, sequence is 5 '-TCTGGCTCAGGATGAACGC-3 ', binding site drops on 20～38bp position of bifidus longum bb 16SrDNA sequence; Described reverse primer is Bif-R, and length is 20bp, sequence is 5 '-CACCGTTACACCGGGAATTC-3 ', binding site drops on 655～674bp position of bifidus longum bb 16S rDNA sequence.
3. reagent kit for detecting live bifidobacteriums in the milk-product according to claim 1 and 2, it is characterized in that: described molecular beacon probe sequence is: 5 '-FAM-CCAGGCATCCGGCATTACC ACCCGTCCTGG-3 '-DABCYL, wherein 5 ' end mark fluorescent group FAM, 6-hydroxyl fluorescein, the fluorescent emission peak value is at the 518nm place, 3 ' end mark quenching group DABCYL, 4-4 '-oxane amino-benzene azobenzoic acid, the fluorescent emission peak value is at the 491nm place.
4. reagent kit for detecting live bifidobacteriums in the milk-product according to claim 1 and 2, it is characterized in that: described PCR reaction solution is to be that 10 μ mol/L, downstream primer concentration are 10 μ mol/L according to upstream primer concentration, and molecular beacon probe concentration is 5 μ mol/L; With upstream primer, downstream primer each 200 μ L, molecular beacon probe 200 μ L with do not contain Mg
2+10 * PCR Buffer250 μ L, Mg
2+250 μ L, dNTP mixture 200 μ L, it is even to join in the centrifuge tube of aseptic brown 1.5mL thorough mixing under aseptic condition, described Taq archaeal dna polymerase concentration is 2.5U/ μ L, described EMA solution, be under the condition of lucifuge, the EMA powder to be dissolved in the DMF solution, final concentration is 1 μ g/ μ L, and described ROX reference dye strength is 0.02mol/L.
5. the method that live bifidobacterium bacteria detects in the milk-product, at first sampling is characterized in that: after the sampling, the EMA solution of using in the described test kit carries out the EMA processing to sample, extract the DNA of sample, the reaction solution of using again in the test kit carries out the PCR in real time detection, calculates the content of bifidus bacillus.
6. the method that live bifidobacterium bacteria detects in the milk-product according to claim 5, it is characterized in that: described sampling is in aseptic technique the sample that contains the bifidus bacillus goods of abundant mixing to be put into the even diluent that the sterilization Erlenmeyer flask that contains the PBS damping fluid was made 1: 10, and room temperature leaves standstill hydration 30min.
7. the method that detects according to live bifidobacterium bacteria in claim 5 or the 6 described milk-product, it is characterized in that: it is to get the sample diluting liquid that contains the bifidus bacillus goods that mixes that described EMA handles sample, adding concentration is 1 μ g/ μ L EMA solution, abundant mixing, lucifuge reaction 5min adopts the halogen lamp of 650W to shine 2min, and sample is apart from light source 20cm, operate on ice and carry out, each sample do two parallel.
8. the method that detects according to live bifidobacterium bacteria in claim 5 or the 6 described milk-product, it is characterized in that: described DNA extraction process is: get the sample of 1mL after EMA handles, the Trisodium Citrate and 100 μ L 4% (w/v) sodium hydroxide that add 150 μ L 18% (w/v), the centrifugal 2min of 10000r/min, collect somatic cells, add 1mL PBS damping fluid piping and druming precipitation, in the centrifugal 2min of 11500g, abandon supernatant, add 1mL deionized water piping and druming precipitation again, in the centrifugal 2min of 11500g, abandon supernatant, extract test kit according to bacterial genomes again and operate.
9. the method that detects according to live bifidobacterium bacteria in claim 5 or the 6 described milk-product, it is characterized in that: the method that described PCR in real time detects is: get PCR reaction solution 26 μ L, ROX reference dyestuff 2 μ L, Taq archaeal dna polymerase 0.5 μ L, template DNA 5 μ L, adding deionized water mends to 50 μ L, mix, carrying out PCR in real time detects, the annealing temperature of fluorescent signal collection is 40 ℃, loop parameter is: 94 ℃ * 3min → 94 ℃ * 30s → 40 ℃ * 35s → 59 ℃ * 30s → 72 ℃ * 1min, cycle number is 40.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910072460A CN101812503A (en) | 2009-07-03 | 2009-07-03 | Reagent kit for detecting live bifidobacteriums in dairy products |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910072460A CN101812503A (en) | 2009-07-03 | 2009-07-03 | Reagent kit for detecting live bifidobacteriums in dairy products |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101812503A true CN101812503A (en) | 2010-08-25 |
Family
ID=42619870
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910072460A Pending CN101812503A (en) | 2009-07-03 | 2009-07-03 | Reagent kit for detecting live bifidobacteriums in dairy products |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101812503A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102605091A (en) * | 2012-04-09 | 2012-07-25 | 四川省畜牧科学研究院 | EMA-PCR (Ethidium Monoazide-Polymerase Chain Reaction) detection method of viable pathogen in farm environment |
| CN103575580A (en) * | 2012-08-08 | 2014-02-12 | 内蒙古伊利实业集团股份有限公司 | Aseptic acquisition and inspection method for packing material |
| CN105349669A (en) * | 2015-11-30 | 2016-02-24 | 武汉轻工大学 | Quick qualitative and quantitative detection reagent kit and method for bifidobacteria added in feed and application |
| CN108277265A (en) * | 2017-01-05 | 2018-07-13 | 周兰嗣 | A method of living bacterial cells in one sample of identification |
| CN109652509A (en) * | 2018-12-27 | 2019-04-19 | 新乡学院 | A kind of method of lactic acid bacterium number in detection fermented feed |
-
2009
- 2009-07-03 CN CN200910072460A patent/CN101812503A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102605091A (en) * | 2012-04-09 | 2012-07-25 | 四川省畜牧科学研究院 | EMA-PCR (Ethidium Monoazide-Polymerase Chain Reaction) detection method of viable pathogen in farm environment |
| CN103575580A (en) * | 2012-08-08 | 2014-02-12 | 内蒙古伊利实业集团股份有限公司 | Aseptic acquisition and inspection method for packing material |
| CN103575580B (en) * | 2012-08-08 | 2016-02-17 | 内蒙古伊利实业集团股份有限公司 | The aseptic collection of packaging material and censorship method |
| CN105349669A (en) * | 2015-11-30 | 2016-02-24 | 武汉轻工大学 | Quick qualitative and quantitative detection reagent kit and method for bifidobacteria added in feed and application |
| CN105349669B (en) * | 2015-11-30 | 2019-02-22 | 武汉轻工大学 | Fast qualitative, immue quantitative detection reagent box and the detection method of Bifidobacterium for being added in feed and application |
| CN108277265A (en) * | 2017-01-05 | 2018-07-13 | 周兰嗣 | A method of living bacterial cells in one sample of identification |
| CN109652509A (en) * | 2018-12-27 | 2019-04-19 | 新乡学院 | A kind of method of lactic acid bacterium number in detection fermented feed |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107513561A (en) | Detect primer, kit and the method for P. aeruginosa and C.perfringens | |
| CN101319250B (en) | Method for quick quantitative determination of active bifidobacteria | |
| CN104328212B (en) | The primer sets of ring mediated isothermal amplification method detection B group streptococcus and test kit | |
| CN101812503A (en) | Reagent kit for detecting live bifidobacteriums in dairy products | |
| CN109593868A (en) | It is a kind of for detecting characteristic nucleotide sequence and its specific primer, the kit and detection method of pseudomonas bacterium | |
| CN102094090B (en) | Cholera toxin virulence gene detection kit and detection method thereof | |
| CN107523619A (en) | The PCR detection kit of drug-fast bacteria comprising mcr genes and its application | |
| CN103436602A (en) | Kit and method for simultaneous detection of Staphylococcus aureus gene and Escherichia coli gene by using dual molecular beacon-LAMP process | |
| Savichtcheva et al. | Application of a direct fluorescence‐based live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate of Bacteroides spp. in drinking water | |
| CN102121051B (en) | Multiplex fluorescent quantitative PCR detection method for main pathogenic bacteria in aquatic product | |
| CN111088380A (en) | Brucella LF-RPA detection primer, probe and detection kit | |
| CN102978282B (en) | Typhoid fever salmonella and salmonella paratyphi fluorescent quantitative polymerase chain reaction (PCR) detection kit and application thereof | |
| CN101333557A (en) | Simultaneous quantitative determination process for various enteric pathogenic bacteria in environment water body example | |
| CN102154487A (en) | Reagent for detecting francisella tularensis and complex probe and fluorescent quantitative polymerase chain reaction (PCR) method for detecting francisella tularensis | |
| CN111500751A (en) | Detection method and kit for rapidly detecting high-toxicity Klebsiella pneumoniae | |
| CN102605069B (en) | Enterohemorrhagic Escherichia coli O104: H4 detection kit and use method thereof | |
| CN101712988B (en) | Method for quickly, qualitatively and quantitatively measuring Bifidobacteria in probiotic dairy products | |
| CN103215362A (en) | Establishment of methodology for carrying out joint detection on bacterial genus genes and toxin genes of clostridium difficile by using TaqMan-MGB probe real-time fluorescent quantitative PCR (polymerase chain reaction) technology | |
| CN106520986A (en) | Quintuple PCR (polymerase chain reaction) detection method capable of detecting multiple pathogens simultaneously | |
| CN102417931A (en) | Polymerase chain reaction (PCR) fluorescence detection kit and detection method for candida albicans | |
| CN111996267A (en) | Fluorescent quantitative PCR (polymerase chain reaction) primer group and kit for rapidly detecting AKK (alkyl ketene dimer) bacteria and application of fluorescent quantitative PCR primer group and kit | |
| CN105063172B (en) | A kind of method of quick Screening and Identification Streptococcus suis | |
| CN114196767B (en) | Specific molecular target and method for detecting staphylococcus aureus ST type by using same | |
| CN107447001A (en) | Detect pseudomonas aeruginosa and ExoS primer, kit and method in water | |
| CN107419012A (en) | Detect pseudomonas aeruginosa and lasB primer, kit and method in water |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20100825 |