CN102311997A - Method for detecting staphylococcus aureus live bacteria in milk - Google Patents
Method for detecting staphylococcus aureus live bacteria in milk Download PDFInfo
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- CN102311997A CN102311997A CN201110253191A CN201110253191A CN102311997A CN 102311997 A CN102311997 A CN 102311997A CN 201110253191 A CN201110253191 A CN 201110253191A CN 201110253191 A CN201110253191 A CN 201110253191A CN 102311997 A CN102311997 A CN 102311997A
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Abstract
The invention relates to a method for detecting staphylococcus aureus live bacteria in milk. The invention aims to solve the problem of overestimation of pathogenic bacteria risk due to the ineffective distinction between dead bacteria and live bacteria by present LAMP detection methods. The method provided by the invention comprises the following steps of: 1, pretreating a tested milk sample; 2, extracting DNA of the tested milk sample treated by Step 1 to obtain an extract; 3, performing LAMP amplification by using the extract as a template; 4, loading a LAMP amplification product into agarose gel for electrophoresis and imaging in a gel imaging analysis system to finish the detection. The method provided by the invention has high detection sensitivity with the minimal measminimal detectable limit of staphylococcus aureus being 100fg, and can be widely applied in the field of foodborne pathogens detection.
Description
Technical field
The present invention relates to a kind of method that detects Ruzhong streptococcus aureus viable bacteria.
Background technology
Loop-mediated isothermal amplification technique (LAMP) is a kind of simple, quick, special, the economic novel nucleic acids amplification technique by the exploitation of Japanese Rong Yan Co., Ltd. in 2000; The characteristics of this method are 4 the special primers of 6 zone design to target gene, utilize a kind of strand displacement archaeal dna polymerase under constant temperature, to react dozens of minutes and can accomplish.Characteristics with high specific and isothermal duplication can increase 10 with target gene within the 1h
9～10
10Doubly.When the DNA extension was synthetic, the pyrophosphate ion of from deoxynucleoside triphosphate matrix (dNTPs), separating out combined with the mg ion in the reaction soln, can produce a kind of white precipitate of magnesium pyrophosphate verivate, utilizes these characteristics with the naked eye can whether judge amplification.
After cell loses viability in the environment; Its DNA still has the persistent existence ability; And can not distinguish positive signal based on traditional DNA detection method is from bacterium alive in the target mikrobe or dead bacterium; Can't effectively distinguish dead bacterium and viable bacteria, possibly cause the too high estimation of pathogenic bacterium risk or draw false positive results.
Summary of the invention
The present invention will solve existing LAMP detection method can not effectively distinguish dead bacterium and viable bacteria, causes the problem to the too high estimation of pathogenic bacterium risk, and a kind of method that detects Ruzhong streptococcus aureus viable bacteria is provided.
The present invention detects the method for Ruzhong streptococcus aureus viable bacteria; Carry out according to the following steps: one, examined the pre-treatment of breast appearance: examined the Ruzhong to 1mL and add PMA; The concentration that makes PMA is 3 μ g/mL; Place the 5～30min of dark place placement on ice then, then place, exposure 3～5min apart from 650W halogen lamp 15～20cm place; Two, the breast of being examined after the step 1 processing is carried out DNA extraction, get extract; Three, carry out the LAMP amplification with extract as template, obtain the LAMP amplified production; Four, identify: get 5 μ L LAMP amplified productions, point sample with 100V voltage electrophoresis 30min in 1 * TAE electrophoretic buffer, forms images in gel imaging system in mass concentration is 2% sepharose then, promptly accomplishes and detects.
The present invention is directed to streptococcus aureus heat stable nuclease gene (nuc) and design primer, carry out the LAMP amplification.Have an azido group (N3) in optical dye PMA (Propidium Monoazide) structure that the present invention uses; Under optical excitation, slough and (N2) form active nitrene midbody; Can insert dead cell DNA and its formation covalent linkage (NH-CH2-), makes DNA that amplified reaction can not take place, thereby suppresses the LAMP amplification; Can effectively distinguish the dead bacterium and the bacterium that lives, avoid causing false assessment the streptococcus aureus content of actual survival in the testing sample.
Electrophoresis result of the present invention is used the gel imaging system imaging, i.e. the explanation that can detect amplified band is examined the Ruzhong and is contained streptococcus aureus.Method detection sensitivity of the present invention is high, is 100fg to the minimum detectability of streptococcus aureus.The present invention has opened up new approaches for the detection of pathogenic bacterium in the food.
Description of drawings
Fig. 1 is the detected result electrophorogram of embodiment 14; Fig. 2 is the LAMP amplification electrophorogram that PMA suppresses the dead bacterium of streptococcus aureus in the embodiment 14; Fig. 3 is the figure as a result of optical dye SYBR Green I checking LAMP reaction in the embodiment 14; Fig. 4 is the electrophorogram of LAMP amplification in the embodiment 14 sensitivity tests experiment; Fig. 5 is the electrophorogram of pcr amplification in the embodiment 14 sensitivity tests experiment.
Embodiment
Technical scheme of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: this embodiment detects the method for Ruzhong streptococcus aureus viable bacteria; Carry out according to the following steps: one, examined the pre-treatment of breast appearance: examined the Ruzhong to 1mL and add PMA; The concentration that makes PMA is 3 μ g/mL; Place the 5～30min of dark place placement on ice then, then place, exposure 3～5min apart from 650W halogen lamp 15～20cm place; Two, the breast of being examined after the step 1 processing is carried out DNA extraction, get extract; Three, carry out the LAMP amplification with extract as template, obtain the LAMP amplified production; Four, identify: get 5 μ L LAMP amplified productions, point sample with 100V voltage electrophoresis 30min in 1 * TAE electrophoretic buffer, forms images in gel imaging system in mass concentration is 2% sepharose then, promptly accomplishes and detects.
Embodiment two: what this embodiment and embodiment one were different is: place on ice the dark place to place 5min in the step 1.Other is identical with embodiment one.
Embodiment three: what this embodiment and embodiment one were different is: place on ice the dark place to place 30min in the step 1.Other is identical with embodiment one.
Embodiment four: what this embodiment and embodiment one were different is: place on ice the dark place to place 10～20min in the step 1.Other is identical with embodiment one.
Embodiment five: what this embodiment and embodiment one were different is: place on ice the dark place to place 15min in the step 1.Other is identical with embodiment one.
Embodiment six: what this embodiment was different with one of embodiment one to five is: place in the step 1 apart from 650W halogen lamp 15cm place.Other is identical with one of embodiment one to five.
Embodiment seven: what this embodiment was different with one of embodiment one to five is: place in the step 1 apart from 650W halogen lamp 18cm place.Other is identical with one of embodiment one to five.
Embodiment eight: what this embodiment was different with one of embodiment one to five is: place in the step 1 apart from 650W halogen lamp 20cm place.Other is identical with one of embodiment one to five.
Embodiment nine: what this embodiment was different with one of embodiment one to eight is: 3min makes public in the step 1.Other is identical with one of embodiment one to eight.
Embodiment ten: what this embodiment was different with one of embodiment one to eight is: 4min makes public in the step 1.Other is identical with one of embodiment one to eight.
Embodiment 11: what this embodiment was different with one of embodiment one to eight is: 5min makes public in the step 1.Other is identical with one of embodiment one to eight.
Embodiment 12: what this embodiment and embodiment one to 11 were different is: the method that breast carries out DNA extraction of being examined after described in the step 2 step 1 being handled is: a, after step 1 is handled examined the DNA extraction liquid that the Ruzhong adds 1mL, Proteinase K and the 10 μ L concentration that 10 μ L concentration are 10mg/mL are the N,O-Diacetylmuramidase of 10mg/mL; 1.5h then vibrates under 37 ℃, 200r/min condition; Add 200 μ L mass concentrations again and be 20% SDS; Then in 65 ℃ of water-bath 1h; With the centrifugal 5min of the rotating speed of 13000r/min, get supernatant afterwards; B, the DNA extraction liquid that in deposition, adds 0.75mL and 75 μ L concentration are 20% SDS, behind vibration 10s on the vortex vibrator, in 65 ℃ of water-bath 20min, at room temperature with the centrifugal 5min of 13000r/min, get supernatant then; The supernatant that c, combining step a and step b obtain adds the mixed solution of isopyknic chloroform and primary isoamyl alcohol, in the centrifugal 5min of 13000r/min; Reclaim water,, place 1.5h at-20 ℃ at 4 ℃ Virahol of 0.6 times of volume of aqueous phase adding; In the centrifugal 10min of 13000r/min, discard supernatant then, it is 70% washing with alcohol 3～5 times that deposition is used 4 ℃ volumetric concentration; Dry back adds the TE damping fluid of 300 μ L, is extract; DNA extraction liquid described in step a and the step b is by the Tris-HCl of 0.1mol/L, the EDTA of 0.1mol/L, the Na of 0.1mol/L
3PO
4, 1.5mol/L NaCl and mass concentration be that 1% CTAB forms, the pH value of DNA extraction liquid is 8; Among the step c in the mixed solution of chloroform and primary isoamyl alcohol the volume ratio of chloroform and primary isoamyl alcohol be 24: 1.Other is identical with embodiment one to 11.
Embodiment 13: what this embodiment and embodiment one to 12 were different is: the reaction system of LAMP amplification is 50 μ L reaction systems in the step 3, is made up of following ingredients:
The LAMP amplification condition is: 95 ℃ of 5min place 4 ℃ to add Bst enzyme, 63 ℃ of 60min, 80 ℃ of 2min rapidly.Other is identical with embodiment one to 12.
Embodiment 14: this embodiment detects the method for Ruzhong streptococcus aureus viable bacteria; Carry out according to the following steps: one, examined the pre-treatment of breast appearance: examined the Ruzhong to 1mL and add PMA; The concentration that makes PMA is 3 μ g/mL; Place the 5min of dark place placement on ice then, then place, exposure 3min apart from 650W halogen lamp 20cm place; Two, the breast of being examined after the step 1 processing is carried out DNA extraction, get extract; Three, carry out the LAMP amplification with extract as template, obtain the LAMP amplified production; Four, identify: get 5 μ L LAMP amplified productions, point sample with 100V voltage electrophoresis 30min in 1 * TAE electrophoretic buffer, forms images in gel imaging system in mass concentration is 2% sepharose then, promptly accomplishes and detects.
The method that breast carries out DNA extraction of being examined after described in this embodiment step 2 step 1 being handled is: a, after step 1 is handled examined the DNA extraction liquid that the Ruzhong adds 1mL, Proteinase K and the 10 μ L concentration that 10 μ L concentration are 10mg/mL are the N,O-Diacetylmuramidase of 10mg/mL; 1.5h then vibrates under 37 ℃, 200r/min condition; Add 200 μ L mass concentrations again and be 20% SDS; In 65 ℃ of water-bath 1h, with the centrifugal 5min of the rotating speed of 13000r/min, get supernatant afterwards then; B, the DNA extraction liquid that in deposition, adds 0.75mL and 75 μ L mass concentrations are 20% SDS, behind vibration 10s on the vortex vibrator, in 65 ℃ of water-bath 20min, at room temperature with the centrifugal 5min of 13000r/min, get supernatant then; The supernatant that c, combining step a and step b obtain adds the mixed solution of isopyknic chloroform and primary isoamyl alcohol, in the centrifugal 5min of 13000r/min; Reclaim water,, place 1.5h at-20 ℃ at 4 ℃ Virahol of 0.6 times of volume of aqueous phase adding; In the centrifugal 10min of 13000r/min, discard supernatant then, it is 70% washing with alcohol 3～5 times that deposition is used 4 ℃ volumetric concentration; Dry back adds the TE damping fluid of 300 μ L, is extract; DNA extraction liquid described in step a and the step b is by the Tris-HCl of 0.1mol/L, the EDTA of 0.1mol/L, the Na of 0.1mol/L
3PO
4, 1.5mol/L NaCl and mass concentration be that 1% CTAB forms, the pH value of DNA extraction liquid is 8; Among the step c in the mixed solution of chloroform and primary isoamyl alcohol the volume ratio of chloroform and primary isoamyl alcohol be 24: 1.
The reaction system of LAMP amplification is 50 μ L reaction systems in this embodiment step 3, is made up of following ingredients:
The LAMP amplification condition is: 95 ℃ of 5min place 4 ℃ to add Bst enzyme, 63 ℃ of 60min, 80 ℃ of 2min rapidly.
Use gel imaging system to be U.S. UVP gel imaging system in this embodiment step 4.The detected result of this embodiment is as shown in Figure 1, and M is DNA Marker among Fig. 1, and swimming lane 1 is for receiving inspection breast 1, and swimming lane 2 receives to contain streptococcus aureus in the inspection breast 1 for receiving inspection breast 2, from Fig. 1, can finding out, and receives not contain streptococcus aureus in the inspection breast 2.
For the method that proves this embodiment viable bacteria of can selectivity only increasing, carry out following test: get the living bacterial liquid and dead bacterium liquid of same concentrations, be divided into four groups: the A group is living bacterial liquid with the B group, and it is dead bacterium liquid that the C group is organized with D, and every group bacterial concentration is all identical with volume.Respectively to B group and D rent bacterium liquid carry out the PMA processing according to the method for this embodiment step 1; Then four groups of bacterium liquid are carried out DNA extraction respectively; With DNA is template; Carry out the LAMP amplification according to the method for this embodiment step 3, then product is carried out agarose gel electrophoresis, the result is as shown in Figure 2.M is DNA Marker among Fig. 2; Swimming lane 1 expression negative control (with deionized water as template); The living bacterial liquid (handling) of swimming lane 2 expression A groups without PMA; The living bacterial liquid through the PMA processing of swimming lane 3 expression B groups, swimming lane 4 are represented the dead bacterium liquid (being to handle through PMA) of C group, the dead bacterium liquid that the process PMA that swimming lane 5 expression D organize handles.
Can find out that from Fig. 2 the dead bacterium and the viable bacteria of handling without PMA can both amplify band, and, have only viable bacteria can amplify band, and dead bacterium does not have band through after the PMA processing.The method of hence one can see that this embodiment can suppress the amplification of dead bacterium effectively, only detects viable bacteria, thereby prevents the generation of false positive results.
Utilize optical dye SYBR Green I whether above-mentioned experiment LAMP reaction is verified, after the LAMP reaction finishes, get the product of partial L AMP reaction, the SYBR Green I dyestuff in product after 10 times of dilutions of adding 1 μ L.The color of SYBR Green I dyestuff itself is orange, if the LAMP reaction has taken place, dye colour can be from the orange green that becomes.The result is as shown in Figure 3 in checking; NC representes negative control (with deionized water as template); The living bacterial liquid (handling) of No. 1 pipe expression A group without PMA; The living bacterial liquid that the process PMA of No. 2 pipe expression B groups handles, the dead bacterium liquid (without the PMA processing) of No. 3 pipe expression C groups, the dead bacterium liquid through the PMA processing of No. 4 pipe expression D groups.
Can find out from Fig. 3, the LAMP reaction does not take place in No. 4 pipes, can explain that PMA can suppress the LAMP amplification of dead bacterium.
The method of this embodiment is carried out the test of sensitivity, and specific as follows: the artificial inoculation streptococcus aureus is in sterile milk, in 37 ℃, 200r/min shaking culture 8h.Adjustment Ruzhong cell concentration is 10
8Cuf/mL carries out 10 times of gradient dilutions then, and making concentration is 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10
1With 10
0Cfu/mL carries out PMA respectively and handles back extraction thallus DNA, is that template is carried out LAMP amplification and pcr amplification with the thallus DNA.
The PCR reaction system is 50 μ L, is made up of following ingredients:
The pcr amplification condition is: 94 ℃ of 5min, 30 circulations: 94 ℃ 45s-62 ℃ 45s-72 ℃ of 30s, 72 ℃ of 5min.
The electrophorogram of LAMP amplification is as shown in Figure 4, from left to right is followed successively by marker, 10 among Fig. 4
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10
1, 10
0And deionized water; Pcr amplification result's electrophorogram is as shown in Figure 5, from left to right is followed successively by marker, pure viable bacteria, 10 among Fig. 5
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10
1, 10
0And deionized water.
Can find out that by Fig. 4 and Fig. 5 the PMA+LAMP combination technology can detect 10
0The thalline of cfu/mL, through detecting the absorbancy of DNA under 260nm and 280nm wavelength, the lowest detection amount is 100fg, and the PMA+PCR technology can detect 10
1The thalline of cfu/mL.So the LAMP technology is sensitiveer more than 10 times than round pcr.
Claims (6)
1. method that detects Ruzhong streptococcus aureus viable bacteria; It is characterized in that detecting the method for Ruzhong streptococcus aureus viable bacteria; Carry out according to the following steps: one, examined the pre-treatment of breast appearance: examined the Ruzhong to 1mL and add PMA, the concentration that makes PMA is 3 μ g/mL, places the 5～30min of dark place placement on ice then; Then place apart from 650W halogen lamp 15～20cm place exposure 3～5min; Two, the breast of being examined after the step 1 processing is carried out DNA extraction, get extract; Three, carry out the LAMP amplification with extract as template, obtain the LAMP amplified production; Four, identify: get 5 μ L LAMP amplified productions, point sample with 100V voltage electrophoresis 30min in 1 * TAE electrophoretic buffer, forms images in gel imaging system in mass concentration is 2% sepharose then, promptly accomplishes and detects.
2. a kind of method that detects Ruzhong streptococcus aureus viable bacteria according to claim 1 is characterized in that placing in the step 1 dark place to place 5min.
3. a kind of method that detects Ruzhong streptococcus aureus viable bacteria according to claim 1 and 2 is characterized in that placing in the step 1 apart from 650W halogen lamp 18cm place.
4. a kind of method that detects Ruzhong streptococcus aureus viable bacteria according to claim 3 is characterized in that the 3min that makes public in the step 1.
5. a kind of method that detects Ruzhong streptococcus aureus viable bacteria according to claim 4; The method that breast carries out DNA extraction of being examined after it is characterized in that described in the step 2 step 1 handled is: a, after step 1 is handled examined the DNA extraction liquid that the Ruzhong adds 1mL, Proteinase K and the 10 μ L concentration that 10 μ L concentration are 10mg/mL are the N,O-Diacetylmuramidase of 10mg/mL; 1.5h then vibrates under 37 ℃, 200r/min condition; Add 200 μ L mass concentrations again and be 20% SDS; In 65 ℃ of water-bath 1h, with the centrifugal 5min of the rotating speed of 13000r/min, get supernatant afterwards then; B, the DNA extraction liquid that in deposition, adds 0.75mL and 75 μ L concentration are 20% SDS, behind vibration 10s on the vortex vibrator, in 65 ℃ of water-bath 20min, at room temperature with the centrifugal 5min of 13000r/min, get supernatant then; The supernatant that c, combining step a and step b obtain adds the mixed solution of isopyknic chloroform and primary isoamyl alcohol, in the centrifugal 5min of 13000r/min; Reclaim water,, place 1.5h at-20 ℃ at 4 ℃ Virahol of 0.6 times of volume of aqueous phase adding; In the centrifugal 10min of 13000r/min, discard supernatant then, it is 70% washing with alcohol 3～5 times that deposition is used 4 ℃ volumetric concentration; Dry back adds the TE damping fluid of 300 μ L, is extract; DNA extraction liquid described in step a and the step b is by the Tris-HCl of 0.1mol/L, the EDTA of 0.1mol/L, the Na of 0.1mol/L
3PO
4, 1.5mol/L NaCl and mass concentration be that 1% CTAB forms, the pH value of DNA extraction liquid is 8; Among the step c in the mixed solution of chloroform and primary isoamyl alcohol the volume ratio of chloroform and primary isoamyl alcohol be 24: 1.
6. a kind of method that detects Ruzhong streptococcus aureus viable bacteria according to claim 5 is characterized in that the reaction system of LAMP amplification in the step 3 is 50 μ L reaction systems, is made up of following ingredients:
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103361429A (en) * | 2013-06-28 | 2013-10-23 | 厦门银祥集团有限公司 | LAMP detection primer, kit and detection method for staphylococcus aureus containing internal standard |
| CN103451264A (en) * | 2013-09-13 | 2013-12-18 | 湖南农业大学 | Method for measuring total number of fermented milk living bacteria |
| CN104946780A (en) * | 2015-07-14 | 2015-09-30 | 东北农业大学 | Kit capable of rapidly detecting staphylococcus aureus in meat and application |
| CN108018366A (en) * | 2018-01-04 | 2018-05-11 | 南京农业大学 | Reagent, kit and application for staphylococcus aureus living stems |
| CN108504753A (en) * | 2018-03-30 | 2018-09-07 | 中国农业科学院北京畜牧兽医研究所 | A method of detection staphylococcus aureus live bacteria in milk |
| CN110527681A (en) * | 2018-05-25 | 2019-12-03 | 中国农业科学院北京畜牧兽医研究所 | The extracting method of total microbial DNA in a kind of milk |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361429A (en) * | 2013-06-28 | 2013-10-23 | 厦门银祥集团有限公司 | LAMP detection primer, kit and detection method for staphylococcus aureus containing internal standard |
| CN103361429B (en) * | 2013-06-28 | 2015-06-03 | 厦门银祥集团有限公司 | LAMP detection primer, kit and detection method for staphylococcus aureus containing internal standard |
| CN103451264A (en) * | 2013-09-13 | 2013-12-18 | 湖南农业大学 | Method for measuring total number of fermented milk living bacteria |
| CN103451264B (en) * | 2013-09-13 | 2015-08-26 | 湖南农业大学 | A kind of method measuring fermented-milk total viable count |
| CN104946780A (en) * | 2015-07-14 | 2015-09-30 | 东北农业大学 | Kit capable of rapidly detecting staphylococcus aureus in meat and application |
| CN108018366A (en) * | 2018-01-04 | 2018-05-11 | 南京农业大学 | Reagent, kit and application for staphylococcus aureus living stems |
| CN108018366B (en) * | 2018-01-04 | 2021-09-17 | 南京农业大学 | Reagent and kit for detecting viable staphylococcus aureus and application of reagent and kit |
| CN108504753A (en) * | 2018-03-30 | 2018-09-07 | 中国农业科学院北京畜牧兽医研究所 | A method of detection staphylococcus aureus live bacteria in milk |
| CN110527681A (en) * | 2018-05-25 | 2019-12-03 | 中国农业科学院北京畜牧兽医研究所 | The extracting method of total microbial DNA in a kind of milk |
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Application publication date: 20120111 |