CN104059906A - Stage temperature control rapid extraction method of food-borne pathogenic bacteria DNA - Google Patents
Stage temperature control rapid extraction method of food-borne pathogenic bacteria DNA Download PDFInfo
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- CN104059906A CN104059906A CN201410161363.3A CN201410161363A CN104059906A CN 104059906 A CN104059906 A CN 104059906A CN 201410161363 A CN201410161363 A CN 201410161363A CN 104059906 A CN104059906 A CN 104059906A
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- 244000052616 bacterial pathogen Species 0.000 title claims abstract description 7
- 238000000605 extraction Methods 0.000 title abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 21
- 239000006228 supernatant Substances 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 14
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 230000001580 bacterial effect Effects 0.000 claims abstract description 11
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 13
- 238000001556 precipitation Methods 0.000 claims description 13
- 244000078673 foodborn pathogen Species 0.000 claims description 12
- 241000186779 Listeria monocytogenes Species 0.000 claims description 7
- 241000191967 Staphylococcus aureus Species 0.000 claims description 7
- 235000013305 food Nutrition 0.000 claims description 7
- 239000012154 double-distilled water Substances 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 5
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 238000000197 pyrolysis Methods 0.000 claims description 3
- 241000607142 Salmonella Species 0.000 claims description 2
- 241000607272 Vibrio parahaemolyticus Species 0.000 claims description 2
- 238000011901 isothermal amplification Methods 0.000 claims description 2
- 230000001404 mediated effect Effects 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 230000001717 pathogenic effect Effects 0.000 claims description 2
- 239000013049 sediment Substances 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims 1
- 239000012930 cell culture fluid Substances 0.000 claims 1
- 239000012531 culture fluid Substances 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 3
- 239000000243 solution Substances 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 230000009089 cytolysis Effects 0.000 abstract 2
- 238000007397 LAMP assay Methods 0.000 abstract 1
- 241001052560 Thallis Species 0.000 abstract 1
- 239000003112 inhibitor Substances 0.000 abstract 1
- 239000011534 wash buffer Substances 0.000 abstract 1
- 238000005406 washing Methods 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 6
- 238000009792 diffusion process Methods 0.000 description 6
- 238000003752 polymerase chain reaction Methods 0.000 description 6
- 238000007400 DNA extraction Methods 0.000 description 4
- 235000013336 milk Nutrition 0.000 description 4
- 239000008267 milk Substances 0.000 description 4
- 210000004080 milk Anatomy 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 206010004022 Bacterial food poisoning Diseases 0.000 description 1
- 241000193155 Clostridium botulinum Species 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 238000011059 hazard and critical control points analysis Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 235000020191 long-life milk Nutrition 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
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- 230000002265 prevention Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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- 231100000331 toxic Toxicity 0.000 description 1
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Abstract
The invention relates to a stage temperature control rapid extraction method of food-borne pathogenic bacteria DNA. The method comprises the steps of centrifuging the bacterial liquid, collecting thalli, washing by washing buffer solution, adding lysis solution for rapid oil bath lysis, performing water bath treatment, centrifuging and taking supernatant as template DNA. The purity OD260/OD280 of the DNA extracted by the method is 1.6-1.8, and the DNA can meet the requirements of rapid detection technologies such as PCR, fluorescence PCR, loop-mediated isothermal amplification and the like on molecular detection of food-borne pathogenic bacteria. The method comprehensively utilizes the stage temperature control to crack cells and remove inhibitors such as protein and the like so as to release DNA, has simple operation and short time, does not need special experimental instruments and reagents, and has low experimental cost.
Description
Technical field
The present invention relates to field of molecular biotechnology, be specifically related to the rapid extracting method of food-borne pathogens DNA.
Background technology
Food-safety problem is the problem that national governments and the people are concerned about the most always, and the food origin disease being caused by food-borne pathogens is one of topmost factor affecting food safety.In the foundation and implementation process of HACCP system, pathogenic pathogenic micro-organism is the emphasis of varieties of food items processing industry control always.
Food-borne pathogens is the pathogenic bacteria that causes bacterial food poisoning.Nearly tens kinds of food-borne pathogens, mainly contain streptococcus aureus, Salmonellas, Shigellae and Vibrio parahemolyticus, intestinal bacteria, listeria monocytogenes, Clostridium botulinum etc.Food-borne pathogens mainly refers to microorganism itself and metabolic process thereof, the pollution of meta-bolites to food raw material, the course of processing and product to the impact of HUMAN HEALTH, and this pollution meeting causes damage to food consumption person's health.
The separation and Culture detection method sense cycle of traditional food-borne pathogens is long, needs 4-7d, and complex operation step, poor specificity.Therefore set up quick, sensitive, detection technique detects food-borne pathogens and seems particularly important accurately, polymerase chain reaction (PCR) equimolecular Biological Detection technology is because of its specificity and highly sensitive being widely adopted.And the first step of PCR detection technique is exactly the preparation of template DNA, i.e. the extraction of DNA in sample, directly affects the result that PCR reacts.For a long time, in sample, the extraction of DNA and purifying are consuming time, loaded down with trivial details processes always, and detection speed has seriously slowed down.Therefore, set up one fast DNA extraction method play vital effect to improving PCR detection efficiency.
The known DNA extraction method of the art mainly contains cetyl trimethylammonium bromide (CTAB), kit method etc.The DNA purity that CTAB method is extracted, be 1.8 left and right, apply more extensive, but its complex operation step, reach 8 steps, and in experimentation, use the toxic reagents such as phenol chloroform, and kit method is because the DNA purity of its extraction is up to 1.8-2.0, a kind of method being most widely used at present, but its operation 1-2h that reaches consuming time.
DNA extraction method of the prior art is consuming time reaches a few hours, the inventive method integrated use water-bath and oil bath carry out stage temperature control, by lysate lysing cell, thereby obtain fast DNA, whole leaching process is less than 40min, simply effective.Present method does not need special laboratory apparatus and reagent, and cost is relatively low.Use DNA sample concentration that present method extracts at 50-400ng/uL, the DNA purity of gained is that the ratio of OD260/OD280 is the requirement that the DNA quality of 1.6-1.8. and gained can meet the detection methods such as regular-PCR, fluorescent PCR, ring mediated isothermal amplification.
Table 1 streptococcus aureus DNA concentration and purity
Concentration and the purity of table 2 moscow' paratyphi B DNA
The concentration of listeria monocytogenes DNA and purity in table 3 milk
Summary of the invention
The object of the invention is to solve existing DNA extraction length consuming time, a step difficult problem how, the method of rapid extraction food-borne pathogens DNA a kind of is provided, and the DNA quality of extracting meets the requirement of subsequent detection method, thereby accelerate the detection speed of food-borne pathogens, so that better for prevention and the control of food origin disease are offered help.
Technical scheme of the present invention is to carry out according to the following steps:
(a) get food samples 10-25g (mL) or pathogenic bacterium bacterial strain to suitable liquid nutrient medium and prepare nutrient solution, then get 1-5mL inoculum, the centrifugal 1-4min of 8000-12000rmp, makes bacterial cell precipitation, abandons supernatant;
(b) make toward step (a) bacterial sediment in add 100-500uL aseptic double-distilled water, fully mix, the centrifugal 1-4min of 8000-12000rmp, abandons supernatant;
(c) in the bacterial precipitation making toward step (b), add 100-500uL cell pyrolysis liquid, suspension bacterial precipitation, and in 105 DEG C of-126 DEG C of oil bath 10s-3min;
(d) mixed solution of step (c) gained is moved to 70 DEG C of-95 DEG C of water-bath 5-30min;
(e) after step (d) finishes, 8000-12000rmp is centrifugal, and 1-4min gets supernatant, and supernatant liquor is template DNA, carries out quality examination to extracting gained DNA, and surplus DNA saves backup in-20 DEG C.
brief description of the drawings
Fig. 1: the agarose gel electrophoresis figure of streptococcus aureus DNA.
In Fig. 1, M is 15000bp Maker; 1,2,3 is 105 DEG C of oil bath 10s, 70 DEG C of water-bath 5min.
Fig. 2: the pcr amplification agarose gel electrophoresis figure of streptococcus aureus DNA.
In Fig. 2, M is 2000bp Maker, 1-3 in 1-3 difference corresponding diagram 1.
Fig. 3: the agarose gel electrophoresis figure of moscow' paratyphi B DNA.
In Fig. 3, M is 15000Maker; 1,2,3 is 110 DEG C of oil bath 60s, 70 DEG C of water-bath 30min.
Fig. 4: the pcr amplification agarose gel electrophoresis figure of moscow' paratyphi B DNA.
In Fig. 4, M is 15000Maker; 1-3 in 1-3 difference corresponding diagram 3.
Fig. 5: the agarose gel electrophoresis figure of listeria monocytogenes DNA in milk.
In Fig. 5, M is 15000Maker; 1,2,3 is 105 DEG C of oil bath 10s, 95 DEG C of water-bath 10min.
Fig. 6: the pcr amplification agarose gel electrophoresis figure of listeria monocytogenes DNA in milk.
In Fig. 6, M is 2000Maker; 1-3 in 1-3 difference corresponding diagram 5
Embodiment
The material, the reagent etc. that in following embodiment, use if no special instructions, are the material that can obtain from commercial channels.
Embodiment 1: the extraction of streptococcus aureus DNA:
● streptococcus aureus ATCC25923, in universal liquid substratum, 37 DEG C, is cultivated to 18h.
● choose the bacterium liquid 1mL of above-mentioned cultivation in 1.5mL centrifuge tube, and establish 3 pipes and repeat, the centrifugal 2min of 12000rpm, abandons supernatant.
● by 1mL aseptic double-distilled water Eddy diffusion precipitation, 4 DEG C of centrifugal 2min of 10000rpm, abandon supernatant, as above-mentioned repetition 1～2 time.
3, in precipitation, add the lysis buffer of 200 μ L5%Chelex-100 to vibrate, make bacterium liquid Eddy diffusion, and in 105 DEG C of oil bath 10s.
4, mixed solution is moved to 70 DEG C of water-bath 5min, complete reaction.
5,12000rpm is centrifugal, gets supernatant, can obtain DNA, and-20 DEG C save backup.
6, agarose gel electrophoresis detects concentration and the purity of DNA.
7, after survey OD260/OD280 or pcr amplification product, detect with agarose gel electrophoresis.
Embodiment 2: the extraction of moscow' paratyphi B DNA:
(a) moscow' paratyphi B CMCC50094 is inoculated in universal liquid substratum, cultivates 18h for 37 DEG C.
(b) the bacterium liquid 1mL that chooses above-mentioned cultivation in 1.5mL centrifuge tube, and establish 3 pipes repeat, the centrifugal 2min of 12000rpm, abandons supernatant.
(c) by 1mL aseptic double-distilled water Eddy diffusion precipitation, 4 DEG C of centrifugal 2min of 10000rpm, abandon supernatant, as above-mentioned repetition 1～2 time.
3, in precipitation, add 200 μ L aseptic double-distilled water vibrations to mix, make bacterium liquid Eddy diffusion, and in 110 DEG C of oil bath 60s.
4, mixed solution is moved to 70 DEG C of water-bath 30min, complete reaction.
5, the centrifugal protein macromolecule precipitation that makes sex change of 12000rpm, gets supernatant, can obtain DNA, and-20 DEG C save backup.
6, agarose gel electrophoresis detects concentration and the purity of DNA.
7, after survey OD260/OD280 or pcr amplification product, detect with agarose gel electrophoresis.
Embodiment 3: the extraction of Listeria monocytogenes DNA in milk:
(a) by 25mL sterilized milk in suitable substratum, 37 DEG C cultivate 18h.
(b) the bacterium liquid 1mL that chooses above-mentioned cultivation in 1.5mL centrifuge tube, and establish 3 pipes repeat, the centrifugal 2min of 12000rpm, abandons supernatant.
(c) by 1mL aseptic double-distilled water Eddy diffusion precipitation, 4 DEG C of centrifugal 2min of 10000rpm, abandon supernatant, as above-mentioned repetition 1～2 time.
3, in precipitation, add 200 μ L1%chelex-100 vibrations to mix, make bacterium liquid Eddy diffusion, and in 126 DEG C of oil bath 10s.
4, mixed solution is moved to 95 DEG C of water-bath 10min, complete reaction.
5,12000rpm is centrifugal, gets supernatant, can obtain DNA, and-20 DEG C save backup.
6, agarose gel electrophoresis detects concentration and the purity of DNA.
7, survey after OD260/OD280 or the suitable pcr amplification product of Listeria monocytogenes and detect with agarose gel electrophoresis.
Claims (5)
1. a stage temperature control rapid extracting method of food-borne pathogens DNA, the method comprises the steps:
(a) get 1-5mL food-borne pathogenic bacteria culture fluid, the centrifugal 1-4min of 8000-12000rmp, makes bacterial cell precipitation, abandons supernatant;
(b) in the bacterial sediment making toward step (a), add 100-500uL aseptic double-distilled water, fully mix, the centrifugal 1-4min of 8000-12000rmp, abandons supernatant;
(c) in the bacterial precipitation making toward step (b), add 100-500uL cell pyrolysis liquid, suspension bacterial precipitation, and in 105 DEG C of-126 DEG C of oil bath 10s-3min;
(d) mixed solution of step (c) gained is moved to water-bath 5-30min in 70 DEG C-95 DEG C immediately;
(e) after step (d) finishes, 8000-12000rmp is centrifugal, and 1-4min gets supernatant, and supernatant liquor is template DNA, and to extracting gained DNA for analyzing and testing, surplus DNA saves backup in-20 DEG C.
2. food-borne pathogens according to claim 1 comprises streptococcus aureus, Salmonellas, Shigellae, listeria monocytogenes, Vibrio parahemolyticus etc.
3. according to claim 1 in step (a), cell culture fluid, for getting food samples 10-25g (mL) or pathogenic bacterium bacterial strain to suitable liquid nutrient medium, is cultivated 12-20h in the suitableeest culture temperature.
4. according to claim 1 in step (c), cell pyrolysis liquid is: the 0-10%chelex-100 aqueous solution.
5. described in step (e), DNA is carried out to analyzing and testing according to claim 1, it is characterized in that, described DNA concentration is by agarose gel electrophoresis or measure OD260, and OD260/OD280 ratio obtains.Gained DNA of pathogenic concentration is at 50-400ng/uL, and the DNA purity OD260/OD280 ratio of gained is 1.6-1.8; And the DNA quality of gained can meet the requirement of the detection methods such as regular-PCR, fluorescent PCR, ring mediated isothermal amplification.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531563A (en) * | 2018-02-05 | 2018-09-14 | 深圳市尚维高科有限公司 | The purposes and lysate of porous microsphere and the application method of lysate |
| CN109811034A (en) * | 2019-03-30 | 2019-05-28 | 西北农林科技大学 | A kind of preparation method of high-purity DNA profiling |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101285090A (en) * | 2008-05-21 | 2008-10-15 | 厦门市疾病预防控制中心 | Detection process for food-borne pathogenic bacteria |
| CN101497923A (en) * | 2008-01-29 | 2009-08-05 | 陈福生 | Method for fast detecting Saimonella |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101497923A (en) * | 2008-01-29 | 2009-08-05 | 陈福生 | Method for fast detecting Saimonella |
| CN101285090A (en) * | 2008-05-21 | 2008-10-15 | 厦门市疾病预防控制中心 | Detection process for food-borne pathogenic bacteria |
Non-Patent Citations (2)
| Title |
|---|
| 徐义刚等: "食源性致病菌基因组DNA的高效提取方法", 《东北林业大学学报》, vol. 37, no. 2, 28 February 2009 (2009-02-28), pages 74 - 2 * |
| 熊丽莎等: "食品中致病菌总DNA快速提取方法比较", 《安徽农业科学》, vol. 41, no. 34, 31 December 2013 (2013-12-31), pages 13392 - 13394 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531563A (en) * | 2018-02-05 | 2018-09-14 | 深圳市尚维高科有限公司 | The purposes and lysate of porous microsphere and the application method of lysate |
| CN109811034A (en) * | 2019-03-30 | 2019-05-28 | 西北农林科技大学 | A kind of preparation method of high-purity DNA profiling |
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