CN109632733A - A method of utilizing bacillus cereus in ATP bioluminescence reaction detection food - Google Patents
A method of utilizing bacillus cereus in ATP bioluminescence reaction detection food Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Abstract
The invention discloses a kind of methods using bacillus cereus in ATP bioluminescence reaction detection food, centrifugation obtains initial supernatant liquid after this method first mixes sample to be detected with PBS buffer solution, digestive juice is then added and carries out heating in water bath for reaction, digestion supernatant is obtained after being centrifuged again and is filtered, filter membrane is immersed into mechanical shaking extraction in ATP release liquid, obtain ATP extracting solution, then the luminescence-producing reaction liquid being prepared is mixed in luminescence-producing reaction pipe with ATP extracting solution, it is placed in ATP fluorescence detector and detects luminous intensity, to realize the quick detection to bacillus cereus in food.Method easy quick, high sensitivity of the present invention using bacillus cereus in ATP bioluminescence reaction detection food, it can accurately detect to be greatly promoted the raising of food safety detection technology whether containing bacillus cereus and relative amount in food.
Description
Technical field
It is the present invention relates to biological detecting method technical field, in particular to a kind of to utilize ATP bioluminescence reaction detection food
The method of bacillus cereus in product.
Background technique
Bacillus cereus (Bacillus cereus) it is a kind of sporiferous gram-positive bacteria of energy, it is widely distributed
In air, sewage and all kinds of raw and cooked foods.Currently, isolating the bacterium from numerous food, including parched rice meal, meat, newborn class
It is common food-borne pathogens, it is most likely that endanger people's health with fish etc..Clinically, bacillus cereus causes
Food poisoning can be divided into diarrhea-type and vomiting type two types, be the diarrhea toxin generated by the bacterium and vomitoxin institute respectively
It causes, the former is sensitive with trypsase to heat, thus can prevent；And the latter is to thermostabilization, activity is not by the interference of pepsin, i.e.,
Make trace expression that can also have larger harm to human body, be not easy to take precautions against, often plays fulminant food poisoning.Therefore, for the pre- of the bacterium
Prevent and detects extremely important.
The conventional method of microorganism detection has medium therapy, PCR Molecular Detection method, enzyme linked immunosorbent assay and ATP biology
Luminescence method etc..Conventional medium method time-consuming is too long, and PCR molecular detecting method needs special instrument and equipment, need to specially extract
The DNA of sample, sample pre-treatments complex steps, operator need to have technical skill.Enzyme linked immunosorbent assay is made with antibody
To identify molecule, influence of the antibody vulnerable to external condition especially temperature requires harshness to storage conditions, largely
Limit the flexible Application of method.In addition, Antibody preparation is needed by zoopery or cell experiment, it is cumbersome bothersome, preparation
Antibody is at high cost, and the method testing cost of development is also corresponding higher.
ATP biloluminescence method is a kind of detection technique to grow up the 1980s, and this method is based on firefly
Principle of luminosity, under the catalytic action of luciferase, ATP and luciferin reaction discharge fluorescence, by test luminous intensity come real
The quick detection of existing microorganism.ATP is both the required substrate and the movable energy of all biological lifes of luciferase catalytic luminescence
Source is measured, the ATP content in every kind of microbial cell is constant.By the ATP concentration in measurement sample, work can be extrapolated
Bacterium number.It is to study a kind of more micro- life both at home and abroad at present because microculture, easy to operate, detection speed is fast without carrying out
Object detecting method.But for ATP biloluminescence method, the burst size of ATP will have a direct impact on test result in microorganism, if
ATP release not exclusively, then can make test result relatively low.And ATP is a kind of degradable energy matter, if release reagent can be broken
It is relatively low to also result in test result by bad ATP.Therefore, select suitable ATP release reagent most important for different test sources.
Summary of the invention
In view of the above-mentioned problems existing in the prior art and demand, it is sent out the object of the present invention is to provide a kind of using ATP biology
The method that light reaction detects bacillus cereus in food, after this method first mixes sample to be detected with PBS buffer solution
Centrifugation obtains initial supernatant liquid, and digestive juice is then added and carries out heating in water bath for reaction, and digestion supernatant is obtained after being centrifuged again simultaneously
Filter, by filter membrane immerse ATP release liquid in mechanical shaking extraction, obtain ATP extracting solution, then by the luminescence-producing reaction liquid being prepared with
ATP extracting solution mixes in luminescence-producing reaction pipe, is placed in ATP fluorescence detector and detects luminous intensity, to realize in food
The quick detection of bacillus cereus.It is of the present invention to utilize bacillus cereus in ATP bioluminescence reaction detection food
Method is easy quickly, high sensitivity, whether can accurately detect in food containing bacillus cereus and relative amount, pole
The earth promotes the raising of food safety detection technology.
Technical solution: to achieve the goals above, food is detected using ATP bioluminescence reaction the invention discloses a kind of
The method of middle bacillus cereus, comprising the following steps:
(1) the liquid/solid sample to be detected of 50 g is mixed with 1 × PBS buffer solution of 950 g, it is ground/to stir to get
Suspension/mixed liquor, is centrifuged off insoluble matter, and 200 μ L digestive juices are added into obtained initial supernatant liquid, are put into after mixing
15~20 min of digestion reaction is carried out in 40 degree of water-baths, is then centrifuged 1~2 min with 3000 g, is digested after abandoning precipitating
Supernatant；
(2) the digestion supernatant for taking 100 mL steps (1) to obtain is filtered, and is then immersed in filter membrane and is contained with 50 mL
In the beaker of ATP release liquid, the ATP release liquid includes Triton X-100, trimethylamino methane, lithium chloride, N- laurel
Acylsarcosine sodium, guanidinium isothiocyanate, urea are then added 15 μ L apyrases, beaker are transferred to 40 DEG C of water
Bath medium-rate mechanical shaking extraction, obtains ATP extracting solution；
(3) by 10 μ L of luciferase, 30 mg of soluble inorganic magnesium salts, dithiothreitol (DTT) 15 mg and 50 mL in gnotobasis
Sterile water mixing, oscillation mix, and obtain luminescence-producing reaction liquid；
(4) for the ATP extracting solution that 0.2 mL step (2) of absorption obtains in luminescence-producing reaction pipe, pH 7.0~9.0 is added contains 0.13
The Tris-HCl buffering of~0.15 mol/L NaCl, 0.02~0.03 mol/L EDTA, 0.06~0.08 mol/L cyclodextrin
Total volume is complemented to 0.9 mL by liquid, adds the luminescence-producing reaction liquid of 0.1 mL step (3), is sufficiently placed in mixed liquor after oscillation
Luminous intensity is detected in ATP fluorescence detector, the detection response time is 20~30 s.
Preferably, the digestive juice composition in the step (1) are as follows: 0.1~0.3 mol/L Triton X-100,0.05~
0.07 mol/L disodium ethylene diamine tetraacetate, 0.02~0.04 mol/L Proteinase K, 0.5~0.9 mol/L dodecyl sulphur
Sour sodium.
Preferably, the pore size of filter membrane is 0.25~0.35 μm in the step (2).
Preferably, in the step (2) ATP release liquid composition are as follows: 0.04~0.06 mol/L Triton X-100,
0.05~0.07 mol/L trimethylamino methane, 0.03~0.05 mol/L lithium chloride, 2~4 mmol/L N- lauroyl
Sodium sarcosinate, 13~15 mmol/L guanidinium isothiocyanates, 8~12 mmol/L urea.
Preferably, the rate of mechanical shaking extraction is 200 r/min in the step (2), and the time of mechanical shaking extraction is 8~10
min。
Preferably, soluble inorganic magnesium salts is any one in magnesium chloride, magnesium nitrate and magnesium sulfate in the step (3)
Kind is several.
Preferably, it is specifically with 10~20 min of rate oscillation of 150 r/min that the oscillation in the step (3), which mixes,.
Preferably, the pH of Tris-HCl buffer is 8.0 in the step (4).
Preferably, when mixed liquor is placed in ATP fluorescence detector the detection response for detecting luminous intensity in the step (4)
Between be 25 s.
Compared with prior art, the invention has the following beneficial effects:
(1) method of the present invention using bacillus cereus in ATP bioluminescence reaction detection food it is easy quickly,
High sensitivity can accurately detect whether contain bacillus cereus and relative amount in food.
(2) method of the present invention using bacillus cereus in ATP bioluminescence reaction detection food is directed to institute
The special object bacillus cereus of detection devises dedicated ATP release liquid, can carry out for bacillus cereus special
Property cracking, sufficiently discharge the ATP in bacillus cereus, avoid the interference of ATP in other miscellaneous bacterias, it is special to substantially increase detection
Property.
Specific embodiment
It is of the invention below by way of combining following specific embodiments to further illustrate.It should be pointed out that real in detail below
It applies mode for explaining only the invention, is not used to be defined the contents of the present invention.
ATP release liquid provided by the invention, by by lithium chloride, N- sodium lauroyl sarcosine, guanidinium isothiocyanate, urea with
Triton X-100, trimethylamino methane in traditional ATP release liquid are compounded, and produce gemma for bacillus cereus,
Gemma is round or cylindricality, middle life or it is close in it is raw, sporangiocyst is without obviously expanding, this Morphological Features of no pod membrane and its cell wall class
Type belongs to the characteristics of gram-positive bacteria, by lithium chloride, N- sodium lauroyl sarcosine, guanidinium isothiocyanate, urea jointly with it is waxy
The thallus of bacillus chemically reacts, and the cell wall of bacillus cereus, cell membrane is made to decompose, to sufficiently discharge
Intracellular ATP realizes the raising of detection sensitivity and accuracy.
Below by embodiment, the present invention is furture elucidated.It should be pointed out that the present invention is not intended to be limited to the reality
Apply example.
Embodiment 1
(1) it is mixed after being crushed the flesh of fish to be detected of 50 g with 1 × PBS buffer solution of 950 g, it is agitated to obtain suspension,
It is centrifuged off insoluble matter, 200 μ L digestive juices, digestive juice composition are as follows: 0.1 mol/L are added into obtained initial supernatant liquid
Triton X-100,0.05 mol/L disodium ethylene diamine tetraacetate, 0.02 mol/L Proteinase K, 0.5 mol/L dodecyl
Sodium sulphate is put into progress 15 min of digestion reaction in 40 degree of water-baths, is then centrifuged 1 min with 3000 g, abandons precipitating after mixing
After obtain digestion supernatant；
(2) the digestion supernatant for taking 100 mL steps (1) to obtain is filtered, and the pore size of filter membrane is 0.25 μm, then will
Filter membrane is immersed in the beaker for being contained with 50 mL ATP release liquids, the composition of the ATP release liquid are as follows: 0.04 mol/L
Triton X-100,0.05 mol/L trimethylamino methane, 0.03 mol/L lithium chloride, 2 mmol/L N- lauroyl fleshes
15 μ L apyrases are then added in propylhomoserin sodium, 13 mmol/L guanidinium isothiocyanates, 8 mmol/L urea, will
Beaker is transferred to 40 DEG C of water-bath medium-rate mechanical shaking extractions, and the rate of mechanical shaking extraction is 200 r/min, and the time of mechanical shaking extraction is
8 min obtain ATP extracting solution；
(3) by 10 μ L of luciferase, 30 mg of soluble inorganic magnesium salts, dithiothreitol (DTT) 15 mg and 50 mL in gnotobasis
Sterile water mixing, is mixed with 10 min of rate oscillation of 150 r/min, obtains luminescence-producing reaction liquid；
(4) for the ATP extracting solution that 0.2 mL step (2) of absorption obtains in luminescence-producing reaction pipe, pH 8.0 is added contains 0.13 mol/
L NaCl, 0.02 mol/L EDTA, 0.06 mol/L cyclodextrin Tris-HCl buffer total volume is complemented into 0.9 mL,
The luminescence-producing reaction liquid of 0.1 mL step (3) is added, mixed liquor is placed in detection in ATP fluorescence detector sufficiently after oscillation and is shone
Intensity, detection response time are 25 s.
Embodiment 2
(1) pork to be detected of 50 g is mixed with 1 × PBS buffer solution of 950 g, ground to obtain suspension, centrifugation removes
Insoluble matter is removed, 200 μ L digestive juices, digestive juice composition are as follows: 0.2 mol/L Triton are added into obtained initial supernatant liquid
X-100,0.06 mol/L disodium ethylene diamine tetraacetate, 0.03 mol/L Proteinase K, 0.7 mol/L lauryl sodium sulfate,
It is put into progress 18 min of digestion reaction in 40 degree of water-baths after mixing, is then centrifuged 1.5 min with 3000 g, is obtained after abandoning precipitating
Digest supernatant；
(2) the digestion supernatant for taking 100 mL steps (1) to obtain is filtered, and the pore size of filter membrane is 0.30 μm, then will
Filter membrane is immersed in the beaker for being contained with 50 mL ATP release liquids, the composition of the ATP release liquid are as follows: 0.05 mol/L
Triton X-100,0.06 mol/L trimethylamino methane, 0.04 mol/L lithium chloride, 3 mmol/L N- lauroyl fleshes
15 μ L apyrases are then added in propylhomoserin sodium, 14 mmol/L guanidinium isothiocyanates, 10 mmol/L urea, will
Beaker is transferred to 40 DEG C of water-bath medium-rate mechanical shaking extractions, and the rate of mechanical shaking extraction is 200 r/min, and the time of mechanical shaking extraction is
9 min obtain ATP extracting solution；
(3) by 10 μ L of luciferase, 30 mg of soluble inorganic magnesium salts, dithiothreitol (DTT) 15 mg and 50 mL in gnotobasis
Sterile water mixing, is mixed with 15 min of rate oscillation of 150 r/min, obtains luminescence-producing reaction liquid；
(4) for the ATP extracting solution that 0.2 mL step (2) of absorption obtains in luminescence-producing reaction pipe, pH 8.0 is added contains 0.14 mol/
L NaCl, 0.025 mol/L EDTA, 0.07 mol/L cyclodextrin Tris-HCl buffer total volume is complemented into 0.9 mL,
The luminescence-producing reaction liquid of 0.1 mL step (3) is added, mixed liquor is placed in detection in ATP fluorescence detector sufficiently after oscillation and is shone
Intensity, detection response time are 25 s.
Embodiment 3
(1) milk to be detected of 50 g is mixed with 1 × PBS buffer solution of 950 g, agitated to obtain mixed liquor, centrifugation removes
Insoluble matter is removed, 200 μ L digestive juices, digestive juice composition are as follows: 0.3 mol/L Triton are added into obtained initial supernatant liquid
X-100,0.07 mol/L disodium ethylene diamine tetraacetate, 0.04 mol/L Proteinase K, 0.9 mol/L lauryl sodium sulfate,
It is put into progress 20 min of digestion reaction in 40 degree of water-baths after mixing, is then centrifuged 2 min with 3000 g, is disappeared after abandoning precipitating
Change supernatant；
(2) the digestion supernatant for taking 100 mL steps (1) to obtain is filtered, and the pore size of filter membrane is 0.35 μm, then will
Filter membrane is immersed in the beaker for being contained with 50 mL ATP release liquids, the composition of the ATP release liquid are as follows: 0.06 mol/L
Triton X-100,0.07 mol/L trimethylamino methane, 0.05 mol/L lithium chloride, 4 mmol/L N- lauroyl fleshes
15 μ L apyrases are then added in propylhomoserin sodium, 15 mmol/L guanidinium isothiocyanates, 12 mmol/L urea, will
Beaker is transferred to 40 DEG C of water-bath medium-rate mechanical shaking extractions, and the rate of mechanical shaking extraction is 200 r/min, and the time of mechanical shaking extraction is
10 min obtain ATP extracting solution；
(3) by 10 μ L of luciferase, 30 mg of soluble inorganic magnesium salts, dithiothreitol (DTT) 15 mg and 50 mL in gnotobasis
Sterile water mixing, is mixed with 20 min of rate oscillation of 150 r/min, obtains luminescence-producing reaction liquid；
(4) for the ATP extracting solution that 0.2 mL step (2) of absorption obtains in luminescence-producing reaction pipe, pH 8.0 is added contains 0.15 mol/
L NaCl, 0.03 mol/L EDTA, 0.08 mol/L cyclodextrin Tris-HCl buffer total volume is complemented into 0.9 mL,
The luminescence-producing reaction liquid of 0.1 mL step (3) is added, mixed liquor is placed in detection in ATP fluorescence detector sufficiently after oscillation and is shone
Intensity, detection response time are 25 s.
Comparative example (in ATP release liquid with bacteriolyze enzymes extraction lithium chloride, N- sodium lauroyl sarcosine, guanidinium isothiocyanate and
Urea)
(1) pork to be detected of 50 g is mixed with 1 × PBS buffer solution of 950 g, agitated to obtain suspension, centrifugation removes
Insoluble matter is removed, 200 μ L digestive juices, digestive juice composition are as follows: 0.2 mol/L Triton are added into obtained initial supernatant liquid
X-100,0.06 mol/L disodium ethylene diamine tetraacetate, 0.03 mol/L Proteinase K, 0.7 mol/L lauryl sodium sulfate,
It is put into progress 18 min of digestion reaction in 40 degree of water-baths after mixing, is then centrifuged 1.5 min with 3000 g, is obtained after abandoning precipitating
Digest supernatant；
(2) the digestion supernatant for taking 100 mL steps (1) to obtain is filtered, and the pore size of filter membrane is 0.30 μm, then will
Filter membrane is immersed in the beaker for being contained with 50 mL ATP release liquids, the composition of the ATP release liquid are as follows: 0.05 mol/L
15 μ L atriphos are then added in Triton X-100,0.06 mol/L trimethylamino methane, 0.5 g/L lysozyme
Beaker is transferred to 40 DEG C of water-bath medium-rate mechanical shaking extractions by bisphosphatase, and the rate of mechanical shaking extraction is 200 r/min, oscillation
The time of extraction is 9 min, obtains ATP extracting solution；
(3) by 10 μ L of luciferase, 30 mg of soluble inorganic magnesium salts, dithiothreitol (DTT) 15 mg and 50 mL in gnotobasis
Sterile water mixing, is mixed with 15 min of rate oscillation of 150 r/min, obtains luminescence-producing reaction liquid；
(4) for the ATP extracting solution that 0.2 mL step (2) of absorption obtains in luminescence-producing reaction pipe, pH 8.0 is added contains 0.14 mol/
L NaCl, 0.025 mol/L EDTA, 0.07 mol/L cyclodextrin Tris-HCl buffer total volume is complemented into 0.9 mL,
The luminescence-producing reaction liquid of 0.1 mL step (3) is added, mixed liquor is placed in detection in ATP fluorescence detector sufficiently after oscillation and is shone
Intensity, detection response time are 25 s.
Reference examples
Embodiment 1-3 sample detected and comparative example sample detected are carried out according to conventional PCR Molecular Detection method flat
Row detection, to separately verify ATP bioluminescence reaction method used in ATP bioluminescence reaction method and comparative example used in the present invention
Detection accuracy.
Embodiment 1-3 is obtained into corresponding bacillus cereus sum through conversion with the fluorescence radiation intensity value of comparative example,
Specific conversion method are as follows: in the luminous intensity of test sample, while blank control is detected using sterile water as blank control
Luminous intensity, sample luminous value take logarithm after subtracting blank luminous value, and it is total to calculate the bacterium in water sample to be measured by luminosity curve
Number, total number of bacteria TBC=N × (505 × 10(A+BLogRLU))/3 × 10-16(a/liter), N=extension rate, RLU are relative light unit,
After A, B are the standard ATP luminosity curve test of ATP luminescence-producing reaction liquid, ATP concentration and net relative light unit obtain after taking double-log
Linear regression luminous equation coefficient.Obtained bacillus cereus sum is examined with the conventional PCR molecule through reference examples
The bacillus cereus sum that survey method is measured is compared one by one, the waxy bud measured with conventional PCR Molecular Detection method
On the basis of spore bacillus sum, calculates embodiment 1-3 and the deviation percentage of bacillus cereus sum that comparative example is measured, tie
Fruit is shown in Table 1.As it can be seen that the method for the invention using bacillus cereus in ATP bioluminescence reaction detection food is only aided with
The ATP release liquid of respective specific component is just able to achieve high-precision detection effect, this is never to report in the prior art, right
The sensitivity for further increasing ATP bioluminescence reaction detection method plays a significant role.
Table 1
| Deviation percentage (%) | |
| Embodiment 1 | -2.03% |
| Embodiment 2 | -1.94% |
| Embodiment 3 | -1.98% |
| Comparative example | -5.57% |
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but can not
Therefore it is construed as limiting the scope of the patent.It should be pointed out that for those of ordinary skill in the art,
Under the premise of not departing from present inventive concept, various modifications and improvements can be made, and these are all within the scope of protection of the present invention.
Claims (9)
1. a kind of method using bacillus cereus in ATP bioluminescence reaction detection food, which is characterized in that including following
Step:
(1) the liquid/solid sample to be detected of 50 g is mixed with 1 × PBS buffer solution of 950 g, it is ground/to stir to get
Suspension/mixed liquor, is centrifuged off insoluble matter, and 200 μ L digestive juices are added into obtained initial supernatant liquid, are put into after mixing
15~20 min of digestion reaction is carried out in 40 degree of water-baths, is then centrifuged 1~2 min with 3000 g, is digested after abandoning precipitating
Supernatant；
(2) the digestion supernatant for taking 100 mL steps (1) to obtain is filtered, and is then immersed in filter membrane and is contained with 50 mL
In the beaker of ATP release liquid, the ATP release liquid includes Triton X-100, trimethylamino methane, lithium chloride, N- laurel
Acylsarcosine sodium, guanidinium isothiocyanate, urea are then added 15 μ L apyrases, beaker are transferred to 40 DEG C of water
Bath medium-rate mechanical shaking extraction, obtains ATP extracting solution；
(3) by 10 μ L of luciferase, 30 mg of soluble inorganic magnesium salts, dithiothreitol (DTT) 15 mg and 50 mL in gnotobasis
Sterile water mixing, oscillation mix, and obtain luminescence-producing reaction liquid；
(4) for the ATP extracting solution that 0.2 mL step (2) of absorption obtains in luminescence-producing reaction pipe, pH 7.0~9.0 is added contains 0.13
The Tris-HCl buffering of~0.15 mol/L NaCl, 0.02~0.03 mol/L EDTA, 0.06~0.08 mol/L cyclodextrin
Total volume is complemented to 0.9 mL by liquid, adds the luminescence-producing reaction liquid of 0.1 mL step (3), is sufficiently placed in mixed liquor after oscillation
Luminous intensity is detected in ATP fluorescence detector, the detection response time is 20~30 s.
2. the method according to claim 1 using bacillus cereus in ATP bioluminescence reaction detection food, special
Sign is that the digestive juice in the step (1) forms are as follows: 0.1~0.3 mol/L Triton X-100,0.05~0.07
Mol/L disodium ethylene diamine tetraacetate, 0.02~0.04 mol/L Proteinase K, 0.5~0.9 mol/L lauryl sodium sulfate.
3. the method according to claim 1 using bacillus cereus in ATP bioluminescence reaction detection food, special
Sign is that the pore size of filter membrane is 0.25~0.35 μm in the step (2).
4. the method according to claim 1 using bacillus cereus in ATP bioluminescence reaction detection food, special
Sign is, the composition of ATP release liquid in the step (2) are as follows: 0.04~0.06 mol/L Triton X-100,0.05~
0.07 mol/L trimethylamino methane, 0.03~0.05 mol/L lithium chloride, 2~4 mmol/L N- Hamposyl Ls
Sodium, 13~15 mmol/L guanidinium isothiocyanates, 8~12 mmol/L urea.
5. the method according to claim 1 using bacillus cereus in ATP bioluminescence reaction detection food, special
Sign is that the rate of mechanical shaking extraction is 200 r/min in the step (2), and the time of mechanical shaking extraction is 8~10 min.
6. the method according to claim 1 using bacillus cereus in ATP bioluminescence reaction detection food, special
Sign is that soluble inorganic magnesium salts is any one or a few in magnesium chloride, magnesium nitrate and magnesium sulfate in the step (3).
7. the method according to claim 1 using bacillus cereus in ATP bioluminescence reaction detection food, special
Sign is that it is specifically with 10~20 min of rate oscillation of 150 r/min that the oscillation in the step (3), which mixes,.
8. the method according to claim 1 using bacillus cereus in ATP bioluminescence reaction detection food, special
Sign is that the pH of Tris-HCl buffer is 8.0 in the step (4).
9. the method according to claim 1 using bacillus cereus in ATP bioluminescence reaction detection food, special
Sign is, it is 25 that mixed liquor, which is placed in ATP fluorescence detector and detects the detection response time of luminous intensity, in the step (4)
s。
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1740186A (en) * | 2005-09-01 | 2006-03-01 | 上海交通大学 | Fast extraction method of adnascent microbe community total DNA of sponge |
| CN101565700A (en) * | 2009-05-08 | 2009-10-28 | 广州中医药大学 | Reagent for extracting RNA or DNA virus in body fluid |
| CN101748176A (en) * | 2008-12-08 | 2010-06-23 | 中华人民共和国黑龙江出入境检验检疫局检验检疫技术中心 | High-efficiency extracting method of food-borne pathogen nucleic acid |
| CN102183648A (en) * | 2011-01-26 | 2011-09-14 | 中国科学院上海微系统与信息技术研究所 | Detection method and detection kit for detecting special pathogenic bacteria by bioluminescence |
| CN103146806A (en) * | 2013-03-18 | 2013-06-12 | 中国人民解放军军事医学科学院微生物流行病研究所 | Method for detecting bacillus anthracis |
| CN105624152A (en) * | 2016-03-01 | 2016-06-01 | 中国人民解放军第二军医大学 | Instrument-free yeast-like fungus DNA extraction method used for nucleic acid amplification |
| CN107119039A (en) * | 2016-02-24 | 2017-09-01 | 北京百泰克生物技术有限公司 | It is a kind of to organize not grinding the method for directly extracting nucleic acid |
| CN108587863A (en) * | 2018-04-28 | 2018-09-28 | 山东森芃生物科技有限公司 | The method and kit of spermatid DNA in assault sexually case sample is extracted in a kind of differentiation cracking |
-
2018
- 2018-11-30 CN CN201811451396.6A patent/CN109632733A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1740186A (en) * | 2005-09-01 | 2006-03-01 | 上海交通大学 | Fast extraction method of adnascent microbe community total DNA of sponge |
| CN101748176A (en) * | 2008-12-08 | 2010-06-23 | 中华人民共和国黑龙江出入境检验检疫局检验检疫技术中心 | High-efficiency extracting method of food-borne pathogen nucleic acid |
| CN101565700A (en) * | 2009-05-08 | 2009-10-28 | 广州中医药大学 | Reagent for extracting RNA or DNA virus in body fluid |
| CN102183648A (en) * | 2011-01-26 | 2011-09-14 | 中国科学院上海微系统与信息技术研究所 | Detection method and detection kit for detecting special pathogenic bacteria by bioluminescence |
| CN103146806A (en) * | 2013-03-18 | 2013-06-12 | 中国人民解放军军事医学科学院微生物流行病研究所 | Method for detecting bacillus anthracis |
| CN107119039A (en) * | 2016-02-24 | 2017-09-01 | 北京百泰克生物技术有限公司 | It is a kind of to organize not grinding the method for directly extracting nucleic acid |
| CN105624152A (en) * | 2016-03-01 | 2016-06-01 | 中国人民解放军第二军医大学 | Instrument-free yeast-like fungus DNA extraction method used for nucleic acid amplification |
| CN108587863A (en) * | 2018-04-28 | 2018-09-28 | 山东森芃生物科技有限公司 | The method and kit of spermatid DNA in assault sexually case sample is extracted in a kind of differentiation cracking |
Non-Patent Citations (1)
| Title |
|---|
| 王建荣: "微生物检验中ATP发光法的应用", 《健康必读》 * |
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