CN105203578B - A kind of method for detecting microorganism - Google Patents
A kind of method for detecting microorganism Download PDFInfo
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- CN105203578B CN105203578B CN201510632772.1A CN201510632772A CN105203578B CN 105203578 B CN105203578 B CN 105203578B CN 201510632772 A CN201510632772 A CN 201510632772A CN 105203578 B CN105203578 B CN 105203578B
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Abstract
The present invention relates to field of detection of food safety, discloses a kind of method for detecting microorganism, including：1) biomolecular is mixed with the testing sample of liquid form, obtains mixed material；The condition of mixing make it that when microorganism in testing sample be present biomolecular specifically can be coupled to obtain with reference to the microorganism, the biomolecular that specifically can be combined with microorganism by antibody and magnetic ball；2) the mixed material is placed in sorting magnetic field and carries out Magneto separate, removing is not associated with the material on biomolecular；3) sediment that Magneto separate obtains is resuspended, and obtained re-suspension liquid is subjected to nuclear magnetic resonance and detects T2, the content of the microorganism in testing sample is judged according to T2.The present invention can fast and effeciently detect the content of microorganism in sample (particularly bacterium, fungi or virus etc.), and operation is simple and high specificity, high sensitivity, in the great application prospect in trace microorganism detection field.
Description
Technical field
The present invention relates to field of detection of food safety, in particular it relates to which a kind of detection microorganism (is particularly harmful to micro- life
Thing) method.
Background technology
Food security is great livelihood issues.Food-borne microbial pathogens are effectively monitored for capital food quality control
Make and supervise and be particularly important.For the pathogenic microorganism (such as bacterium, fungi and virus) in food and other harmful organisms
(such as parasite), traditional detection method are isolated culture.Increase bacterium, increasing bacterium, biochemical identification or serum before being separately cultured needs
Identification is learned, whole detection process needs the time of even 5-6 days 2-3 days to complete, and the culture needed for detection process
Benchmark is standby, flat board culture, bacterium colony counting, biochemical identification etc. make it that Detection task labor intensity is big, takes, it is impossible in time, quick
The security of microorganism in food is evaluated, is unfavorable for fast reaction of the supervision department to problem food.The micro- life of traditional detection cause of disease
Thing method and step is more, and required time length, speed is slow, it is difficult to adapts to food and quickly circulates the needs of detection；Modern microbiological detects
Although method detection speed is fast, missing inspection is easily caused in the case where bacterial concentration is relatively low, sensitivity is low.
The content of the invention
The purpose of the present invention is overcome the deficiencies in the prior art, there is provided one kind is simple to operate, detection time is short, high sensitivity
Detection microorganism method.
The present inventor has carried out substantial amounts of research, finds with the use of immune magnetic separation technique and nuclear magnetic resonance skill
Art is that the high-sensitivity detection of microorganism can be achieved.Therefore, to achieve these goals, the invention provides one kind to detect micro- life
The method of thing, this method comprise the following steps：
(1) biomolecular is mixed with the testing sample of liquid form, obtains mixed material；The condition of mixing
So that when microorganism in testing sample be present, biomolecular can specifically with reference to the microorganism, it is described can be special
The biomolecular that ground is combined with microorganism is coupled to obtain by antibody and magnetic ball；
(2) the mixed material is placed in sorting magnetic field and carries out Magneto separate, removing is not associated with biomolecular
Material；
(3) sediment that Magneto separate obtains is resuspended, and obtained re-suspension liquid is subjected to nuclear magnetic resonance and detects transverse relaxation
Time, the content of the microorganism in testing sample is judged according to T2.
Pass through above-mentioned technical proposal, the present invention can fast and effeciently detect microorganism in sample (particularly bacterium, fungi
Or virus etc.) content, operation is simple and high specificity, high sensitivity, in the great application in trace microorganism detection field
Prospect.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Brief description of the drawings
Accompanying drawing is for providing a further understanding of the present invention, and a part for constitution instruction, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the content and △ T of the microorganism measured according to one embodiment of the present invention2Graph of a relation between value；
Fig. 2 is the content and △ T of the microorganism measured according to another embodiment of the invention2Relation between value
Figure；
Fig. 3 is the content and △ T of the microorganism measured according to another embodiment of the invention2Relation between value
Figure.
Embodiment
The embodiment of the present invention is described in detail below.It is it should be appreciated that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to limit the invention.
In the present invention, in the case where not making opposite explanation, the unit " CFU/mL " that uses means to be measured with every milliliter
The CFU numbers of bacterial strain on the basis of sample；The term " biomolecular " used refers to antibody binding to magnetic by coupling reaction
On the surface of microballoon (or magnetic ball), the immune magnetic microsphere of formation；Term " T2 " refers to that two are in a spacing
Claim from core interaction interior, that precession frequency is identical, precession orientation is different, positive energy exchange, the time needed for change precession direction
For T2 (or spin spin relaxation time, T2)；The liquid volume used in the present invention is the number at 20 DEG C
Value；The term " room temperature " being related to refers to indoor temperature, usually 5-40 DEG C.
The method of detection microorganism provided by the invention comprises the following steps：
(1) biomolecular is mixed with the testing sample of liquid form, obtains mixed material；The condition of mixing
So that when microorganism in testing sample be present, biomolecular can specifically with reference to the microorganism, can specifically with
The biomolecular that microorganism combines is coupled to obtain by antibody and magnetic ball；
(2) the mixed material is placed in sorting magnetic field and carries out Magneto separate, removing is not associated with biomolecular
Material；
(3) sediment that Magneto separate obtains is resuspended, and obtained re-suspension liquid is subjected to nuclear magnetic resonance and detects transverse relaxation
Time, the content of the microorganism in testing sample is judged according to T2.
The detection method of the present invention is preferably external detection method.
According to the present invention, the microorganism can be various common microorganisms, particularly harmful microorganism (or cause a disease
Bacterium), such as bacterium, fungi or virus.The antibody can be selected according to the specific species of microorganism, as long as being capable of specificity
Ground is with reference to the microorganism so as to realizing immune Magneto separate.Under preferable case, the microorganism is salmonella
(Salmonella), the antibody is the KPL polyclonal antibodies that numbering is 01-91-99.
According to the present invention, the method that antibody obtains the biomolecular (or immunomagnetic beads) with the coupling of magnetic ball can be conventional
Method, for example, can (prepared by Liu Huirong etc., simple and effective separation cell novel immune magnetic bead, China is public to be defended according to document
It is raw, 2008,11:Method in 1349-1351) is carried out.
Preferably, relative to every gram of magnetic ball, the dosage of the antibody for 10-150mg (more preferably 10mg, 30mg,
50mg, 70mg, 80mg, 90mg, 95mg, 100mg, 105mg, 110mg, 120mg, 140mg, 150mg or above-mentioned any two
Scope between numerical value).
Preferably, relative to the testing sample of every milliliter of liquid form, the dosage of the biomolecular is 100-600 μ g
(100 μ g, 200 μ g, 300 μ g, 380 μ g, 390 μ g, 400 μ g, 410 μ g, 420 μ g, 450 μ g, 500 μ g, 550 μ g, 600 μ g or
Scope between above-mentioned any two numerical value).
The magnetic ball can be the various magnetic balls conventionally used for Magneto separate is immunized, for example, can be surface modification carboxyl
The Fe of polyethylene glycol (PEG) parcel3O4Complex microsphere and/or surface modification have the Fe of the glucan parcel of carboxyl3O4It is compound micro-
Ball.The magnetic ball can be by commercially available, for example, 79-56-201,79-56-102 and 79- of Micromod Co., Ltds
At least one of 56-501.
Under preferable case, the particle diameter of the magnetic ball for 10-200nm (such as 10nm, 20nm, 30nm, 35nm, 40nm, 45nm,
50nm、55nm、60nm、65nm、70nm、75nm、80nm、90nm、100nm、110nm、120nm、130nm、140nm、150nm、
Scope between 160nm, 170nm, 180nm, 190nm, 200nm or above-mentioned any two numerical value).
According to the present invention, in step (1), the condition of the mixing is not required particularly, as long as microorganism can be made
Specifically combined by the biomolecular.The condition of the mixing can include：Temperature is room temperature, time 25-
35min.Mixing can be carried out in phosphate buffer, and the phosphate buffer contains 0.144-0.162mol/L phosphoric acid
The sodium dihydrogen phosphate of disodium hydrogen and 0.038-0.056mol/L.
According to the present invention, being placed in sorting magnetic field the Magneto separate that carries out can be by immune magnetic separation system (such as magnetic force
Magnetic separation system is immunized in frame or Matrix Pathatrix) carry out, to the condition of the Magneto separate, there is no particular limitation, preferably
Ground, in step (2), the condition of the Magneto separate includes：Temperature is room temperature, time 25-35min.
According to the present invention, in step (3), those skilled in the art can be selected the dosage of solution used in resuspension,
For example, relative to every milligram of sediment, the dosage of solution used in resuspension is 0.5-1.5mL.Solution used, which is resuspended, to be
The solution of the conventional biomolecular that is used to suspend, preferably phosphate buffer, the phosphate buffer contain 0.144-
0.162mol/L disodium hydrogen phosphate and 0.038-0.056mol/L sodium dihydrogen phosphate.The phosphoric acid preferably used in step (1)
Salt buffer can be with identical or different with the phosphate buffer preferably used in step (3).
According to the present invention, the condition of the nuclear magnetic resonance includes：Scanning times (scans) are 4-8 times, the duplicate delays time
(recycle delay) is 1.5-2.5s, and receiver multiplication factor (gain) is 50-60dB, the duplicate measurements time
(repetition time) is 10s.
In the present invention, the △ T of magnetic resonance detection2Value (the T that testing sample measures with blank sample2Difference) with combine
The content (St) for having the biomolecular of microorganism is in proportionate relationship (△ T2=alg (St)+b, a and b is constant), therefore, inspection
Standard sample known to content of microorganisms is surveyed, obtains △ T2After relation between content, you can according to the △ T of testing sample2Value
Judge the content of the microorganism in testing sample.
According to the preferred embodiment of the present invention, the microorganism is salmonella, and the testing sample is egg, described
The particle diameter of magnetic ball is 30-80nm (such as 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm
Or the scope between above-mentioned any two numerical value).It was found by the inventors of the present invention that use magnetic ball system of the particle diameter for 30-80nm
The biomolecular obtained carries out Magneto separate and coordinates nuclear magnetic resonance technique particularly effectively to detect the salmonella in egg.
The method according to the invention, wherein, testing sample can be food and/or medicine.Specifically, can be according to country
Standard GB 4789.1-2010《National food safety standard food microbiological examination general provisions》In method obtain testing sample.
The method of the present invention has higher sensitivity, is 1-10 especially suitable for content of microorganisms6CFU/mL's is to be measured
The detection of sample.
The present invention will be described in detail by way of examples below.In following examples, it will be obtained commercially available from KPL companies
Arrive, article number (Cat.#) is 01-91-99 antibody as specific antibody；Magnetic ball is purchased from Micromod Co., Ltds, 20nm
The article No. of magnetic ball is 79-56-201, and the article No. of 50nm magnetic balls is 79-56-102, and the article No. of 100nm magnetic balls is 79-56-501.
Prepare embodiment 1
This preparation embodiment 1 prepares the biomolecular used in embodiment using specific antibody.
1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides (EDC) and n-hydroxysuccinimide will be passed through
(NHS) Fe of the glucan parcel of activation3O4Complex microsphere (is purchased from Micromod Co., Ltds, concentration 5mg/mL, particle diameter is
50nm, surface modification have carboxyl functional group) used as magnetic ball, 2mg magnetic balls are taken, are disperseed in phosphate buffer solution, in dispersion liquid
The concentration of magnetic ball is 2mg/mL.0.2mg specific antibodies (being dissolved in 1mL phosphate buffer) are taken to be mixed with 2mg magnetic balls, 25 DEG C
Under 3h is kept in the shaking table (200r/min), then cleaned by Magneto separate, removal is not coupled to the special of magnetic ball surface
Property antibody.Then with the bovine serum albumin solution (being dissolved in phosphate buffer) that concentration is 1% (w/v), closed at 37 DEG C
30min, then cleaned by Magneto separate, wash away unnecessary bovine serum albumin solution, that is, obtain biomolecular.
Embodiment 1
Using phosphate buffer (PBS, purchased from Biotopped) and salmonella (salmonella typhimurium,
Salmonella enteritidis, purchased from ATCC, article No. 14028) prepare various concentrations the sample solution containing salmonella
(100-106CFU/mL), take 1mL sample solutions to be added to 400 μ g to prepare in biomolecular made from embodiment 1, be placed in blending instrument
Upper (25 DEG C) incubation 30min of room temperature.Then, reaction solution is placed in room temperature on magnetic frame (25 DEG C) Magneto separate 30min, carefully taken
Go out supernatant, be resuspended with PBS.Re-suspension liquid is added in nuclear magnetic tube the nuclear magnetic resonance (condition that is used for：Scanning times are 4 times,
The duplicate delays time is 2s, and receiver multiplication factor is 56dB, and the duplicate measurements time is 10s) detect T2Value, while do blank
Control test T20Value, so as to calculate △ T2It is worth (T2- T20), obtain the concentration of microorganism and △ T2Between value graph of a relation (referring to
Fig. 1).
It will be seen from figure 1 that △ T2Value and the concentration (St) of salmonella are in proportionate relationship, △ T2=0.65908lg (St)
+ 7.2851, R2=0.99681, therefore, can be according to T2The concentration of value detection salmonella.
Embodiment 2
The T of the different sample solutions containing salmonella is detected in the way of embodiment 12Value, unlike, the magnetic used
The particle diameter of ball is 20nm, as a result (△ T as shown in Figure 22=0.26625lg (St)+2.00597, R2=0.98341).
Embodiment 3
The T of the different sample solutions containing salmonella is detected in the way of embodiment 12Value, unlike, the magnetic used
The particle diameter of ball is 100nm, as a result (△ T as shown in Figure 32=0.08763lg (St)+1.4692, R2=0.98094).
From embodiment 1-3 can be seen that the present invention method have quick, sensitive, simple, time it is short (<It is 1h) etc. obvious
Advantage, and its detection limit is relatively low, suitable for tackling emergency case, has good development in fields such as food security, environmental sanitations
Prospect.In addition, compare Fig. 1-3 as can be seen that can further improve the degree of accuracy of detection using the magnetic ball of preferable particle size.
Embodiment 4
The present embodiment is used for the method for illustrating the salmonella of the method detection egg eggshell surface according to the present invention.
Egg is placed in 30mL PBS and stands 5min, taken 1mL sample solutions to be added to made from preparation embodiment 1 and exempt from
In epidemic disease magnetic ball, remaining step is carried out according to the method described in embodiment 1, the △ T measured2It is worth for 9.034ms, sample is calculated
The content of salmonella is 446CFU/mL in product solution.
According to (the national food safety standard food microbiological examination Salmonella of method specified in GB 4789.4-2010
Bacterium is examined), the thalline number that colony counting method is carried out to sample solution detects, and finally gives containing for salmonella in sample solution
Measure as 451CFU/mL.
This example demonstrates that the method for the present invention has the higher degree of accuracy.
The preferred embodiment of the present invention described in detail above, still, the present invention are not limited in above-mentioned embodiment
Detail, in the range of the technology design of the present invention, a variety of simple variants can be carried out to technical scheme, this
A little simple variants belong to protection scope of the present invention.
It is further to note that each particular technique feature described in above-mentioned embodiment, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should equally be considered as content disclosed in this invention.
Claims (8)
- A kind of 1. method for detecting microorganism, it is characterised in that this method comprises the steps of：(1) biomolecular is mixed with the testing sample of liquid form, obtains mixed material；The condition of mixing causes When microorganism in testing sample be present, biomolecular can specifically with reference to the microorganism, it is described can specifically with The biomolecular that microorganism combines is coupled to obtain by antibody and magnetic ball, and the testing sample is food and/or medicine, the magnetic ball Particle diameter be 10-80nm；(2) the mixed material is placed in sorting magnetic field and carries out Magneto separate, removing is not associated with the thing on biomolecular Matter；(3) sediment that Magneto separate obtains is resuspended, and obtained re-suspension liquid is subjected to nuclear magnetic resonance and detects T2, The content of the microorganism in testing sample is judged according to T2；Wherein, the microorganism is salmonella (Salmonella), and the antibody is that the KPL that numbering is 01-91-99 is polyclonal Antibody；The magnetic ball is the Fe for the polyethylene glycol parcel that surface modification has carboxyl3O4Complex microsphere and/or surface modification have carboxyl The Fe of glucan parcel3O4Complex microsphere.
- 2. according to the method for claim 1, wherein, relative to every gram of magnetic ball, the dosage of the antibody is 10-150mg.
- 3. the method according to claim 11, wherein, it is described immune relative to the testing sample of every milliliter of liquid form The dosage of magnetic ball is 100-600 μ g.
- 4. according to the method for claim 1, wherein, the particle diameter of the magnetic ball is 30-80nm.
- 5. according to the method for claim 1, wherein, in step (1), the condition of the mixing includes：Temperature is room temperature, when Between be 25-35min.
- 6. according to the method for claim 1, wherein, in step (2), the condition of the Magneto separate includes：Temperature is room temperature, Time is 25-35min.
- 7. according to the method for claim 1, wherein, in step (3), it is phosphate buffer that solution used, which is resuspended, institute State disodium hydrogen phosphate that phosphate buffer contains 0.144-0.162mol/L and 0.038-0.056mol/L sodium dihydrogen phosphate.
- 8. according to the method for claim 1, wherein, the microorganism is salmonella, the testing sample is egg, institute The particle diameter for stating magnetic ball is 30-80nm.
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| US7172904B2 (en) * | 2002-07-31 | 2007-02-06 | Freescale Semiconductor, Inc. | High sensitivity sensor for tagged magnetic bead bioassays |
| DE10331439B3 (en) * | 2003-07-10 | 2005-02-03 | Micromod Partikeltechnologie Gmbh | Magnetic nanoparticles with improved magnetic properties |
| WO2012054638A2 (en) * | 2010-10-22 | 2012-04-26 | T2 Biosystems, Inc. | Nmr systems and methods for the detection of analytes |
| CN103207198B (en) * | 2013-01-23 | 2015-09-30 | 南昌大学 | A kind of based on Fe 3o 4the food-borne pathogens NMR detection method of nano material |
| CN103175858B (en) * | 2013-03-17 | 2016-06-22 | 南昌大学 | A kind of based on Fe3O4The NMR food-borne pathogen rapid detection of nano particle indirect enrichment |
| CN104155325B (en) * | 2014-07-09 | 2017-01-11 | 上海美吉医学检验有限公司 | Sample-transfer-free and low-field nuclear magnetic resonance (NMR) rapid rare cell detection method based on magnetic microspheres |
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| 表面羧基化Fe3O4磁性纳米粒子的快捷制备和表征;苏鹏飞;《高等学校化学学报》;20110731;第32卷(第7期);1472-1477 * |
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