CN104651479A - Method for detecting living bacteria body in sample - Google Patents
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- CN104651479A CN104651479A CN201310582552.3A CN201310582552A CN104651479A CN 104651479 A CN104651479 A CN 104651479A CN 201310582552 A CN201310582552 A CN 201310582552A CN 104651479 A CN104651479 A CN 104651479A
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Abstract
The invention discloses a method for detecting living bacteria bodies in a sample. The method includes following steps: (1) coupling an antibody capable of being specifically combined with the living bacteria bodies onto magnetic balls to obtain immune magnetic balls, wherein the antibody capable of being specifically combined with the living bacteria bodies cannot be combined with dead bacteria bodies; (2) mixing the immune magnetic balls with a liquid sample to obtain a mixture, wherein the mixing condition is described as that when the living bacteria bodies exists in the sample, the immune magnetic balls can specifically adsorb the living bacteria bodies; (3) placing the mixture in a separation magnetic field for immobilizing the immune magnetic balls and eluting substances being not adsorbed on the immune magnetic balls to obtain eluted immune magnetic balls; (4) performing DNA extraction to substances adsorbed onto the immune magnetic balls to obtain a template sample; (5) performing PCR amplification to the template sample with a specific primer aiming to the living bacteria bodies, and determining the content of the living bacteria bodies in the sample according to the content of targeted fragments in a PCR amplification product.
Description
Technical field
The present invention relates to food, environment and biomedicine field, particularly, relate to a kind of method detecting viable bacteria body in sample.
Background technology
Pathogenic bacterium (Pathogenic bacteria) can cause the microorganism of disease to be called pathogenic micro-organism or pathogenic bacterium.Pathogenic micro-organism comprises bacterium, virus, spirochete, Rickettsiae, chlamydozoan, mycoplasma, fungi and actinomycetes etc.General said pathogenic bacterium refer to the bacterium in pathogenic micro-organism.Pathogenic and its virulence of bacterium, invade quantity and portal entry relevant.Although most bacterium is harmless even useful, a large portion can be caused a disease.Conditioned pathogen is only caused a disease under given conditions, bacterium can be allowed to enter blood if any wound, or when immunizing power reduces.Such as, streptococcus aureus and suis are also normal microfloras, often may reside in skin surface, nasal cavity and do not cause disease, but potentially can cause skin infections, as pneumonia (pneumonia), meningitis (meningitis) and septicemia (sepsis) etc.
At present, the method detecting pathogenic bacterium can only detect the total number of the pathogenic bacterium in sample, and the quantity of the viable bacteria body can not distinguished in sample and dead thalline is respectively how many.
Summary of the invention
In order to the dead thalline in testing sample and viable bacteria tagma not come, the invention provides a kind of method detecting viable bacteria body in sample, the method comprises: the antibody coupling that (1) can be combined with viable bacteria body specifically, on magnetic ball, obtains biomolecular; The described antibody that can be combined with viable bacteria body specifically can not be combined with dead thalline; (2) biomolecular is mixed with the testing sample of liquid form, obtain mixed material; The condition of mixing makes when there is viable bacteria body in testing sample, and biomolecular can adsorb viable bacteria body specifically; (3) described mixed material is placed in sorting magnetic field with fixing biomolecular, and wash-out is not adsorbed on the material on biomolecular, obtains the biomolecular after wash-out; (4) material be adsorbed on biomolecular is carried out the operation of extracting DNA, obtain template sample; (5) use the Auele Specific Primer for described viable bacteria body to carry out pcr amplification to template sample, and judge the content of the viable bacteria body in testing sample according to the content of the object fragment in pcr amplification product
By technique scheme, the dead thalline in testing sample and viable bacteria tagma can not come by the present invention effectively.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
In the present invention, when not doing contrary explanation, the term " biomolecular " of use to refer to by linked reaction by antibodies on the surface of magnetic microsphere, the immune magnetic microsphere of formation.Term " PCR " refers to polymerase chain reaction.Term " primer " comprises at least one upstream primer and at least one downstream primer.The liquid volume used in the present invention is the numerical value at 20 DEG C.
The invention provides a kind of method detecting viable bacteria body in sample, the method comprises: the antibody coupling that (1) can be combined with viable bacteria body specifically, on magnetic ball, obtains biomolecular; The described antibody that can be combined with viable bacteria body specifically can not be combined with dead thalline; (2) biomolecular is mixed with the testing sample of liquid form, obtain mixed material; The condition of mixing makes when there is viable bacteria body in testing sample, and biomolecular can adsorb viable bacteria body specifically; (3) described mixed material is placed in sorting magnetic field with fixing biomolecular, and wash-out is not adsorbed on the material on biomolecular, obtains the biomolecular after wash-out; (4) material be adsorbed on biomolecular is carried out the operation of extracting DNA, obtain template sample; (5) use the Auele Specific Primer for described viable bacteria body to carry out pcr amplification to template sample, and judge the content of the viable bacteria body in testing sample according to the content of the object fragment in pcr amplification product.
Wherein, the mode of the antibody that acquisition can be combined with viable bacteria body specifically can be: will be detected the antibody determining to be combined with thalline specifically by cross reaction, carry out viable bacteria body neutralization reaction to detect, the antibody that removal can be combined with dead thalline, remaining antibody is can not be combined with dead thalline but the antibody that can be combined with viable bacteria body specifically.
Wherein, the method for antibody coupling on magnetic ball can be carried out according to method well known in the art, such as, according to document (Liu Huirong etc., prepared by simple and effective isolated cell Novel immune magnetic bead, Chinese public health, 2008,11:1349-1351) in method carry out.
Wherein, the condition of mixing can comprise: temperature is 35-37 DEG C, and the time is 180min; Mixing can be carried out in phosphoric acid buffer, and described phosphoric acid buffer contains the Sodium phosphate dibasic of 0.144-0.162mol/L and the SODIUM PHOSPHATE, MONOBASIC of 0.038-0.056mol/L.
Wherein, as the particularly preferred a kind of embodiment of the present invention, described viable bacteria body is Escherichia coli O 157 (Escherichia coli, O157:H7), the described antibody that can be combined with viable bacteria body is specifically the KPL polyclonal antibody being numbered 01-95-90, this antibody can be commercially available from KPL company, and article number (Cat.#) is 01-95-90.
Wherein, as the particularly preferred a kind of embodiment of the present invention, described viable bacteria body is Salmonellas (Salmonella typhimurium (Salmonella typhimurium), the described antibody that can be combined with viable bacteria body is specifically the KPL polyclonal antibody being numbered 01-95-99, this antibody can be commercially available from KPL company, and article number (Cat.#) is 01-95-99.
Wherein, as the particularly preferred a kind of embodiment of the present invention, described viable bacteria body is Shigellae (shigella dysenteriae, Shigella dysenteriae), the described antibody that can be combined with viable bacteria body is specifically the Meridian polyclonal antibody being numbered B65901R, this antibody can be commercially available from Meridian company, and article number (Cat.#) is B65901R.
Wherein, as the particularly preferred a kind of embodiment of the present invention, described viable bacteria body is streptococcus aureus (Staphyloccocus aureus), the described antibody that can be combined with viable bacteria body is specifically the KPL polyclonal antibody being numbered 01-90-05, this antibody can be commercially available from KPL company, and article number (Cat.#) is 01-90-05.
Wherein, under preferable case, the magnetic ball relative to every milligram, the consumption of described antibody is 0.05-2mg.
Wherein, under preferable case, the testing sample of the liquid form relative to every milliliter, with the weighing scale of magnetic ball, the consumption of described biomolecular is 10-25 μ g.
Wherein, in step (3), wash-out liquid used is phosphoric acid buffer, and described phosphoric acid buffer contains the Sodium phosphate dibasic of 0.144-0.162mol/L and the SODIUM PHOSPHATE, MONOBASIC of 0.038-0.056mol/L.
Wherein, as the particularly preferred a kind of embodiment of the present invention, when described viable bacteria body is Escherichia coli O 157 (Escherichia coli, O157:H7), time, pcr amplification primer used comprises forward primer as shown in SEQ ID NO:1 (5 '-AATAGCCTGGTAGTCTTGT-3 ') and the reverse primer as shown in SEQ ID NO:2 (5 '-GTTTGATAAATCGTGGTCT-3 '); Or, when described viable bacteria body is Salmonellas, pcr amplification primer used comprises forward primer as shown in SEQ ID NO:3 (5 '-TCCTCCGCTCTGTCTACTTA-3 ') and the reverse primer as shown in SEQ ID NO:4 (5 '-ACCGAAATATTCATTGACGTT-3 '); Or, when described viable bacteria body is Shigellae, pcr amplification primer used comprises forward primer as shown in SEQ ID NO:5 (5 '-CATGGCTGGAAAAACTCAGTGC-3 ') and the reverse primer as shown in SEQ ID NO:6 (5 '-CTCATACTTCTGCTCTTCTGCC-3 '); Or, when described viable bacteria body is streptococcus aureus, pcr amplification primer used comprises forward primer as shown in SEQ ID NO:7 (5 '-GCGATTGATGGTGATACGGTT-3 ') and the reverse primer as shown in SEQ ID NO:8 (5 '-AGCCAAGCCTTGACGAACTAAAGC-3 ').
Wherein, under preferable case, described magnetic ball is the superparamagnetism Fe that finishing has amino or carboxyl
3o
4polystyrene complex microsphere.
According to method of the present invention, wherein, testing sample can be food and/or medicine.Particularly, testing sample can be obtained according to the method in standard GB/T 4789.1-2010 " national food safety standard food microbiological analysis general provisions ".
Below will be described the present invention by embodiment.In following examples, described phosphoric acid buffer is the aqueous solution of the SODIUM PHOSPHATE, MONOBASIC of Sodium phosphate dibasic containing 0.153mol/L and 0.047mol/L.To be commercially available from KPL company, the antibody that article number (Cat.#) is 01-95-90 is as antibody 1.To be commercially available from KPL company, the antibody that article number (Cat.#) is 01-95-99 is as antibody 2.To be commercially available from Meridian company, the antibody that article number (Cat.#) is B65901R is as antibody 3.To be commercially available from KPL company, the antibody that article number (Cat.#) is 01-90-05 is as antibody 4.
Preparation embodiment 1
This is prepared embodiment 1 and uses antibody 1-antibody 4 to prepare method of the present invention biomolecular used respectively.
The finishing activated by 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC) and N-hydroxy-succinamide (NHS) there is the superparamagnetism Fe of carboxyl
3o
4polystyrene complex microsphere is (purchased from Aorun Weina New Material Science and Technology Co., Ltd., Shanghai, PM3-020 type polymkeric substance magnetic ball, concentration is 10mg/mL, particle diameter is 180nm, finishing has carboxyl functional group) use as magnetic ball, get 2mg magnetic ball, in dispersion phosphate buffer solution, in dispersion liquid, the concentration of magnetic ball is 2mg/mL.Get 0.2mg specific antibody (being dissolved in the phosphoric acid buffer of 1mL) to mix with 2mg magnetic ball, in shaking table (200r/min), keep 3h under room temperature, then cleaned by Magneto separate, remove the specific antibody not being coupled to magnetic ball surface.Then be 1%(w/v by concentration) bovine serum albumin solution (being dissolved in phosphoric acid buffer), at 37 DEG C, close 30min, then cleaned by Magneto separate, wash away unnecessary bovine serum albumin solution, namely obtain biomolecular.
This prepares the biomolecular 1-biomolecular 4 that embodiment is used antibody 1-antibody 4 to prepare respectively.
Embodiment 1
Escherichia coli O 157 (Escherichia coli, O157:H7) (purchased from ATCC, article No. 43895) is cultured to logarithmic phase in LB substratum (Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L), with LB substratum adjustment cell concentration to 10
3individual/mL, as the positive containing Escherichia coli O 157 viable bacteria body.
Above-mentioned positive is obtained after high pressure deactivation (121 DEG C, 15min) not containing Escherichia coli O 157 viable bacteria body, but containing 10
3the negative sample of the dead thalline of individual/mL.
Positive and negative sample are carried out equal-volume mix, namely obtain containing 5 × 10
2the dead thalline of individual/mL and 5 × 10
2the biased sample of the viable bacteria body of individual/mL.
Respectively by the positive of 1mL, negative sample and biased sample and biomolecular 1(with the weighing scale of magnetic ball, 15mg) mix, obtain mixed material, 30min is hatched at 27 DEG C, then in sorting magnetic field, biomolecular is fixed, wash-out is not adsorbed on the material on biomolecular, the material obtaining the biomolecular after wash-out He elute.
Use the bacterial genomes DNA extraction kit (DP302) purchased from Tian Gen biochemical technology company limited, according to the working method of its specification sheets, the operation of extracting DNA is carried out respectively to the biomolecular after wash-out and the material eluted, obtain respectively being enriched in the DNA sample in the material on immunomagnetic beads and the DNA sample in the remaining material of enrichment, using them as template sample.Forward primer as shown in SEQID NO:1 (5 '-AATAGCCTGGTAGTCTTGT-3 ') and the reverse primer as shown in SEQ IDNO:2 (5 '-GTTTGATAAATCGTGGTCT-3 ') is used to carry out pcr amplification (real-time quantitative PCR to template sample, positive Ct value is proofreaded outside using), result is as shown in table 1.
According to the method specified in GB/T4789.36-2008 (microbiological test of food hygiene colon bacillus O157:H7/NM checks first method general culture method), respectively the material on immunomagnetic beads and the remaining material of enrichment are carried out to the viable bacteria body number detection of colony counting method, result is as shown in table 1.
Table 1
Visible according to the data of table 1, the change of the viable bacteria body number that PCR quantifiable signal value and colony counting method obtain is completely the same.
Embodiment 2
By Salmonellas (Salmonella typhimurium, Salmonella enteritidis) (purchased from ATCC, article No. 14028) at LB substratum (Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L) in be cultured to logarithmic phase, with LB substratum adjustment cell concentration to 10
3individual/L, as the positive containing Salmonellas viable bacteria body.
Above-mentioned positive is obtained after high pressure deactivation (121 DEG C, 15min) not containing Salmonellas viable bacteria body, but containing 10
3the negative sample of the dead thalline of individual/L.
Positive and negative sample are carried out equal-volume mix, namely obtain containing 5 × 10
2the dead thalline of individual/L and 5 × 10
2the biased sample of the viable bacteria body of individual/L.
Respectively by the positive of 1mL, negative sample and biased sample and biomolecular 2(with the weighing scale of magnetic ball, 15mg) mix, obtain mixed material, 30min is hatched at 28 DEG C, then in sorting magnetic field, biomolecular is fixed, wash-out is not adsorbed on the material on biomolecular, the material obtaining the biomolecular after wash-out He elute.
Use the bacterial genomes DNA extraction kit (DP302) purchased from Tian Gen biochemical technology company limited, according to the working method of its specification sheets, the operation of extracting DNA is carried out respectively to the biomolecular after wash-out and the material eluted, obtain respectively being enriched in the DNA sample in the material on immunomagnetic beads and the DNA sample in the remaining material of enrichment, using them as template sample.Forward primer as shown in SEQID NO:3 (5 '-TCCTCCGCTCTGTCTACTTA-3 ') and the reverse primer as shown in SEQ IDNO:4 (5 '-ACCGAAATATTCATTGACGTT-3 ') is used to carry out pcr amplification (real-time quantitative PCR to template sample, positive Ct value is proofreaded outside using), result is as shown in table 2.
According to the method specified in GB4789.4-2010 (inspection of national food safety standard food microbiological analysis Salmonellas), respectively the material on immunomagnetic beads and the remaining material of enrichment are carried out to the viable bacteria body number detection of colony counting method, result is as shown in table 2.
Table 2
Visible according to the data of table 2, the change of the viable bacteria body number that PCR quantifiable signal value and colony counting method obtain is completely the same.
Embodiment 3
By Shigellae (shigella dysenteriae, Shigella dysenteriae) (purchased from ATCC, article No. 27853) at LB substratum (Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L) in be cultured to logarithmic phase, with LB substratum adjustment cell concentration to 10
3individual/L, as the positive containing Shigellae viable bacteria body.
Above-mentioned positive is obtained after high pressure deactivation (121 DEG C, 15min) not containing Shigellae viable bacteria body, but containing 10
3the negative sample of the dead thalline of individual/L.
Positive and negative sample are carried out equal-volume mix, namely obtain containing 5 × 10
2the dead thalline of individual/L and 5 × 10
2the biased sample of the viable bacteria body of individual/L.
Respectively by the positive of 1mL, negative sample and biased sample and biomolecular 3(with the weighing scale of magnetic ball, 15mg) mix, obtain mixed material, 30min is hatched at 25 DEG C, then in sorting magnetic field, biomolecular is fixed, wash-out is not adsorbed on the material on biomolecular, the material obtaining the biomolecular after wash-out He elute.
Use the bacterial genomes DNA extraction kit (DP302) purchased from Tian Gen biochemical technology company limited, according to the working method of its specification sheets, the operation of extracting DNA is carried out respectively to the biomolecular after wash-out and the material eluted, obtain respectively being enriched in the DNA sample in the material on immunomagnetic beads and the DNA sample in the remaining material of enrichment, using them as template sample.Forward primer as shown in SEQID NO:5 (5 '-CATGGCTGGAAAAACTCAGTGC-3 ') and the reverse primer as shown in SEQ ID NO:6 (5 '-CTCATACTTCTGCTCTTCTGCC-3 ') is used to carry out pcr amplification (real-time quantitative PCR to template sample, positive Ct value is proofreaded outside using), result is as shown in table 3.
According to the method specified in GB 4789.5-2012 (inspection of national food safety standard food microbiological analysis Shigellae), respectively the material on immunomagnetic beads and the remaining material of enrichment are carried out to the viable bacteria body number detection of colony counting method, result is as shown in table 3.
Table 3
Visible according to the data of table 1, the change of the viable bacteria body number that PCR quantifiable signal value and colony counting method obtain is completely the same.
Embodiment 4
Streptococcus aureus (Staphyloccocus aureus) (purchased from ATCC, article No. 29213) is cultured to logarithmic phase in LB substratum (Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L), with LB substratum adjustment cell concentration to 10
3individual/L, as the positive containing streptococcus aureus viable bacteria body.
Above-mentioned positive is obtained after high pressure deactivation (121 DEG C, 15min) not containing streptococcus aureus viable bacteria body, but containing 10
3the negative sample of the dead thalline of individual/L.
Positive and negative sample are carried out equal-volume mix, namely obtain containing 5 × 10
2the dead thalline of individual/L and 5 × 10
2the biased sample of the viable bacteria body of individual/L.
Respectively by the positive of 1mL, negative sample and biased sample and biomolecular 4(with the weighing scale of magnetic ball, 15mg) mix, obtain mixed material, 30min is hatched at 29 DEG C, then in sorting magnetic field, biomolecular is fixed, wash-out is not adsorbed on the material on biomolecular, the material obtaining the biomolecular after wash-out He elute.
Use the bacterial genomes DNA extraction kit (DP302) purchased from Tian Gen biochemical technology company limited, according to the working method of its specification sheets, the operation of extracting DNA is carried out respectively to the biomolecular after wash-out and the material eluted, obtain respectively being enriched in the DNA sample in the material on immunomagnetic beads and the DNA sample in the remaining material of enrichment, using them as template sample.Forward primer as shown in SEQID NO:7 (5 '-GCGATTGATGGTGATACGGTT-3 ') and the reverse primer as shown in SEQID NO:8 (5 '-AGCCAAGCCTTGACGAACTAAAGC-3 ') is used to carry out pcr amplification (real-time quantitative PCR to template sample, positive Ct value is proofreaded outside using), result is as shown in table 4.
According to the method specified in GB 4789.10-2010 (inspection of food microbiological analysis streptococcus aureus), respectively the material on immunomagnetic beads and the remaining material of enrichment are carried out to the viable bacteria body number detection of colony counting method, result is as shown in table 4.
Table 4
Visible according to the data of table 4, the change of the viable bacteria body number that PCR quantifiable signal value and colony counting method obtain is completely the same.
Visible by the result showing 1-4 in embodiment 1-4, dead thalline quantity in testing sample and the difference of viable bacteria body quantity can come by the present invention effectively, because method of the present invention does not need to carry out yeast culture to testing sample, thus greatly accelerate and simplify the detection of the pathogenic bacterium in sample.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode, in order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. detect a method for viable bacteria body in sample, the method comprises:
(1) antibody coupling that can be combined with viable bacteria body specifically, on magnetic ball, obtains biomolecular; The described antibody that can be combined with viable bacteria body specifically can not be combined with dead thalline;
(2) biomolecular is mixed with the testing sample of liquid form, obtain mixed material; The condition of mixing makes when there is viable bacteria body in testing sample, and biomolecular can adsorb viable bacteria body specifically;
(3) described mixed material is placed in sorting magnetic field with fixing biomolecular, and wash-out is not adsorbed on the material on biomolecular, obtains the biomolecular after wash-out;
(4) material be adsorbed on biomolecular is carried out the operation of extracting DNA, obtain template sample;
(5) use the Auele Specific Primer for described viable bacteria body to carry out pcr amplification to template sample, and judge the content of the viable bacteria body in testing sample according to the content of the object fragment in pcr amplification product.
2. method according to claim 1, wherein, described viable bacteria body is Escherichia coli O 157 (Escherichia coli, O157:H7), and the described antibody that can be combined with viable bacteria body is specifically the KPL polyclonal antibody being numbered 01-95-90.
3. method according to claim 1, wherein, described viable bacteria body is Salmonellas, and the described antibody that can be combined with viable bacteria body is specifically the KPL polyclonal antibody being numbered 01-95-99.
4. method according to claim 1, wherein, described viable bacteria body is Shigellae, and the described antibody that can be combined with viable bacteria body is specifically the Meridian polyclonal antibody being numbered B65901R.
5. method according to claim 1, wherein, described viable bacteria body is streptococcus aureus, and the described antibody that can be combined with viable bacteria body is specifically the KPL polyclonal antibody being numbered 01-90-05.
6. according to the method in claim 1-5 described in any one, wherein, the magnetic ball relative to every milligram, the consumption of described antibody is 0.05-2mg.
7. according to the method in claim 1-5 described in any one, wherein, the testing sample of the liquid form relative to every milliliter, with the weighing scale of magnetic ball, the consumption of described biomolecular is 10-25 μ g.
8. according to the method in claim 1-5 described in any one, wherein, in step (3), wash-out liquid used is phosphoric acid buffer, and described phosphoric acid buffer contains the Sodium phosphate dibasic of 0.144-0.162mol/L and the SODIUM PHOSPHATE, MONOBASIC of 0.038-0.056mol/L.
9. method according to claim 1, wherein, when described viable bacteria body is Escherichia coli O 157 (Escherichia coli, O157:H7), pcr amplification primer used comprises the forward primer as shown in SEQ ID NO:1 and the reverse primer as shown in SEQ ID NO:2;
Or when described viable bacteria body is Salmonellas, pcr amplification primer used comprises the forward primer as shown in SEQ IDNO:3 and the reverse primer as shown in SEQ ID NO:4;
Or when described viable bacteria body is Shigellae, pcr amplification primer used comprises the forward primer as shown in SEQ IDNO:5 and the reverse primer as shown in SEQ ID NO:6;
Or when described viable bacteria body is streptococcus aureus, pcr amplification primer used comprises the forward primer as shown in SEQ ID NO:7 and the reverse primer as shown in SEQ ID NO:8.
10. method according to claim 1, wherein, described magnetic ball is the superparamagnetism Fe that finishing has amino or carboxyl
3o
4polystyrene complex microsphere.
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