CN104651481B - Method for detecting living bacteria body of listeria monocytogenes in sample - Google Patents
Method for detecting living bacteria body of listeria monocytogenes in sample Download PDFInfo
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- CN104651481B CN104651481B CN201310583132.7A CN201310583132A CN104651481B CN 104651481 B CN104651481 B CN 104651481B CN 201310583132 A CN201310583132 A CN 201310583132A CN 104651481 B CN104651481 B CN 104651481B
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Abstract
The invention discloses a method for detecting living bacteria bodies of listeria monocytogenes in a sample. The method includes following steps: (1) coupling an antibody capable of being specifically combined with the living bacteria bodies of listeria monocytogenes onto magnetic balls to obtain immune magnetic balls, wherein the antibody capable of being specifically combined with the living bacteria bodies of listeria monocytogenes 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 of listeria monocytogenes exists in the sample, the immune magnetic balls can specifically adsorb the living bacteria bodies of listeria monocytogenes; (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 of listeria monocytogenes, and determining the content of the living bacteria bodies of listeria monocytogenes in the sample according to the content of targeted fragments in a PCR amplification product.
Description
Technical field
The present invention relates to biomedicine field, in particular it relates to unicellular hyperplasia Listeria is lived in a kind of detection sample
The method of thalline.
Background technology
Pathogenic bacterium (Pathogenic bacteria) can cause the microorganism of disease to be referred to as pathogenic microorganism or pathogenic bacterium.
Pathogenic microorganism includes antibacterial, virus, spirillum, rickettsia, chlamydia, mycoplasma, fungus and actinomycetes etc..General institute
The pathogenic bacterium said refer to the antibacterial in pathogenic microorganism.Pathogenic and its virulence of antibacterial, intrusion quantity and portal of entry have
Close.Although most antibacterials are harmless the most useful, but a large portion can be caused a disease.Conditioned pathogen is only spy
Cause a disease under fixed condition, antibacterial can be allowed to enter blood if any wound, or when immunity reduces.Such as, Staphylococcus aureus
Bacterium and streptococcus are also normal floras, often may reside in skin surface, nasal cavity and do not cause disease, but potential can cause skin
Skinfeel contaminates, such as pneumonia (pneumonia), meningitis (meningitis) and septicemia (sepsis) etc..
Unicellular monocytogenes (Listeria monocytogenes) is called for short Listeria monocytogenes, is a kind of people
The pathogen that poultry is the most ill.It can cause the disease of Lee Salmonella of people and animals, mainly shows as septicemia, meningitis and monokaryon after infection
Cytosis.It is widely present in nature, and present in food, single increasing Lee Salmonella has danger to the safety of the mankind, this bacterium
In the environment of 4 DEG C still can growth and breeding, be chilled food one of the main pathogenic fungi that threatens human health, therefore, at food
In sanitary microorganism inspection, it is necessary to paid attention to.
At present, the method for detection pathogenic bacterium can only detect the total number of the pathogenic bacterium in sample, and cannot distinguish between sample
In unicellular hyperplasia Listeria viable bacteria body and the quantity of dead thalline be respectively how many.
Summary of the invention
In order to the dead thalline quantity in testing sample and unicellular hyperplasia Listeria viable bacteria body quantity are distinguished, this
Invention provides and a kind of detects the method for unicellular hyperplasia Listeria viable bacteria body in sample, and the method includes: (1) can be special
The antibody coupling that strange land is combined with unicellular hyperplasia Listeria viable bacteria body, on magnetic ball, obtains biomolecular；Described can be special
The antibody that strange land is combined with unicellular hyperplasia Listeria viable bacteria body can not be combined with dead thalline；(2) by biomolecular and liquid
The testing sample of form mixes, and obtains mixed material；The condition of mixing makes when existing in testing sample unicellular
During hyperplasia Listeria viable bacteria body, biomolecular can specifically adsorb unicellular hyperplasia Listeria viable bacteria body；(3) by institute
State mixed material to be placed in sorting magnetic field with fixing biomolecular, and the unadsorbed material on biomolecular of eluting,
Biomolecular after eluting；(4) material adsorbed on biomolecular carries out extracting the operation of DNA, obtain template sample；
(5) use the specific primer for described unicellular hyperplasia Listeria viable bacteria body that template sample carries out PCR amplification, and root
The content of unicellular hyperplasia Listeria viable bacteria body in testing sample is judged according to the content of the purpose fragment in pcr amplification product
By technique scheme, the present invention can be effectively by the dead thalline quantity in testing sample and unicellular hypertrophy
Listerella viable bacteria body quantity is distinguished.
Other features and advantages of the present invention will be described in detail in detailed description of the invention part subsequently.
Detailed description of the invention
Hereinafter the detailed description of the invention of the present invention is described in detail.It should be appreciated that described herein specifically
Embodiment is merely to illustrate and explains the present invention, is not limited to the present invention.
In the present invention, in the case of illustrating on the contrary, the term " biomolecular " of use refers to by coupling anti-
Should be by antibodies to the surface of magnetic microsphere, the immune magnetic microsphere of formation.Term " PCR " refers to polymerase chain reaction
Should.Term " primer " includes at least one forward primer and at least one downstream primer.The liquid volume used in the present invention is equal
It is the numerical value at 20 DEG C.
The invention provides and a kind of detect the method for unicellular hyperplasia Listeria viable bacteria body in sample, the method includes:
(1) by the antibody coupling that can be combined with unicellular hyperplasia Listeria viable bacteria body specifically on magnetic ball, obtain
Biomolecular；The described antibody that can be combined with unicellular hyperplasia Listeria 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
Making when there is unicellular hyperplasia Listeria viable bacteria body in testing sample, biomolecular can specifically adsorb unicellular
Hyperplasia Listeria viable bacteria body；
(3) described mixed material is placed in sorting magnetic field with fixing biomolecular, and eluting is unadsorbed in immunity
Material on magnetic ball, obtains the biomolecular after eluting；
(4) material adsorbed on biomolecular carries out extracting the operation of DNA, obtain template sample；
(5) use the specific primer for described unicellular hyperplasia Listeria viable bacteria body that template sample is carried out PCR
Amplification, and judge that the unicellular hyperplasia Listeria in testing sample is lived according to the content of the purpose fragment in pcr amplification product
The content of thalline.
Wherein, as a kind of embodiment specifically preferred according to the invention, described can specifically with unicellular hypertrophy Li Si
The California Bioscience monoclonal antibody that antibody is numbered MC0007 that special bacterium viable bacteria body combines, this antibody can
To be commercially available from California Bioscience company, article number is MC0007.
Wherein, the magnetic ball relative to every milligram, the consumption of described antibody can be 0.05-2mg.
Wherein, the testing sample of the liquid form relative to every milliliter, in terms of the weight of magnetic ball, the use of described biomolecular
Amount can be 10-25 μ g.
Wherein, in step (3), the liquid used by eluting can be phosphate buffer, and described phosphate buffer contains
The disodium hydrogen phosphate of 0.144-0.162mol/L and the sodium dihydrogen phosphate of 0.038-0.056mol/L.
Wherein, as a kind of embodiment specifically preferred according to the invention, for unicellular hyperplasia Listeria viable bacteria body,
Primer used by PCR amplification includes forward primer (the 5 '-CCTAAGACCCCAATCGAAAAGAAA-as shown in SEQ ID NO:1
Reverse primer 3 ') and as shown in SEQ ID NO:2 (5 '-TAGTTCTACATCAACTGAGACAGA-3 ').
Wherein, described magnetic ball can be superparamagnetism Fe that surface is modified with amino or carboxyl3O4Polystyrene is combined micro-
Ball.
The method according to the invention, wherein, testing sample can be food and/or medicine.Specifically, can be according to country
Method in standard GB4789.1-2010 " national food safety standard food microbiological examination general provisions " obtains testing sample.
Hereinafter will be described the present invention by embodiment.In following example, described phosphate buffer is for containing
There are the disodium hydrogen phosphate of 0.153mol/L and the aqueous solution of the sodium dihydrogen phosphate of 0.047mol/L.Will be from California
Bioscience company is commercially available, and article number is that the antibody of MC0007 is as antibody 1.
Preparation embodiment 1
This is prepared embodiment 1 and uses antibody 1 to prepare the biomolecular used by the method for the present invention.
Will be by 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and N-hydroxy-succinamide
(NHS) surface activated is modified with superparamagnetism Fe of carboxyl3O4Polystyrene complex microsphere (moistens micro-nano green wood purchased from Shanghai Austria
Material Science and Technology Ltd., PM3-020 type polymer magnetic ball, concentration is 10mg/mL, and particle diameter is 180nm, and surface is modified with carboxyl official
Can roll into a ball) use as magnetic ball, take 2mg magnetic ball, in dispersion phosphate buffer solution, in dispersion liquid, the concentration of magnetic ball is 2mg/mL.Take
0.2mg specific antibody (being dissolved in the phosphate buffer of 1mL) mixes with 2mg magnetic ball, under room temperature in shaking table (200r/min)
Keep 3h, be then carried out by Magneto separate, remove the specific antibody not being coupled to magnetic ball surface.Then it is 1% by concentration
(w/v) bovine serum albumin solution (being dissolved in phosphate buffer), is closed 30min at 37 DEG C, is then carried out by Magneto separate
Clean, wash away unnecessary bovine serum albumin solution, i.e. obtain biomolecular.
This is prepared embodiment and obtains using the biomolecular 1 of antibody 1 preparation.
Embodiment 1
Unicellular monocytogenes (Listeria monocytogenes) (purchased from ATCC, article No. 35152) is existed
LB culture medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L) is cultivated to logarithmic (log) phase, cultivates keynote with LB
Whole cell concentration is to 103Individual/L, as the positive containing unicellular monocytogenes viable bacteria body.
Obtain not containing unicellular hypertrophy Listeria after high pressure inactivation (121 DEG C, 15min) by above-mentioned positive
Bacterium viable bacteria body, but containing 103The negative sample of the dead thalline of individual/L.
With negative sample, positive is carried out equal-volume mix, i.e. obtain containing 5 × 102The dead thalline of individual/L and 5 ×
102The biased sample of the viable bacteria body of individual/L.
Respectively by the positive of 1mL, negative sample and biased sample with biomolecular 1(in terms of the weight of magnetic ball,
15mg) mixing, obtains mixed material, hatches 30min at 29 DEG C, then fixes biomolecular in sorting magnetic field, washes
De-unadsorbed material on biomolecular, obtains the biomolecular after eluting and the material eluted.
Use the bacterial genomes DNA extraction kit (DP302) purchased from Tian Gen biochemical technology company limited, say according to it
The operational approach of bright book, carries out extracting the operation of DNA to the biomolecular after eluting respectively with the material eluted, obtains respectively
To the DNA sample being enriched in the material on immunomagnetic beads and the DNA sample being enriched with in remaining material, using them as template
Sample.Use forward primer as shown in SEQ ID NO:1 (5 '-CCTAAGACCCCAATCGAAAAGAAA-3 ') and such as SEQ
It is (real that reverse primer shown in ID NO:2 (5 '-TAGTTCTACATCAACTGAGACAGA-3 ') carries out PCR amplification to template sample
Time quantitative PCR, use and outer proofread positive Ct value), result is as shown in table 1.
According to method (the food microbiological examination Listeria Monocytogenes of regulation in GB4789.30-2010
Inspection), respectively the material on immunomagnetic beads and the remaining material of enrichment are carried out the viable bacteria body number detection of colony counting method,
Result is as shown in table 1.
Table 1
Data according to table 1 are visible, and the change of the viable bacteria body number that PCR quantifiable signal value and colony counting method obtain is complete
Unanimously.
Visible by the result of table 1 in embodiment 1, the present invention can be effectively by hypertrophy Li Si unicellular in testing sample
Dead thalline quantity and the viable bacteria body quantity of special Salmonella are distinguished, owing to the method for the present invention need not testing sample is carried out bacterium
Body is cultivated, thus greatly accelerates and simplify the detection of the pathogenic bacterium in sample.
The preferred embodiment of the present invention described in detail above, but, the present invention is not limited in above-mentioned embodiment
Detail, in the technology concept of the present invention, technical scheme can be carried out multiple simple variant, this
A little simple variant belong to protection scope of the present invention.
It is further to note that each the concrete technical characteristic described in above-mentioned detailed description of the invention, at not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to various can
The compound mode of energy illustrates the most separately.
Additionally, combination in any can also be carried out between the various different embodiment of the present invention, as long as it is without prejudice to this
The thought of invention, it should be considered as content disclosed in this invention equally.
Claims (4)
1. detecting a method for unicellular hyperplasia Listeria viable bacteria body in sample, the method includes:
(1) by the antibody coupling that can be combined with unicellular hyperplasia Listeria viable bacteria body specifically on magnetic ball, immunity is obtained
Magnetic ball；The described antibody that can be combined with unicellular hyperplasia Listeria viable bacteria body specifically can not be combined with dead thalline and for compiling
Number it is the California Bioscience monoclonal antibody of MC0007, the magnetic ball relative to every milligram, the consumption of described antibody
For 0.05-2mg；
(2) biomolecular is mixed with the testing sample of liquid form, obtain mixed material；The condition of mixing makes
When there is unicellular hyperplasia Listeria viable bacteria body in testing sample, biomolecular can specifically adsorb unicellular hypertrophy
Listerella viable bacteria body, testing sample is food and/or medicine；
(3) described mixed material is placed in sorting magnetic field with fixing biomolecular, and eluting is unadsorbed at biomolecular
On material, obtain the biomolecular after eluting；
(4) material adsorbed on biomolecular carries out extracting the operation of DNA, obtain template sample；
(5) use the specific primer for described unicellular hyperplasia Listeria viable bacteria body that template sample is carried out PCR amplification,
And judge unicellular hyperplasia Listeria viable bacteria body in testing sample according to the content of the purpose fragment in pcr amplification product
Content, the primer used by PCR amplification includes the forward primer as shown in SEQ ID NO:1 and as shown in SEQ ID NO:2
Reverse primer.
Method the most according to claim 1, wherein, the testing sample of the liquid form relative to every milliliter, with magnetic ball
Weight meter, the consumption of described biomolecular is 10-25 μ g.
Method the most according to claim 1, wherein, in step (3), the liquid used by eluting is phosphate buffer, described
Phosphate buffer contains disodium hydrogen phosphate and the sodium dihydrogen phosphate of 0.038-0.056 mol/L of 0.144-0.162 mol/L.
4. according to the method described in any one in claim 1-3, wherein, described magnetic ball is that surface is modified with amino or carboxyl
Superparamagnetism Fe3O4Polystyrene complex microsphere.
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| CN101381777B (en) * | 2008-10-20 | 2013-01-23 | 中华人民共和国上海出入境检验检疫局 | Listeria monocytogenes detection kit and detection method thereof |
| CN102816850B (en) * | 2012-08-28 | 2015-01-21 | 无锡中德伯尔生物技术有限公司 | Method for simultaneously detecting salmonella typhimurium, escherichia coli O157:H7 and listeria monocytogenesis |
| CN103439495B (en) * | 2013-08-13 | 2015-04-15 | 南昌大学 | Listeria monocytogenes enrichment and rapid detection method |
| CN104374916A (en) * | 2013-08-15 | 2015-02-25 | 广州万孚生物技术股份有限公司 | Listeria monocytogenes fluorescence quantitative determination immunochromatography kit |
| CN103468823A (en) * | 2013-09-30 | 2013-12-25 | 山东出入境检验检疫局检验检疫技术中心 | Listeria monocytogenes LAMP detection primer and detection method, kit and preparation method |
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