CN103468823A - Listeria monocytogenes LAMP detection primer and detection method, kit and preparation method - Google Patents

Listeria monocytogenes LAMP detection primer and detection method, kit and preparation method Download PDF

Info

Publication number
CN103468823A
CN103468823A CN201310464599XA CN201310464599A CN103468823A CN 103468823 A CN103468823 A CN 103468823A CN 201310464599X A CN201310464599X A CN 201310464599XA CN 201310464599 A CN201310464599 A CN 201310464599A CN 103468823 A CN103468823 A CN 103468823A
Authority
CN
China
Prior art keywords
saccade
listeria monocytogenes
primer
magnetic bead
reaction
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201310464599XA
Other languages
Chinese (zh)
Inventor
李正义
贾俊涛
梁成珠
徐彪
姜英辉
赵丽青
张晓梅
马云
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Inspection and Quarantine Technology Center of Shandong Entry Exit Inspection and Quarantine Bureau
Original Assignee
Inspection and Quarantine Technology Center of Shandong Entry Exit Inspection and Quarantine Bureau
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Inspection and Quarantine Technology Center of Shandong Entry Exit Inspection and Quarantine Bureau filed Critical Inspection and Quarantine Technology Center of Shandong Entry Exit Inspection and Quarantine Bureau
Priority to CN201310464599XA priority Critical patent/CN103468823A/en
Publication of CN103468823A publication Critical patent/CN103468823A/en
Pending legal-status Critical Current

Links

Images

Landscapes

  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a Listeria monocytogenes LAMP detection primer and detection method, a kit and a preparation method, and belongs to the field of biotechnology. Listeria monocytogenes-FIP is an upstream inner primer, Listeria monocytogenes-BIP is a downstream inner primer, Listeria monocytogenes-F3 is an upstream outer primer, and the Listeria monocytogenes-B3 is a downstream outer primer. The kit comprises (1) nanometer immunomagnetic beads with chemical bonds coupled with Listeria monocytogenes monoclonal antibodies, (2) LAMP reaction liquid, (3) 8U/muL of BstDNA polymerase:, (4) negative control, (5) positive control and (6) a color reagent. The kit is used for detecting Listeria monocytogenes in food, is high in sensitivity, good in specificity, low in cost, easy and convenient to operate, suitable for laboratory rapid detection and portable on-site detection, and wide in promotion prospect.

Description

Listeria monocytogenes LAMP detects primer, detection method, test kit and preparation method thereof
Technical field
The present invention relates to a kind of LAMP detection kit and detection method thereof of Listeria monocytogenes, belong to biological technical field.
Background technology
Listeria monocytogenes (Listeria monocytogenes, LM) is called for short Listeria monocytogenes.Listeria monocytogenes can infect numerous food product, comprises livestock meat, fermented sausages and sea-food etc.WHO is listed as the large food-borne pathogens nineties four in 20th century by itself and Escherichia coli O 157: H7, Salmonellas, streptococcus aureus.It is the strongest bacterium of virulence in listeria.
Traditional biochemical reaction experiment and the hemolytic tests etc. of adopting of the evaluation of Listeria monocytogenes at present, additive method provides critical technique means as methods such as PCR method, enzyme-linked immunosorbent assays for the rapid detection Listeria monocytogenes more.
The traditional biological credit is the gold standard of detection of pathogens from cultivating with biochemical identification.In routine testing, at first to increase in advance bacterium by detecting in the sample liquid medium within, carry out afterwards separation and Culture on solid medium, finally carry out biochemical identification.The method required time is long, generally needs 5-7 days, is difficult to meet the needs of Rapid identification.Round pcr depend on traditional method before increase the bacterium step, often contain PCR inhibitor so sensitvity constraint in enrichment liquid.Enzyme-linked immunosorbent assay is quick, sensitive as screening method, but the test kit used is external product, expensive, and needs special instrument, promotes limited.Ring mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) is based on a kind of new amplification technique that detects hereditary material DNA.This technology depends on primer and a kind of archaeal dna polymerase with strand displacement characteristic that can identify 6 special zones on target sequence, can be efficiently under isothermal condition, fast, high amplified target sequence specifically, do not need expensive complicated instrument, only need a simple well heater, as common water-bath gets final product, therefore be beneficial in food inspection and apply.
The pathogenic micro-organism of nanometer magnetic bead in separating food has significant application value: efficiently concentrating; Can be moved it fast and be separated under additional the action of a magnetic field; Antibody is easy to mark.Listeria monocytogenes content in food is lower, and can cause damage in various degree to the LM cell in the course of processing of food, even but cell is impaired, also there is potential infection ability.
Summary of the invention
The invention provides a kind of LAMP detection kit and detection method thereof that detects low levels Listeria monocytogenes in food.
For achieving the above object, the present invention is by the following technical solutions:
The invention provides one group of LAMP primer that detects Listeria monocytogenes, this group primer is according to (the NCBI gene number of including: JN703915) design of Listeria monocytogenes reference culture ATCC19114 hemolysin gene.
Saccade-F3:5'-TAAACTTCGGCGCAATCA-3',
Saccade-B3:5'-CAAATAAACTTGACGGCCAT-3',
Saccade-FIP:5'-GGAAGGTCTTGTAGGTTCATTAACAGGAAAATGCAAGAAGAAGTCA-3',
Saccade-BIP:5'-TCGGCAAAGCTGTTACTAAAGAGCTTGAGATATATGCAGGAGGA-3',
The invention provides a kind of LAMP detection kit of Listeria monocytogenes, it comprises following material:
(1) the chemical bond coupling has the nano immune magnetic bead of Listeria monocytogenes monoclonal antibody;
Wherein, the monoclonal antibody of Listeria monocytogenes can be disclosed Listeria monocytogenes monoclonal antibody in prior art, and nanometer magnetic bead can be oneself development or commercial magnetic nanoparticle; By using the chemical bond coupling to prepare the nano immune magnetic bead of Listeria monocytogenes monoclonal antibody, effectively the Listeria monocytogenes in enrichment food to be checked, improve the sensitivity detected.
(2) ring mediated isothermal amplification (LAMP) reaction solution:
Comprising 10 * Thermopol reaction buffer, 1.0～2.0mmol/L dNTP, 2～10mmol/L sal epsom (MgSO 4), 0.5～2.0 μ mol/L upstream inner primer (saccade-FIP), 0.5～2.0 μ mol/L downstream inner primer (saccade-BIP), 0.1～0.5 μ mol/L upstream outer primer (saccade-F3), 0.1～0.5 μ mol/L downstream outer primer (saccade-B3) and 1～5mol/L trimethyl-glycine;
Contain 200mmol/L Tris-HCl (pH8.8), 100mmol/LKCl, 100mmol/L (NH in wherein said 10 * Thermopol reaction buffer 4) 2sO 4, 20mmol/L MgSO 4with 1%Triton X-100;
(3) Bst archaeal dna polymerase: 8U/ μ L;
(4) developer: fluorescence dye SYBR Green I;
(5) negative control: sterile purified water;
(6) positive control: the nucleic acid extracted by Listeria monocytogenes reference culture ATCC19114, as positive control, adopts the gram positive bacterium genome DNA extracting reagent kit and extracts lmL bacteria suspension genomic dna by its operation instructions.
The present invention also provides a kind of detection method of Listeria monocytogenes, and the method comprises the following steps:
(1) nano immune magnetic bead enrichment thalline: get 1mg nano immune magnetic bead in centrifuge tube, after being placed in magnetic separator frame absorption 2min, abandon supernatant, immunomagnetic beads is resuspended with 100 μ L sterile purified waters; Getting lmL food to be checked or bacteria suspension adds in the resuspended liquid of nano immune magnetic bead, slowly put upside down and mix, incubated at room 20min, then be placed on magnetic frame and adsorb 3min, abandon supernatant, nano immune magnetic bead sterile purified water washed twice, each 10s, finally use the resuspended magnetic bead of 100 μ L distilled water, the thalline sample after the acquisition enrichment;
(2) DNA of the thalline sample that extraction step (1) obtains, template DNA, can utilize method well known in the prior art or test kit to extract, for example gram positive bacterium genome DNA extracting reagent kit or equivalent agent box.
(3) carry out the LAMP reaction, this LAMP reaction system sees the following form:
LAMP reaction system (25 μ L):
Form Institute adds volume Final concentration
Saccade-FIP/ saccade BIP 4μL 1.6μmol/L
Saccade-F3/ saccade-B3 0.5μL 0.2μmol/L
Trimethyl-glycine 5μL 1mol/L
dNTP 1.4μL 1.4mmol/L
MgSO 4 2μL 8mmol/L
10 * Thermopol reaction buffer 2.5μL 1×
Template DNA 2μL /
The Bst archaeal dna polymerase 1μL 8U
ddH2O Polishing to 25 μ L /
(4) LAMP reaction conditions:
95 ℃ of denaturation 10min, 4 ℃ of 5min; Add Bst archaeal dna polymerase 1 μ L, 63 ℃ of sex change 60min, 80 ℃ of annealing 10min.
(5) result is judged:
Colour-change: add 1 μ L developer SYBR Green I in each reaction tubes to be checked, the colour-change that directly detects by an unaided eye, positive reaction is fluorescent green, and negative reaction keeps the fluorescent orange of dyestuff.
Electrophoresis detection: the amplified production of LAMP method is the stem ring texture DNA of various different lengthss, so positive reaction detects and to be trapezoid-shaped strips by 1.5% agarose electrophoresis, and negative reaction does not have trapezoidal amplified band to occur.
The invention has the beneficial effects as follows:
The present invention's application Listeria monocytogenes monoclonal antibody specific chemical coupling nanometer magnetic bead, be nano immune magnetic bead (Nano-Immunomagnetic Beads, Nano-IMB), it is good that it has selectivity, high specificity, can play the effect of separation, enrichment, the low Listeria monocytogenes of content in the energy enriched food, effectively avoid or reduce undetected.
The present invention designs the identification in four distinguished sequence districts of four primer pair target sequences, has guaranteed the high degree of specificity of LAMP amplification, and has not needed expensive PCR instrument, only needs common water-bath to get final product, simple and quick.
The LAMP detection reagent of Listeria monocytogenes of the present invention adopts the nano immune magnetic bead to combine with the LAMP technology, set up the method for quick of Listeria Monocytogenes In Food: Nano-IMB-LAMP, can detect the Listeria monocytogenes of lower concentration in food, and only need a simple well heater, there is very strong application prospect, be specially adapted to applying of feeler mechanism of basic unit.
The accompanying drawing explanation
The LAMP detection kit specificity experimental result picture that Fig. 1 is Listeria monocytogenes.M wherein: molecular weight standard, 100bp ladder; 1 Wei Shi listeria spp ATCC35897 nucleic acid amplification result; 2 Ying Nuoke listeria bacteria ATCC33090 nucleic acid amplification results; 3 listeria grayi ATCC700545 nucleic acid amplification results; 4 Salmonella enteritidis ATCC13076 nucleic acid amplification results; 5 Proteus mirabilis ATCC25933 nucleic acid amplification results; 6 intestinal bacteria ATCC51755 nucleic acid amplification results; 7 smell Serratia ATCC33077 nucleic acid amplification results; 8 enterobacter cloacae ATCC700323 nucleic acid amplification results; 9 Song Shi Shigellae ATCC51592 nucleic acid amplification results; 10 Vibrio parahaemolyticus ATCC17802 nucleic acid amplification results; 11 Vibrio harveyi CGMCC1.1616 nucleic acid amplification results; 12 Maxwell vibrios ATCC7708 nucleic acid amplification results; 13 singly increase listeria spp ATCC13932 nucleic acid amplification result; 14 singly increase listeria spp ATCC19115 nucleic acid amplification result; 15 singly increase listeria spp ATCC19114 nucleic acid amplification result; 16 singly increase listeria spp ATCC19112 nucleic acid amplification result.
Fig. 2 is the LAMP detection kit sensitivity experiment figure as a result of Listeria monocytogenes.M wherein: molecular weight standard, D2000; Empty: negative contrast; 1～7: 10 times of serial dilution bacteria suspension amplifications of Listeria monocytogenes ATCC19114 reference culture.Fig. 3 is that embodiment 4 detects artificial contamination's chicken meat sample test-results figure.M wherein: molecular weight standard, D2000; Empty: negative contrast; T: artificial contamination's chicken meat sample Nano-IMB-LAMP detected result.
Embodiment
The following example further illustrates the present invention, but the restriction of the present invention of should not opposing.
Embodiment 1
Make the LAMP detection kit of Listeria monocytogenes by following composition:
(1) coupling has the nano immune magnetic bead of Listeria monocytogenes monoclonal antibody;
The preparation of nano immune magnetic bead: the method that the commercial nanometer magnetic bead of 2mg (particle diameter is 20～100nm) separates by magnetic in the phosphate buffer solution (PBS) of 6mL pH7.0 is cleaned magnetic bead twice, add two kinds of activation linking agents: 3mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (being abbreviated as EDCHCl) and 2mg sulfonic group succinimide (sulfo-NHS) contain in the phosphate buffer solution of 2mg commercialization nanometer magnetic bead to 6mL, room temperature revolving reaction 30min; The method of separating by magnetic is cleaned magnetic bead twice, and the resuspended magnetic bead of phosphate buffer solution with 1mL pH7.0, add 50 μ g Listeria monocytogenes monoclonal antibodies, room temperature revolving reaction 5h; The method that coupling separates by magnetic after finishing is cleaned magnetic bead twice, adds in the phosphate buffer solution that 1mL bovine serum albumin (BSA) mass percent is 2% room temperature revolving reaction 1h; Method that nano immune magnetic bead after sealing separates by magnetic is cleaned magnetic bead once, joins in the phosphate buffer solution that 10mL bovine serum albumin (BSA) mass percent is 0.2% 4 ℃ of refrigerations standby.
(2) LAMP reaction solution:
The mixture that contains 2.5 μ L10 * Thermopol reaction buffer, tetra-kinds of thymus nucleic acids of 1.4 μ L25mmol/L dNTP(), 4.0 μ L10 μ mol/L upstream inner primers (saccade-FIP), 4.0 μ L10 μ mol/L downstream inner primers (saccade-BIP), 0.5 μ L10 μ mol/L upstream outer primer (saccade-F3), 0.5 μ L10 μ mol/L downstream outer primer (saccade-B3), 2 μ L100mmol/LMgSO4,5 μ L5M trimethyl-glycines and 2.1 μ L H2O(aqua sterilisas).
Wherein said upstream inner primer (saccade-FIP):
5'-GGAAGGTCTTGTAGGTTCATTAACAGGAAAATGCAAGAAGAAGTCA-3'
Downstream inner primer (saccade-BIP):
5'-TCGGCAAAGCTGTTACTAAAGAGCTTGAGATATATGCAGGAGGA-3'
Upstream outer primer (saccade-F3):
5'-TAAACTTCGGCGCAATCA-3'
Downstream outer primer (saccade-B3):
5'-CAAATAAACTTGACGGCCAT-3'
(3) Bst archaeal dna polymerase: 100 μ L (8U/ μ L), purchased from NEB company;
(4) negative control: aqua sterilisa, 1mL;
(5) positive control: as positive control, OD260/OD280 can reach 1.8 to the genomic dna (100 μ L) extracted by Listeria monocytogenes reference culture ATCC19114, and concentration reaches 20ng/ μ L.
(6) nitrite ion: SYBR Green I dyestuff (200 μ L), purchased from Invitrogen company.
(7) the magnetic separator frame is 1, Tianjin biochip company limited.
Embodiment 2
The Nano-IMB-LAMP detection kit specific test of Listeria monocytogenes:
Listed bacterial strain in following table is carried out to the Nano-IMB-LAMP detection, and process is as follows:
(1) the nano immune magnetic bead is caught mycetocyte: get nano immune magnetic bead that 200 μ L couplings have the Listeria monocytogenes monoclonal antibody in aseptic centrifuge tube, after being placed in magnetic separator frame absorption 2min, abandon supernatant, immunomagnetic beads is resuspended with 100 μ L sterile purified waters; Getting lmL mycetocyte suspension adds in the resuspended liquid of nano immune magnetic bead, slowly put upside down and mix, incubated at room 20min, then be placed on magnetic frame and adsorb 3min, abandon supernatant, nano immune magnetic bead sterile purified water washed twice, each 10s, finally use the resuspended magnetic bead of 100 μ L distilled water, the thalline sample after the acquisition enrichment;
(2) extraction of enrichment thalline sample DNA: use the bacterial genomes DNA extraction test kit of border biological gene Science and Technology Ltd. of the village, Beijing ally to extract sample DNA, OD260/OD280 can reach 1.8, and concentration reaches 20ng/ μ L.
Experiment bacterial strain list
Sequence number Strain name Numbering
1 The Wei Shi listeria spp ATCC35897
2 The Ying Nuoke listeria bacteria ATCC33090
3 Listeria grayi ATCC700545
4 Salmonella enteritidis ATCC13076
5 Proteus mirabilis ATCC25933
6 Intestinal bacteria ATCC51755
7 The smell Serratia ATCC33077
8 Enterobacter cloacae ATCC700323
9 The Song Shi Shigellae ATCC51592
10 Vibrio parahaemolyticus ATCC17802
11 Vibrio harveyi CGMCC1.1616
12 The Maxwell vibrios ATCC7708
13 Singly increase listeria spp ATCC13932
14 Singly increase listeria spp ATCC19115
15 Singly increase listeria spp ATCC19114
16 Singly increase listeria spp ATCC19112
(3) LAMP reaction:
Add 2 μ L template DNA to be checked in the reaction tubes that 22 μ L LAMP reaction solutions are housed, after 95 ℃ of placement 10min, be placed in immediately 4 ℃ and place 5min; Add again 1 μ L Bst archaeal dna polymerase in reaction tubes, and in water bath with thermostatic control 63 ℃ of amplified reaction 60min; Water-bath is transferred to 80 ℃ of stopped reactions, takes out to be checked after 10min;
(4) result is judged:
Electrophoresis detection: positive reaction detects and is trapezoid-shaped strips by 1.5% agarose electrophoresis, and negative reaction does not have trapezoidal amplified band to occur.
Result as shown in Figure 1, only have and singly increase listeria spp and amplified band occurs, show that four primers that the present invention designs have high degree of specificity, the LAMP detection kit high specificity of Listeria monocytogenes of the present invention, can play the effect of separation, enrichment, the low Listeria monocytogenes of content in the energy enriched food, effectively avoid or reduce undetected.
Embodiment 3
The Nano-IMB-LAMP detection kit sensitivity test of Listeria monocytogenes:
To the Listeria monocytogenes reference culture, ATCC19114 is inoculated into nutrient broth medium, incubated overnight, prepare 10 times of serial dilution bacteria suspensions, get respectively each gradient dilution liquid of 1mL and carry out enrichment thalline, DNA extraction and LAMP reaction according to the method for implementing in 2 by the Nano-IMB-LM detection kit.Detected result is shown in Fig. 2: it is 10 that the Nano-IMB-LM detection kit can detect extent of dilution -5the time bacteria suspension, corresponding Listeria monocytogenes bacteria concentration is 6.3 * 10 2cFU/mL, the sensitivity of applying this detection kit is 6.3 * 10 2cFU/mL.
Embodiment 4
The Nano-IMB-LAMP test kit of Listeria monocytogenes detects the test of artificial contamination's chicken meat sample:
Containing carrying out artificial the interpolation in the chicken of Listeria monocytogenes, do not testing.In the 25g chicken meat sample, add the Listeria monocytogenes bacteria suspension that the 1mL cell concentration is 120CFU/mL, 25g artificial contamination chicken meat sample joins in the 225mL nutrient broth, cultivate 16～20h for 25 ℃ ± 1 ℃, get 1mL and increase bacterial context soup and carry out enrichment thalline, DNA extraction and LAMP according to the method in 2 implemented by the Nano-IMB-LM detection kit and react.Detected result is shown in Fig. 3: after the chicken meat sample artificial contamination, sample after increasing bacterium, available Nano-IMB-LAMP detection method detects Listeria monocytogenes.
Obviously, the above embodiment of the present invention is only for example of the present invention clearly is described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here can't give all embodiments exhaustive.Every still row in protection scope of the present invention of apparent variation that technical scheme of the present invention extends out or change that belong to.
The nucleotides sequence list
<110 > Inspection and Quarantine Technology Center, Shandong Inspection and Quarantine
<120 > Listeria monocytogenes LAMP detects primer, detection method, test kit and preparation method thereof
<160> 4
<210> 1
<211> 46
<212> DNA
<213 > artificial sequence
<221> prim_bind
<222> (1)...(46)
<400> 1
GGAAGGTCTTGTAGGTTCATTAACAGGAAAATGCAAGAAGAAGTCA 46
<210> 2
<211> 44
<212> DNA
<213 > artificial sequence
<221> prim_bind
<222> (1)...(44)
<400> 2
TCGGCAAAGCTGTTACTAAAGAGCTTGAGATATATGCAGGAGGA 44
<210> 3
<211> 18
<212> DNA
<213 > artificial sequence
<221> prim_bind
<222> (1)...(18)
<400> 3
TAAACTTCGGCGCAATCA 18
<210> 4
<211> 20
<212> DNA
<213 > artificial sequence
<221> prim_bind
<222> (1)...(20)
<400> 4
CAAATAAACTTGACGGCCAT 20

Claims (5)

1. a LAMP primer that detects Listeria monocytogenes, is characterized in that, saccade-FIP is the upstream inner primer, and saccade-BIP is the downstream inner primer, and saccade-F3 is the upstream outer primer, and saccade-B3 is the downstream outer primer;
Concrete primer is:
Saccade-FIP:5'-GGAAGGTCTTGTAGGTTCATTAACAGGAAAATGCAAGAAGAAGTCA-3';
Saccade-BIP:5'-TCGGCAAAGCTGTTACTAAAGAGCTTGAGATATATGCAGGAGGA-3';
Saccade-F3:5'-TAAACTTCGGCGCAATCA-3';
Saccade-B3:5'-CAAATAAACTTGACGGCCAT-3'.
2. the LAMP detection kit of a Listeria monocytogenes, is characterized in that, this test kit comprises following material:
(1) the chemical bond coupling has the nano immune magnetic bead of Listeria monocytogenes monoclonal antibody;
(2) ring mediated isothermal amplification (LAMP) reaction solution,
Comprising 10 * Thermopol reaction buffer, 1.0～2.0mmol/L dNTP, 2～10mmol/L sal epsom (MgSO 4), 0.5～2.0 μ mol/L upstream inner primer (saccade-FIP), 0.5～2.0 μ mol/L downstream inner primer (saccade-BIP), 0.1～0.5 μ mol/L upstream outer primer (saccade-F3), 0.1～0.5 μ mol/L downstream outer primer (saccade-B3) and 1～5mol/L trimethyl-glycine;
Contain 200mmol/L Tris-HCl (pH8.8), 100mmol/LKCl, 100mmol/L (NH in wherein said 10 * Thermopol reaction buffer 4) 2sO 4, 20mmol/L MgSO 4with 1%Triton X-100;
(3) Bst archaeal dna polymerase: 8U/ μ L;
(4) negative control: sterile purified water;
(5) positive control: the nucleic acid extracted by Listeria monocytogenes reference culture ATCC19114, as positive control, adopts the gram positive bacterium genome DNA extracting reagent kit and extracts lmL bacteria suspension genomic dna by its operation instructions;
(6) nitrite ion: fluorescence dye SYBR Green I.
3. test kit according to claim 2, is characterized in that, in described LAMP reaction solution, the use final concentration of various compositions is 1 * ThermoPol damping fluid, 1.4mmol/L dNTP, 8.0mmol/L MgSO 4, 0.2 μ mol/L upstream outer primer saccade-F3,0.2 μ mol/L downstream outer primer saccade-B3,1.6 μ mol/L upstream inner primer saccade-FIP, 1.6 μ mol/L downstream inner primer saccade-BIP.
4. the preparation method of the LAMP detection kit of Listeria monocytogenes, is characterized in that, comprises the following steps:
(1) coupling has the nano immune magnetic bead of Listeria monocytogenes monoclonal antibody:
Get the method that the commercial nanometer magnetic bead of 2mg (particle diameter is 20～100nm) separates by magnetic in the phosphate buffer solution (PBS) of 6mL pH7.0 and clean magnetic bead twice, add two kinds of activation linking agents: 3mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (being abbreviated as EDCHCl) and 2mg sulfonic group succinimide (sulfo-NHS) contain in the phosphate buffer solution of 2mg commercialization nanometer magnetic bead to 6mL, room temperature revolving reaction 30min; The method of separating by magnetic is cleaned magnetic bead twice, and the resuspended magnetic bead of phosphate buffer solution with 1mL pH7.0, add 50 μ g Listeria monocytogenes monoclonal antibodies, room temperature revolving reaction 5h; The method that coupling separates by magnetic after finishing is cleaned magnetic bead twice, adds in the phosphate buffer solution that 1mL bovine serum albumin (BSA) mass percent is 2% room temperature revolving reaction 1h; Method that nano immune magnetic bead after sealing separates by magnetic is cleaned magnetic bead once, joins in the phosphate buffer solution that 10mL bovine serum albumin (BSA) mass percent is 0.2% 4 ℃ of refrigerations standby;
(2) LAMP reaction solution:
The mixture that contains 2.5 μ L10 * Thermopol reaction buffer, tetra-kinds of thymus nucleic acids of 1.4 μ L25mmol/L dNTP(), 4.0 μ L10 μ mol/L upstream inner primers (saccade-FIP), 4.0 μ L10 μ mol/L downstream inner primers (saccade-BIP), 0.5 μ L10 μ mol/L upstream outer primer (saccade-F3), 0.5 μ L10 μ mol/L downstream outer primer (saccade-B3), 2 μ L100mmol/LMgSO4,5 μ L5M trimethyl-glycines and 2.1 μ L H2O(aqua sterilisas);
Wherein said upstream inner primer (saccade-FIP):
5'-GGAAGGTCTTGTAGGTTCATTAACAGGAAAATGCAAGAAGAAGTCA-3'
Downstream inner primer (saccade-BIP):
5'-TCGGCAAAGCTGTTACTAAAGAGCTTGAGATATATGCAGGAGGA-3'
Upstream outer primer (saccade-F3):
5'-TAAACTTCGGCGCAATCA-3'
Downstream outer primer (saccade-B3):
5'-CAAATAAACTTGACGGCCAT-3'
(3) Bst archaeal dna polymerase: 100 μ L (8U/ μ L): purchased from NEB company;
(4) negative control: aqua sterilisa, 1mL;
(5) positive control: as positive control, OD260/OD280 can reach 1.8 to the genomic dna (100 μ L) extracted by Listeria monocytogenes reference culture ATCC19114, and concentration reaches 20ng/ μ L;
(6) nitrite ion: SYBR Green I dyestuff (200 μ L), purchased from Invitrogen company;
(7) the magnetic separator frame is 1, Tianjin biochip company limited.
5. the detection method of a Listeria monocytogenes, is characterized in that, the method comprises the following steps:
1) the nano immune magnetic bead is caught mycetocyte: get 1mg nano immune magnetic bead in centrifuge tube, after being placed in magnetic separator frame absorption 2min, abandon supernatant, immunomagnetic beads is resuspended with 100 μ L sterile purified waters; Getting lmL food to be checked or bacteria suspension adds in the resuspended liquid of nano immune magnetic bead, slowly put upside down and mix, incubated at room 20min, then be placed on magnetic frame and adsorb 3min, abandon supernatant, nano immune magnetic bead sterile purified water washed twice, each 10s, finally use the resuspended magnetic bead of 100 μ L distilled water, the thalline sample after the acquisition enrichment;
2) extraction of enrichment thalline sample DNA;
3) LAMP reaction system (25 μ L) is as follows:
Form Institute adds volume Final concentration Saccade-FIP/ saccade BIP 4μL 1.6μmol/L Saccade-F3/ saccade-B3 0.5μL 0.2μmol/L
Trimethyl-glycine 5μL 1mol/L dNTP 1.4μL 1.4mmol/L MgSO 4 2μL 8mmol/L 10 * Thermopol reaction buffer 2.5μL 1× Template DNA 2μL / The Bst archaeal dna polymerase 1μL 8U ddH2O Polishing to 25 μ L /
4) LAMP reaction; 95 ℃ of denaturation 10min, 4 ℃ of 5min; Add Bst archaeal dna polymerase 1 μ L, 63 ℃ of sex change 60min, 80 ℃ of annealing 10min;
5) result is judged: carry out judgement as a result by reaction system colour-change or electrophoresis detection,
Colour-change: add 1 μ L developer SYBR Green I in each reaction tubes to be checked, the colour-change that directly detects by an unaided eye, positive reaction is fluorescent green, and negative reaction keeps the fluorescent orange of dyestuff;
Electrophoresis detection: the amplified production of LAMP method is the stem ring texture DNA of various different lengthss, so positive reaction detects and to be trapezoid-shaped strips by 1.5% agarose electrophoresis, and negative reaction does not have trapezoidal amplified band to occur.
CN201310464599XA 2013-09-30 2013-09-30 Listeria monocytogenes LAMP detection primer and detection method, kit and preparation method Pending CN103468823A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310464599XA CN103468823A (en) 2013-09-30 2013-09-30 Listeria monocytogenes LAMP detection primer and detection method, kit and preparation method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310464599XA CN103468823A (en) 2013-09-30 2013-09-30 Listeria monocytogenes LAMP detection primer and detection method, kit and preparation method

Publications (1)

Publication Number Publication Date
CN103468823A true CN103468823A (en) 2013-12-25

Family

ID=49793863

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310464599XA Pending CN103468823A (en) 2013-09-30 2013-09-30 Listeria monocytogenes LAMP detection primer and detection method, kit and preparation method

Country Status (1)

Country Link
CN (1) CN103468823A (en)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104328113A (en) * 2014-10-12 2015-02-04 中国疾病预防控制中心传染病预防控制所 Nucleotide sequence composition and application thereof in Listeria monocytogenes identification
CN104651481A (en) * 2013-11-19 2015-05-27 北京市理化分析测试中心 Method for detecting living bacteria body of listeria monocytogenes in sample
CN105567864A (en) * 2016-03-18 2016-05-11 北京农学院 LAMP (loop-mediated isothermal amplification) detection primers and method of Listeria monocytogenes in food
CN104328113B (en) * 2014-10-12 2017-01-04 中国疾病预防控制中心传染病预防控制所 One group of nucleotide sequence and the application in Listeria monocytogenes is identified
CN106771217A (en) * 2016-12-05 2017-05-31 百奥森（江苏）食品安全科技有限公司 A kind of single method for quick for increasing listeria spp
CN108676842A (en) * 2018-07-18 2018-10-19 河南省商业科学研究所有限责任公司 A kind of method of quick detection Listeria monocytogenes and staphylococcus aureus
CN109321637A (en) * 2018-10-31 2019-02-12 武汉利恩达医疗科技有限公司 A kind of pathogen nucleic acid capture amplification detection method
CN110819685A (en) * 2019-10-24 2020-02-21 广东环凯生物科技有限公司 Listeria monocytogenes immunomagnetic bead washing liquid
CN111662804A (en) * 2020-06-17 2020-09-15 清华大学 Digital loop-mediated isothermal amplification micro-fluidic chip for food microorganism detection

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102277422A (en) * 2011-06-20 2011-12-14 黑龙江省乳品工业技术开发中心 Method for rapid detection of Listeria monocytogenes viable bacteria in liquid milk

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102277422A (en) * 2011-06-20 2011-12-14 黑龙江省乳品工业技术开发中心 Method for rapid detection of Listeria monocytogenes viable bacteria in liquid milk

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HUA YANG等: "Nanoparticle-based Immunomagnetic Separation of Listeria monocytogenes", 《INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY》, vol. 118, no. 2, 16 September 2007 (2007-09-16), XP022230590, DOI: doi:10.1016/j.ijfoodmicro.2007.06.019 *
张体银等: "环介导等温扩增技术快速检测食品中的单增李斯特氏菌", 《中国食品学报》, vol. 10, no. 3, 30 June 2010 (2010-06-30), pages 200 - 203 *
钟子清等: "纳米免疫磁珠富集单核增生李斯特菌的研究", 《食品科学 CNKI网络预发表》, 7 May 2013 (2013-05-07) *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104651481A (en) * 2013-11-19 2015-05-27 北京市理化分析测试中心 Method for detecting living bacteria body of listeria monocytogenes in sample
CN104328113A (en) * 2014-10-12 2015-02-04 中国疾病预防控制中心传染病预防控制所 Nucleotide sequence composition and application thereof in Listeria monocytogenes identification
CN104328113B (en) * 2014-10-12 2017-01-04 中国疾病预防控制中心传染病预防控制所 One group of nucleotide sequence and the application in Listeria monocytogenes is identified
CN105567864A (en) * 2016-03-18 2016-05-11 北京农学院 LAMP (loop-mediated isothermal amplification) detection primers and method of Listeria monocytogenes in food
CN106771217A (en) * 2016-12-05 2017-05-31 百奥森（江苏）食品安全科技有限公司 A kind of single method for quick for increasing listeria spp
CN108676842A (en) * 2018-07-18 2018-10-19 河南省商业科学研究所有限责任公司 A kind of method of quick detection Listeria monocytogenes and staphylococcus aureus
CN108676842B (en) * 2018-07-18 2022-04-12 河南省商业科学研究所有限责任公司 Method for rapidly detecting Listeria monocytogenes and staphylococcus aureus
CN109321637A (en) * 2018-10-31 2019-02-12 武汉利恩达医疗科技有限公司 A kind of pathogen nucleic acid capture amplification detection method
CN110819685A (en) * 2019-10-24 2020-02-21 广东环凯生物科技有限公司 Listeria monocytogenes immunomagnetic bead washing liquid
CN111662804A (en) * 2020-06-17 2020-09-15 清华大学 Digital loop-mediated isothermal amplification micro-fluidic chip for food microorganism detection

Similar Documents

Publication Publication Date Title
CN103468823A (en) Listeria monocytogenes LAMP detection primer and detection method, kit and preparation method
Amagliani et al. Development of a magnetic capture hybridization‐PCR assay for Listeria monocytogenes direct detection in milk samples
Miller et al. Real-time reverse-transcriptase PCR for Salmonella Typhimurium detection from lettuce and tomatoes
CN102943113B (en) Loop-mediated isothermal amplification detection primer groups of Escherichia coli 0157, detection method and reagent kit
CN104450940B (en) A kind of nucleotide sequence and detection method and detection kit for detecting Listeria Monocytogenes
CN102417929B (en) Specific PCR detection method of Riemerella anatipestifer
CN104263838B (en) Listeria monocytogenes LAMP-LFD detection kit and detection method thereof
Ruan et al. Rapid and sensitive detection of Cronobacter spp.(previously Enterobacter sakazakii) in food by duplex PCR combined with capillary electrophoresis–laser-induced fluorescence detector
CN102703588A (en) Multiplex PCR-based synchronous and rapid method for detecting 13 pathogenic microorganisms in water
CN110791592A (en) Primer and kit for rapidly detecting African swine fever virus
Gilhuus et al. Characterisation of Dichelobacter nodosus isolates from Norway
CN105316391A (en) Method of detecting salmonella, shigella and staphylococcus aureus
CN103882130A (en) Primer pair for detecting mycobacterium tuberculosis complex group and application of primer pair
CN103160603B (en) LAMP (loop-mediated isothermal amplification) detection kit of vibrio parahaemolyticus and detection method thereof
CN102776283B (en) Visualized loop-mediated isothermal amplification kit for detecting Haemophilus parasuis
Soria et al. Comparison of 3 culture methods and PCR assays forSalmonella gallinarum andSalmonella pullorum detection in poultry feed
Vinayaka et al. Point-of-care diagnosis of invasive non-typhoidal Salmonella enterica in bloodstream infections using immunomagnetic capture and loop-mediated isothermal amplification
CN104342496B (en) A kind of quick detection, identify that Liszt belongs to the method for bacterium
CN101864483A (en) Salmonella and shigella joint detection kit and detection method thereof
CN103805691A (en) Preparation method and application of nucleic acid lateral flow test strip for detecting Shigella
CN100489527C (en) Shinga toxin-producing Escherichia coli nucleic acid purified blood serum identification kit
Kadiroğlu et al. Quantification of Staphylococcus aureus in white cheese by the improved DNA extraction strategy combined with TaqMan and LNA probe-based qPCR
CN108707680B (en) Triple-link seven-PCR (polymerase chain reaction) detection primer group, kit and detection method for streptococcus agalactiae virulence genes
CN103993072B (en) The multiple PCR detection kit of the pathogenic sweetfish pseudomonas of a kind of Rapid identification and method thereof
CN103305613B (en) Giant salamander pathogenic hydrophila gingivalis PCR diagnostic kit

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C12 Rejection of a patent application after its publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20131225