CN103088112B - F0F1-ATPase rotary molecular motor sensor kit for detecting Listeria monocytogenes - Google Patents
F0F1-ATPase rotary molecular motor sensor kit for detecting Listeria monocytogenes Download PDFInfo
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- CN103088112B CN103088112B CN201110336049.0A CN201110336049A CN103088112B CN 103088112 B CN103088112 B CN 103088112B CN 201110336049 A CN201110336049 A CN 201110336049A CN 103088112 B CN103088112 B CN 103088112B
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Abstract
An F0F1-ATPase rotary molecular motor sensor kit for detecting Listeria monocytogenes belongs to the field of the rapid detection of food microbes, and mainly solves an overlong time (about 5 days) problem of traditional detection methods. The kit is mainly characterized in that an F0F1-ATPase rotary molecular motor sensor on a chromatophore is utilized, an F0F1-ATP synthase can rapidly rotate, and the coupling between a catalytic site and proton transfer is established through rotating the F0F1-ATP synthase. A prfA probe is connected to the epsilon subunit of the ATP synthase, a sample to be measured and a negative contrast substance are respectively combined with a biosensor, the ATP outputs 20min after the ATP catalytic synthesis are compared, the ATP synthetic outputs can be measured through the amount of H<+> in the environment, and the H<+> amount can be obtained through the fluorescence intensity of H-DHPE. The kit can realize the rapid, sensitive and high-flux detection of the Listeria monocytogenes in the sample to be measured.
Description
Technical field
The invention relates to the one of fast detection technique of food microorganisms.
Background technology
Traditional list increases listeria spp detection method and requires to carry out the steps such as non-selective increasing bacterium, selective enrichment, separation, screening and identification to each Interventions Requested, and general needs just can provide examining report for 5 days, has a strong impact on quality and the shelf-life of goods.Some new technology still depend on traditional detecting step as detection methods such as round pcr, immunology detection technology, biochip technologies, namely need to carry out increasing bacterium, take time and effort, complete the time that one-time detection needs several days usually.Which not only adds the workload in laboratory, nor be beneficial to the monitoring of Production Flow Chart in foodstuffs industry, the quality control of finished product and government department to the management of food safety and control.
Summary of the invention
With controlled molecular motor technology for core, the technology such as integrated nucleic acid probe identification, fluorescent probe mark and detection, establish the Fast Detection Technique system of a novel concept.Utilize ATP enzyme to carry out detection as carrier to target compound and there is highly sensitive, high specificity, fast feature.At F
0f
1-ATPase molecular motor connects upper special nucleic acid probe, can realize quick, the specificity to certain food microorganism, high-throughout detection.Detection time can be foreshortened to 2 days by the present invention, and detection sensitivity can reach 10
2cFU/ml, meets existing Microbiological detection of foods scope.
Accompanying drawing explanation
Accompanying drawing 1 is molecular motor biosensor mode chart (wherein a, b, c, α, β, δ, γ, ε are atp synthase subunit), 1 is epsilon subunit antibody, 2 is Streptavidin (Strptavidin), 3 is N-biotin, 4 is prfA probe, and 5 is single increasing listeria spp single stranded DNA.
Accompanying drawing 2 is the detected result of chro prfA to bacterial strain not of the same race, and ordinate zou represents fluorescent value, and X-coordinate represents the sample of detection, and wherein 1 is water, and 2 for singly to increase listeria spp, and 3 is Vibrio parahemolyticus, and 4 is Salmonellas, and 5 is vibrio cholerae, and 6 is intestinal bacteria.
Accompanying drawing 3 is that chro prfA increases the detected result of listeria spp sample to 30 strain lists, and ordinate zou represents fluorescent value, and X-coordinate represents the sample of detection, and 1 ~ 30 is food inspection sample number.
Embodiment
Increase listeria spp prfA gene design specific dna probe 5 '-gatacagaaacatcggttggc according to single, 5 ' of its middle probe marks with vitamin H.This probe is connected to above molecular motor by biotin antibody, utilize molecular motor sensor can detect the DNA of sample to be tested, when sample is single increasing listeria spp DNA, can be there is obvious change in the fluorescent value of detection system, can judgement sample be positive by this change.For this reason, we devise a test kit, can increase listeria spp easily and fast detect the list in food.
Test kit forms:
| Numbering | Ingredient names | Quantity | Preservation condition |
| 1 | chro prfA | 20μl | -20℃ |
| 2 | Synthesis buffer | 10ml | Room temperature |
| 3 | ADP(1.6mol/l) | 1ml | -20℃ |
| 4 | 1×PBS | 25ml | Room temperature |
| 5 | Luciferase/luciferin | 100 units | -20℃ |
| 6 | Luciferase/luciferin reassembly buffer liquid | 12ml | -20℃ |
| 7 | Sterilized water | 5ml | Room temperature |
Operation steps is as follows:
1. get 1.5ml EP to manage, add sample to be tested 10 μ l.
2. above-mentioned EP pipe being put into boiling water 3min, then transferring to immediately on ice to cooling completely.
3. get 2 μ l chro prfA, be diluted to certain multiple with synthesis buffer.Get the chro after dilution 10 μ l to add above-mentioned EP and manage.
4. separately getting 1 EP pipe and add 10 μ l sterilized waters, 3min in boiling water, then transferring to immediately on ice to cooling completely, then add 10 μ l synthesis buffer as Background control, short term oscillation makes reaction system mix.
5. in 2 EP pipes, add 30 μ l startup buffer more respectively (start buffer by ADP (2.2mol/l) and synthesis buffer 1: 6 preparation by volume, certain amount preparation is got in each experiment on demand, matching while using), vibration makes reaction system mix, then of short duration centrifugal with the globule removed on cap wall immediately.
6. above-mentioned reaction system is put into 37 DEG C of constant-temperature table incubation 20min.
7. taken out from shaking table by EP pipe, add 450 μ l PBS damping fluids respectively, vibration makes system mix.
8. get one piece of clean 96 orifice plate, end reaction system in 2 pipes is added wherein, every individual system adds 3 holes, every hole application of sample 50 μ l, then (being added by luciferase/luciferin reassembly buffer liquid is equipped with in the Brown Glass Brown glass bottles and jars only of luciferase/luciferin to add to each well the luciferase solution that 30 μ l have configured respectively, cover bottle stopper, repeatedly put upside down and mix several times, can not vibrate.Mixed solution should be placed 1h in room temperature before using), repeatedly blow and beat with rifle and make system mix several times.
9. by machine testing on 96 orifice plates, processing data, averages to each group of data, then deducts with sample values the actual fluorescent value that background numerical value is sample.
By experiment, molecular motor biosensor chro prfA has good specificity, with chro prfA to water, singly increase listeria spp, Vibrio parahemolyticus, Salmonellas, vibrio cholerae, intestinal bacteria detect, single fluorescent value increasing listeria spp is starkly lower than the fluorescent value of water and other contrast bacterium, concrete data see the following form, and the results are shown in accompanying drawing 2.
| Sample (10ng/mL) | Fluorescent value |
| Water | 284901 |
| Singly increase listeria spp | 270174 |
| Vibrio parahemolyticus | 300679 |
| Salmonellas | 294943 |
| Vibrio cholerae | 294649 |
| Intestinal bacteria | 285223 |
Detect 30 strain food samples with chro prfA, all can detect, concrete data see the following form, and the results are shown in accompanying drawing 3.
| Sample number | Fluorescent value | Sample number | Fluorescent value |
| Water | 562689 | 16 | 305267 |
| 1 | 269526 | 17 | 303836 |
| 2 | 288540 | 18 | 335545 |
| 3 | 297241 | 19 | 322881 |
| 4 | 328428 | 20 | 312907 |
| 5 | 308957 | 21 | 313883 |
| 6 | 266580 | 22 | 311252 |
| 7 | 280415 | 23 | 309237 |
| 8 | 278235 | 24 | 296812 |
| 9 | 286678 | 25 | 307311 |
| 10 | 305228 | 26 | 317081 |
| 11 | 317380 | 27 | 317412 |
| 12 | 307364 | 28 | 300856 |
| 13 | 304734 | 29 | 300767 |
| 14 | 290645 | 30 | 286644 |
| 15 | 289172 | —— | —— |
Experimental result shows, this test kit is 1h to single detection time increasing listeria spp DNA, detects and is limited to 10ng/ml.To 30 strain food inspection sample bacterial strains to detect result consistent with the result that PCR detects.By this molecular motor biosensor of a large amount of experimental verifications, there is good specificity and higher sensitivity, and by 96 hole Chemiluminescent plates are high-throughout, sample is detected.
Claims (1)
1. one kind increases the F of listeria spp for detecting list
0f
1-ATPase Rotary molecular motor sensor reagent box, comprises following component:
Listeria spp prfA gene design specific dna probe 5 '-gatacagaaacatcggttggc is increased according to single, 5 ' of its middle probe marks with vitamin H, this probe is connected to above molecular motor by biotin antibody, utilize molecular motor sensor Chro prfA can detect the DNA of sample to be tested, operation steps is as follows:
1) get 1.5mL EP to manage, add sample to be tested 10 μ L;
2) above-mentioned EP pipe being put into boiling water 3min, then transferring on ice to cooling completely immediately;
3) get 2 μ L chro prfA, with synthesis buffer dilution, get the Chro prfA 10 μ L after dilution and add above-mentioned EP and manage;
4) separately getting 1 EP pipe and add 10 μ L sterilized waters, 3min in boiling water, then transferring to immediately on ice to cooling completely, then add 10 μ L synthesis buffer as Background control, short term oscillation makes reaction system mix;
5) in 2 EP pipes, add 30 μ L more respectively start buffer, start buffer by ADP2.2M and synthesis buffer 1:6 preparation by volume, matching while using, vibration makes reaction system mix, then of short duration centrifugal with the globule removed on cap wall immediately;
6) above-mentioned reaction system is put into 37 DEG C of constant-temperature table incubation 20min;
7) taken out from shaking table by EP pipe, add 450 μ L PBS damping fluids respectively, vibration makes system mix;
8) one piece of clean 96 orifice plate is got, add wherein by the end reaction system in 2 pipes, every individual system adds 3 holes, every hole application of sample 50 μ L, then the luciferase solution that 30 μ L have configured is added respectively to each well, repeatedly blow and beat with rifle and make system mix several times; Described luciferase solution is, is added by luciferase/luciferin reassembly buffer liquid and is equipped with in the Brown Glass Brown glass bottles and jars only of luciferase/luciferin, cover bottle stopper, repeatedly put upside down and mix several times, can not vibrate, and before using, mixed solution is placed 1h in room temperature;
9) by machine testing on 96 orifice plates, processing data, averages to each group of data, then deducts with sample values the actual fluorescent value that background numerical value is sample.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201110336049.0A CN103088112B (en) | 2011-10-31 | 2011-10-31 | F0F1-ATPase rotary molecular motor sensor kit for detecting Listeria monocytogenes |
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| CN201110336049.0A CN103088112B (en) | 2011-10-31 | 2011-10-31 | F0F1-ATPase rotary molecular motor sensor kit for detecting Listeria monocytogenes |
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| CN103088112B true CN103088112B (en) | 2015-06-03 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101397586B (en) * | 2008-10-10 | 2011-02-02 | 广东省疾病预防控制中心 | Composite gene chip for food-borne pathogenic bacteria detection |
| CN102135543A (en) * | 2010-01-27 | 2011-07-27 | 中国科学院生物物理研究所 | Real-time and sensitive novel method for detecting biomacromolecule and preparation method for kit |
| CN102154473A (en) * | 2011-01-24 | 2011-08-17 | 杭州市农业科学研究院 | Gene chip and applications thereof in detection of aquatic pathogenic microorganism |
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2011
- 2011-10-31 CN CN201110336049.0A patent/CN103088112B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101397586B (en) * | 2008-10-10 | 2011-02-02 | 广东省疾病预防控制中心 | Composite gene chip for food-borne pathogenic bacteria detection |
| CN102135543A (en) * | 2010-01-27 | 2011-07-27 | 中国科学院生物物理研究所 | Real-time and sensitive novel method for detecting biomacromolecule and preparation method for kit |
| CN102154473A (en) * | 2011-01-24 | 2011-08-17 | 杭州市农业科学研究院 | Gene chip and applications thereof in detection of aquatic pathogenic microorganism |
Non-Patent Citations (3)
| Title |
|---|
| Evaluation of Paramagnetic Beads Coated with Recombinant Listeria Phage Endolysin-Derived Cell-Wall-Binding Domain Proteins for Separation of Listeria monocytogenes from Raw Milk in Combination with Culture-Based and Real-Time Polymerase Chain Reaction;Walcher Georg et al.;《FOODBORNE PATHOGENS AND DISEASE》;20100525;第7卷(第9期);第1020页右栏倒数第3段 * |
| Taq Man-MGB探针Real-timePCR快速检测单增李斯特菌的研究;梅玲玲等;《中国卫生检验杂志》;20070210(第02期);全文 * |
| 实时荧光PCR定量检测食品中单增李斯特菌;熊国华等;《中国食品卫生杂志》;20070531(第03期);全文 * |
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Inventor after: Zhang Jie Inventor after: Lu Xiaoyu Inventor after: Han Jing Inventor after: Liu Yan Inventor after: Gu Dezhou Inventor after: Chen Guangquan Inventor after: Yang Xiangying Inventor after: Zhang Yan Inventor after: Wan Xiaonan Inventor after: Zhang Lei Inventor after: Zhang Huiyuan Inventor after: Wang Qi Inventor after: Lu Lin Inventor after: Zhang Cuan Inventor before: Zhang Jie Inventor before: Chen Guangquan Inventor before: Zhang Huiyuan Inventor before: Wang Qi Inventor before: Lu Lin Inventor before: Zhang Cuan Inventor before: Lu Xiaoyu Inventor before: Han Jing Inventor before: Liu Yan Inventor before: Gu Dezhou |
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