CN104198536A - Method for detecting paralytic shellfish poisoning - Google Patents
Method for detecting paralytic shellfish poisoning Download PDFInfo
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- CN104198536A CN104198536A CN201410369789.8A CN201410369789A CN104198536A CN 104198536 A CN104198536 A CN 104198536A CN 201410369789 A CN201410369789 A CN 201410369789A CN 104198536 A CN104198536 A CN 104198536A
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Abstract
The invention discloses a method for detecting paralytic shellfish poisoning. The method is implemented on a mouse neuroma cell impedance sensor. The method comprises the following steps: inoculating neuro2a cells to sensor chip cell culture holes for culturing; respectively adding a to-be-detected solution into cell culture holes a solution containing ouabain and veratridine; and detecting a CI value of each cell culture hole after the to-be-detected solution is added, and judging the content of the paralytic shellfish poisoning in the to-be-detected solution according to change of the CI value. According to the method, cells are directly taken as detection carriers, the operation is simple, and the cost is low. According to the method, the detection limit of saxitoxin is low to 0.9ng/ml and has obvious progress compared with the detection limit (160ng/ml) of mouse bioassay in a current national standard detection method. In addition, the method is slightly influenced by interference effect of shellfish meat matrixes and reliable in data.
Description
Technical field
The present invention relates to a kind of method that detects paralytic shellfish poisoning (PSP).
Background technology
Paralytic shellfish poisoning (PSP) is a kind of by the biogenic alkyl hydrogenation of marine algae purines micromolecular compound, and it can realize the enrichment process in body of shellfish by the filter food effect of shellfish.Once people have eaten the shellfish meat that is subject to this endotoxin contamination by mistake, human body will produce nerve toxic reaction, as: slight shouting pain, four limbs and lip paralysis, headache fever etc., serious entail dangers to life.These symptoms usually occur in to be eaten by mistake between latter 10 minutes to 15 hours.In addition, paralytic toxin is also that world wide class distributes the most extensively, endangers one of maximum ocean toxin, and there is no up to now special efficacy antidote.Therefore, the detection of paralytic shellfish poisoning (PSP) seems particularly important for the guarantee of human health and food security.
Official's detection method of recommending as AOAC, Mouse bioassay and liquid chromatography fluorescence method have obtained increasing application in recent years, but also expose some problems thereupon.For example, though Mouse bioassay is simple to operate, and without complicated technology and equipment, detectability is higher, and the person's that is subject to animal protection opposition; Liquid chromatography fluorescence method not only exists apparatus expensive, operating personnel and has relatively high expectations etc. outside problem, also exists the drawback that can not find new toxin.Therefore, find new scientific and reasonable detection method and seem extremely necessary.
Cell detection method, utilizes toxin the not same-action of cell to be detected to a kind of detection method of toxin.Because cell has good homogeneity and stability, and easily realize high flux, cell detection method is considered to a very promising technology.A novel detection technique of getting up as development in recent years, cell impedance transducer effectively combines sensor technology and biological cell.Cell impedance transducer can be by the form number of variations of real-time unmarked monitoring cell and the change of growth conditions, thereby realizes the Real-Time Monitoring of cell to paralytic shellfish poisoning (PSP).
Summary of the invention
The object of the invention is for the deficiencies in the prior art, a kind of method that detects paralytic shellfish poisoning (PSP) is provided.
The object of the invention is to be achieved through the following technical solutions: a kind of method that detects paralytic shellfish poisoning (PSP), the method realizes on mouse neuroma cell impedance transducer, and described mouse neuroma cell impedance transducer connects looper by cell culture hole, cell impedance chip erecting bed, chip circuit plate, chip; Cell culture hole is positioned on cell impedance chip erecting bed, and chip circuit plate is connected with cell impedance chip erecting bed, and chip connects looper and is connected with chip circuit plate, and chip connects looper the impedance signal on chip circuit plate is transferred to computer; In cell culture hole, impedance electrodes is installed; The method comprises the following steps:
(1) the neuro2a cell that Fusion of Cells degree is reached to 80-90% is inoculated in 8 cell culture hole (1), and in each cell culture hole (1), nutrient solution cumulative volume is 300 μ l, and number of cells is with 30000; Cell was cultivated after 24 hours, and original fluid is changed with the fresh nutrient solution of 270 μ l in every hole;
(2) choose 4 cell culture hole (1) as experimental group, add the unabain of 15 mM of 10 μ l solution to be measured, 10 μ l and the veratridine solution of 1.5 mM of 10 μ l to each cell culture hole (1);
Remain 4 cell culture hole (1) as a control group, add the unabain of 15 mM of 10 μ l nutrient solutions, 10 μ l and the veratridine solution of 1.5 mM of 10 μ l to each cell culture hole (1);
(3) sensor chip being put back to incubator continues to cultivate;
(4) cell continues to cultivate the CI value that detects in real time each cell culture hole (1) in 24 hours, in the time that the CI of experimental group value is greater than the CI value of cellular control unit culture hole, is judged to be this solution to be measured and contains paralytic shellfish poisoning (PSP); CI value is higher, and paralytic shellfish poisoning (PSP) content is higher;
Described nutrient solution is RPMI1640 nutrient culture media, the hyclone that wherein to have added volume fraction be 10%, and the Sodium Pyruvate that massfraction is 1%, nonessential amino acid, massfraction that massfraction is 1% is that 1% glutamine, massfraction are 1% P/S is dual anti-.
The invention has the beneficial effects as follows: the method is directly taking cell as detecting carrier, simple to operate, with low cost.The detectability of saxitoxin is low to moderate 0.9 ng/ml, compares with detectability of the prior art (160 ng/ml) and has significant progress, and in addition, the method is subject to shellfish sarcenchyma interference effect little, and data are reliable.
Brief description of the drawings
Fig. 1 is mouse neuroma cell impedance transducer structural representation;
Fig. 2 is the schematic diagram that the present invention detects cell number and growth conditions;
Fig. 3 is the result figure that the present invention detects variable concentrations paralytic toxin;
After Fig. 4 is the saxitoxin of variable concentrations of the present invention and unabain, veratridine co-treatment different time, the linear relationship between cell index and toxin concentration;
Fig. 5 is that the present invention detects other saxitoxins and the shellfish extract result figure that does not contain toxin.
In figure, cell culture hole 1, cell impedance chip erecting bed 2, chip circuit plate 3, chip connect looper 4, impedance electrodes 5, cell 6.
Embodiment
As shown in Figure 2, the variation of cell resistance value is relevant with cell number and form.The advantages such as cell impedance transducer is the number that a kind of variation by cell resistance value detects cell, attaches and the change technique such as growth conditions, has real-time detection, unmarked and not damaged detection.In the time there is no Growth of Cells on chip, resistance value Z=Z
0, along with attaching and the growth of cell, resistance value Z=Z
cell.And cell index (CI) is the parameter through being commonly used to characterize cells state, therefore cell index can be according to formula CI=(Z
cell-Z
0)/Z
0calculate.Veratridine is a kind of activator of sodium channel, can promote sodium ion inflow, and unabain is the inhibitor of sodium pump, the direct result of these two kinds of medicine acting in conjunction neuro2a cells make exactly born of the same parents' extracellular sodium ion a large amount of in stream, cause cellular swelling and dead.And paralytic shellfish poisoning (PSP) can with cell membrane on Voltage-gated sodium channels specific binding and stop sodium ion inflow, therefore can suppress the cell death that veratridine and unabain cause.Based on this principle, can detect the paralytic shellfish poisoning (PSP) in shellfish meat by the variation of monitoring cell index.
According to this principle, a kind of method that detects paralytic shellfish poisoning (PSP) of the present invention, the method realizes on mouse neuroma cell impedance transducer, described mouse neuroma cell impedance transducer as shown in Figure 1, connects looper 4 by cell culture hole 1, cell impedance chip erecting bed 2, chip circuit plate 3, chip.Cell culture hole 1 is positioned on cell impedance chip erecting bed 2, and chip circuit plate 3 is connected with cell impedance chip erecting bed 2, and chip connects looper 4 and is connected with chip circuit plate 3, and chip connects looper 4 impedance signal on chip circuit plate 3 is transferred to computer.Impedance electrodes 5 is installed in cell culture hole 1.The method comprises the following steps:
1. before experiment, prepare.
The cultivation of 1.1 Neuro2a cells.
Neuro2a cell is cultivated at 25cm
2culture flask in, cell culture fluid has adopted RPMI1640 nutrient culture media, the hyclone that wherein to have added volume fraction be 10%, the Sodium Pyruvate that massfraction is 1%, nonessential amino acid, massfraction that massfraction is 1% is that 1% glutamine, massfraction are 1% P/S is dual anti-.Neuro2a cell needs every day and changes fresh nutrient culture media, treats that the degrees of fusion of cell reaches 80-90%, uses 0.25% trypsase-EDTA digestion to go down to posterity or be seeded on impedance chip and cultivates.
1.2 mouse neuroma cell impedance transducers check.
Cell is inoculated into mouse neuroma cell impedance transducer and goes forward, and adds the nutrient solution of 100 μ L to carry out testing background resistance value to cell culture hole 1, detects mouse neuroma cell impedance transducer and normally works.In the time that background impedance value is 0, show that system is normal, can detect.
2. the detection of toxin and data processing.
The neuro2a cell that Fusion of Cells degree is reached to 80-90% is inoculated in 9 cell culture hole 1 with the density in 30000, every hole, and in each cell culture hole 1, nutrient solution cumulative volume is 300 μ l.Cell was cultivated after 24 hours, changed fresh nutrient solution 270 μ l.Select the principal ingredient saxitoxin of paralytic toxin, the diarrhoea property principal ingredient okadaic acid of toxin and the representative toxin Euglena toxin of nerve toxin are as the detection toxin of this test.
Choose 7 cell culture hole 1 as experimental group, in each cell culture hole 1, adding respectively concentration is the Euglena toxin 10 μ l of okadaic acid 10 μ l, 300 nM of saxitoxin 10 μ l, 300 nM of saxitoxin 10 μ l, 3000 nM of saxitoxin 10 μ l, the 300nM of saxitoxin 10 μ l, the 30nM of 3nM, the shellfish extract 10 μ l that do not contain toxin, and every hole adds the unabain of 15 mM of 10 μ l and the veratridine solution of 1.5 mM of 10 μ l again.
Choose 2 cell culture hole as a control group, one of them hole adds the unabain of 15 mM of 10 μ l and the veratridine solution of 1.5 mM of 10 μ l, and any processing is not done in another one hole.
In each hole, add nutrient solution, making every hole cumulative volume is 300 μ l.After shaking up gently, sensor chip being put back to incubator continues to cultivate.At whole cell growth process, CI value detected once every 10 minutes, shown in testing result Fig. 3.Pass through the variation of the cell resistance value of different disposal by calculating, obtain toxin concentration and cell CI value change curve (as shown in Figure 4).
As can be seen from Figure 3, the CI value of toxin processed group cell is obviously high than unabain and veratridine control group, illustrate that paralytic toxin can obviously suppress the Apoptosis that unabain and veratridine acting in conjunction cause, and this inhibiting effect in the time that concentration range is 0.1-100 nM and the CI value of cell be dose-response relationship.This sensor can detect the saxitoxin that is low to moderate 0.1 nM, is equivalent to concentration 0.9 ng/ml in shellfish meat tissue.Well below detectability 160 ng/ml of current GB detection method Mouse bioassay.
The variation of the detection cell resistance value that as can be seen from Figure 4, the anti-sensor detecting method of groups of cells of the present invention's use can be real-time.Chosen toxin and processed rear 8h, tri-time points of 16h and 24h carry out data analysis.Can find out the increase along with the time, CI value reduces, but toxin inhibiting effect still continues.
Fig. 5 is that the cell sensor set up of the present invention detects variety classes toxin and there is no the result figure of the shellfish extract of toxin.Wherein the CI value of saxitoxin processed group is apparently higher than control group, and the CI value of other toxin processed group does not all have significant difference with control group, illustrate the method can be sensitive special detect paralytic shellfish poisoning (PSP).CI value and the control group of Toxic extraction liquid processed group do not have difference substantially, are all starkly lower than saxitoxin processed group, illustrate that the method is subject to shellfish sarcenchyma interference effect little.
3. aftertreatment
First with pancreatin, the cell culture hole 1 of cultivating cell is digested 30 minutes, ensureing that all cells are all digested gets off.Under microscope, whether observation of cell all digested, if do not had, use pipettor piping and druming tens of lower after, wash away pancreatin.Then with pure water rinsing chip 3-5 time, then use 75% alcohol immersion chip 10 minutes.Finally take out chip and dry, ultraviolet sterilization 30 minutes adds PBS refrigerator 4 degree of 200 μ l to preserve in cell culture hole, needs to be detected next time.
Claims (1)
1. one kind is detected the method for paralytic shellfish poisoning (PSP), the method realizes on mouse neuroma cell impedance transducer, and described mouse neuroma cell impedance transducer connects looper (4) by cell culture hole (1), cell impedance chip erecting bed (2), chip circuit plate (3), chip; Cell culture hole (1) is positioned on cell impedance chip erecting bed (2), chip circuit plate (3) is connected with cell impedance chip erecting bed (2), chip connects looper (4) and is connected with chip circuit plate (3), and chip connects looper (4) impedance signal on chip circuit plate (3) is transferred to computer; Impedance electrodes (5) is installed in cell culture hole (1); It is characterized in that, the method comprises the following steps:
(1) the neuro2a cell that Fusion of Cells degree is reached to 80-90% is inoculated in 2 cell culture hole (1), and in each cell culture hole (1), nutrient solution cumulative volume is 300 μ l, and number of cells is 30000; Cell was cultivated after 24 hours, and original fluid is changed with the fresh nutrient solution of 270 μ l in every hole;
(2) choose 1 cell culture hole (1) as experimental group, add the unabain of 15 mM of 10 μ l solution to be measured, 10 μ l and the veratridine solution of 1.5 mM of 10 μ l to cell culture hole (1);
Another cell culture hole (1) as a control group, adds the unabain of 15 mM of 10 μ l nutrient solutions, 10 μ l and the veratridine solution of 1.5 mM of 10 μ l to each cell culture hole (1);
(3) sensor chip being put back to incubator continues to cultivate;
(4) detect the CI value of each cell culture hole (1), in the time that the CI of experimental group value is greater than the CI value of cellular control unit culture hole, is judged to be this solution to be measured and contains paralytic shellfish poisoning (PSP); CI value is higher, and paralytic shellfish poisoning (PSP) content is higher;
Described nutrient solution is RPMI1640 nutrient culture media, the hyclone that wherein to have added volume fraction be 10%, and the Sodium Pyruvate that massfraction is 1%, nonessential amino acid, massfraction that massfraction is 1% is that 1% glutamine, massfraction are 1% P/S is dual anti-.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104777299A (en) * | 2015-03-13 | 2015-07-15 | 浙江大学 | Diarrhetic shellfish toxin high throughput detection device and method based on image analysis |
| CN105259292A (en) * | 2015-11-12 | 2016-01-20 | 上海市农业科学院 | Method for measuring paralysis shellfish poison in aquatic products |
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| CN101839913A (en) * | 2009-12-30 | 2010-09-22 | 复旦大学 | Microfluidic chip for rapid detection of saxitoxin and method for preparing same |
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| CN103278539A (en) * | 2013-06-07 | 2013-09-04 | 浙江大学 | Cell impedance sensor apparatus for detecting marine aquatic product diarrheal toxin, and method |
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2014
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Patent Citations (5)
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| CN101839913A (en) * | 2009-12-30 | 2010-09-22 | 复旦大学 | Microfluidic chip for rapid detection of saxitoxin and method for preparing same |
| CN101975768A (en) * | 2010-08-27 | 2011-02-16 | 深圳市疾病预防控制中心 | Method for detecting diarrhea shellfish toxin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104777299A (en) * | 2015-03-13 | 2015-07-15 | 浙江大学 | Diarrhetic shellfish toxin high throughput detection device and method based on image analysis |
| CN105259292A (en) * | 2015-11-12 | 2016-01-20 | 上海市农业科学院 | Method for measuring paralysis shellfish poison in aquatic products |
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Application publication date: 20141210 |