CN102584861B - Diarrhetic shellfish poison standard sample as well as preparation method and application thereof - Google Patents
Diarrhetic shellfish poison standard sample as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a diarrhetic shellfish poison (DSP) standard sample as well as a preparation method and an application thereof. The method comprises the following steps: shelling fresh shellfish containing DSP masculine, taking out intestinal gland, adding organic solvent, homogenizing, freeze-drying primarily to obtain a coarse sample, grinding and screening the coarse sample to obtain freeze-dried powder, dispersing the freeze-dried powder in organic solvent again, and freeze-drying to obtain the DSP standard sample. The DSP standard sample has better uniformity and stability, can be used for proficiency testing activities in DSP testing projects in laboratories and for qualitative and quantitative detection of DSP, and can also be used for verification of detection methods, calibration of testing instruments and quality control and evaluation of testing results. The invention has the advantages that the raw materials cost is low, the preparation method is simple, and the obtained standard sample is a substantial standard sample, is uniform and stable, is easy to store, and is beneficial to the effective monitoring to shellfish poisons.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of saxitoxin standard model and its preparation method and application, specifically, relate to a kind of research of diarrhetic shellfish poisons standard model and its preparation method and application.
Background technology
Saxitoxin belongs to the marine natural organism, and in its formation and ocean, poisonous algae red tide is closely related.According to the literature, micro-algae that can form red tide approximately has 184～267 kinds, and wherein poisonous micro-algae is about 60～78 kinds.The toxin that poisonous algae produces often enters in body of shellfish by food chain, thereby usually claims that these toxin are shellfish poison, and people are edible to cause poisoning with the shellfish of shellfish poison.The kind of saxitoxin is a lot, and that finds at present mainly contains paralytic shellfish poisoning (PSP), research of diarrhetic shellfish poisons (diarrhetic shellfish poisons, DSP), nervosa saxitoxin (NSP) and the large class of Toxin-Domoic Acid (ASP) four.
Research of diarrhetic shellfish poisons (DSP) is the fat-soluble secondary metabolite of a class of algae or microorganisms in ocean, and in the shellfish body, character is highly stable, and general cooking heating can not make its destruction.The mankind eat by mistake to produce take the toxicity symptom that diarrhoea is principal character, and jaundice, acute atrophy hepatic necrosis can appear in severe patient.The shellfish of being poisoned by DSP is relevant with local pollution of the sea, close with Relation With Red Tide.The shellfish that can be poisoned by DSP is bivalve shellfish, is mainly scallop, mussel, variegated clam, clam, oyster etc., no matter what shellfish, DSP generally all is confined to mid intestinal gland.In recent years, by the caused food poisoning of DSP, report is constantly arranged all over the world, DSP, in marine biotoxins, especially occupies critical role in saxitoxin, has become serious problems that affect shellfishery and food sanitation.
Saxitoxin has become an important public health problem in recent years, and existing a plurality of countries limit the quantity of and controlled shellfish poison in food, and WHO has also issued about the harm of prevention shellfish poison, protected sanitarian draft.China is that the marine economy shellfish produces and big export country, produces approximately 1,100 ten thousand tons of shellfishes per year, accounts for world's total amount 70%.During the nearly last ten years, because the reasons such as industrial pollution cause the China coastal seas frequent occurrence, marine aquaculture development and the export trade have been had a strong impact on.European Union, after nineteen ninety-five, edible Chinese import mussel poisoning event occurred, has taked close the border indefinitely to the shellfish import of China; The states such as Korea S, Japan, the U.S. have also formulated strict restriction to China import shellfish and related products.Saxitoxin has become large public hazards that hinder the development of China coast fishery economic, and the safety of shellfish food is great public health problem, is an important factor of restriction China marine fishery Economic development.
At present, people also can't effectively control red tide and saxitoxin, and therefore prevention and forecast are the effective means that alleviates its harm.The most effective prevention method that WHO recommends is that the control monitoring facilities is carried out in water source or results zone.The links of producing shellfish, the variation of monitoring at any time saxitoxin is vital.Set up the method for a lot of detection DSP, as bioassay method, immunoassay, cytotoxicity detection method and chemical analysis etc., but each method all must the Application standard sample or quality-control sample do Quality Control and quantitative.The saxitoxin standard model is difficult to obtain, and first because it is highly toxic substance, there is strict control and management regulation countries in the world to the entry and exit of saxitoxin; Saxitoxin is of a great variety in addition, extremely unstable, is difficult to preserve.This certainly will greatly limit shellfish poison has effectively been monitored, thereby causes irreparable damage can to shellfish culture enterprise.China for thering is tracing to the source, card is arranged, the research of diarrhetic shellfish poisons standard model of matter sample or quality control sample technology of preparing be blank, there is not yet the development report of relevant research of diarrhetic shellfish poisons standard model.Development research of diarrhetic shellfish poisons material standard sample can meet the market requirement to the continuous expanding day of saxitoxin standard model, solve the demand of the famine saxitoxin standard model of domestic and international seashell products testing laboratory, to improving food safety detection technology, technical solution barrier, ensuring food safety and play great effect, also improve China's influence power in the world simultaneously.
Summary of the invention
One object of the present invention is to provide a kind of preparation method of research of diarrhetic shellfish poisons standard model.
This research of diarrhetic shellfish poisons standard model that provides present method to prepare is provided another purpose of the present invention.
The 3rd purpose of the present invention is to provide the application of this research of diarrhetic shellfish poisons standard model.
In order to achieve the above object, the preparation method of research of diarrhetic shellfish poisons standard model of the present invention comprises the following steps:
A) shellfish sample decon is got to mid intestinal gland;
B) add distilled water in described mid intestinal gland sample, add methanol-acetone-ether mixing solutions, homogeneous, obtain homogenate after mixing;
C) obtain study by after described homogenate precooling, carrying out lyophilize;
D) sieve after grinding described study, obtain shellfish freeze-drying powder;
E) add distilled water in described shellfish freeze-drying powder, add the mixing solutions of methanol-acetone-ether, mix, again carry out lyophilize after precooling and get standard samples;
F) encapsulate described standard model;
G) check homogeneity and the stability of described standard model;
H) the DSP content of described standard model carried out to definite value;
Wherein, the shellfish sample that described shellfish sample comprises the DSP positive or negative.
Under optimal way, methyl alcohol in methanol-acetone in described step B-ether mixing solutions: acetone: the ether volume ratio is 3～6:2～5:1, and the volume of described mixing solutions is to add 1/10～1/5 of distilled water volume; The add-on of distilled water and described enteraden sample equal-volume.In described step e, in described shellfish freeze-drying powder, add pentaploid to amass distilled water, add the mixing solutions of the long-pending methanol-acetone-ether of monoploid; Methanol-acetone in described step e-ether mixing solutions methyl alcohol: acetone: the ether volume ratio is 3～6:2～5:1.Under optimum way, methanol-acetone in described step e-ether mixing solutions methyl alcohol: acetone: the ether volume ratio is 3:2:1.
In addition, under optimal way, the precooling temperature in described step C is-196 ℃～-20 ℃, and corresponding pre-cool time is 30min～24h; Precooling temperature in described step e is-196 ℃～-20 ℃, and corresponding pre-cool time is 10min～6h.In described step D, the order number sieved is 30～60 orders.
Research of diarrhetic shellfish poisons standard model prepared by the inventive method, its toxin component comprises OA, PTX2; The DSP total content is 1.0～15 μ g/g.
The invention provides research of diarrhetic shellfish poisons standard model prepared by above-mentioned preparation method.
Research of diarrhetic shellfish poisons standard model prepared by preparation method of the present invention, ability verification for research of diarrhetic shellfish poisons test event between laboratory, also for quantitative or qualitative detection research of diarrhetic shellfish poisons, and for detection of the quality control of the calibration of the checking of method, testing tool, test result and examination etc.
Research of diarrhetic shellfish poisons standard model of the present invention and preparation method thereof, fill up domestic and international blank, raw materials cost is low, and the preparation method is simple, do not relate to expensive processing unit, the standard model obtained is the material standard sample, uniform and stable, easily preserve, not only solved the difficult problem that the DSP composition is very easily degraded, and the price that certainly will make the research of diarrhetic shellfish poisons standard model that obtains is far smaller than the price of the research of diarrhetic shellfish poisons standard sterling of selling in the market, guarantee the validity that shellfish poison detects, be conducive to shellfish poison is effectively monitored.Research of diarrhetic shellfish poisons standard model of the present invention can be used for diarrhetic shellfish poison power of test checking between laboratory, for quantitative or qualitative detection paralytic shellfish poisoning (PSP), being suitable for each department such as food, fishing prison, environmental protection, quality inspection, fishery, health uses, can promote aquaculture, processing and Trade Development, guarantee food safety, will bring significant Social benefit and economic benefit.
The accompanying drawing explanation
Fig. 1 is the chemical structural formula of research of diarrhetic shellfish poisons OA component;
Fig. 2 is the chemical structural formula of research of diarrhetic shellfish poisons PTX2 component;
Fig. 3 is research of diarrhetic shellfish poisons preparation of standard sample process flow diagram of the present invention;
Fig. 4-1st, OA standard substance LC-MS spectrogram;
Fig. 4-2nd, PTX2 standard substance LC-MS spectrogram;
Fig. 4-3 and Fig. 4-4th, the LC-MS spectrogram of research of diarrhetic shellfish poisons standard model of the present invention.
Wherein, std refers to standard substance, and Sample refers to research of diarrhetic shellfish poisons standard model of the present invention.
Embodiment
The chemical structural formula of research of diarrhetic shellfish poisons as depicted in figs. 1 and 2.
As shown in Figure 3, the preparation method of research of diarrhetic shellfish poisons standard model of the present invention comprises the following steps: (A) sampling, decon; (B) homogenate; (C) lyophilize; (D) grind, sieve; (E) mixed sample, lyophilize again; (F) encapsulation; (G) homogeneity and stability test; (H) determine DSP content.
In a kind of preferred implementation, the preparation method of research of diarrhetic shellfish poisons standard model of the present invention comprises the following steps:
(A) get the positive mussel of DSP, with clear water, thoroughly clean the shellfish shell, cut off closed shell flesh, open shell, with inner silt and other foreign matters removed of clear water drip washing, take out shellfish meat (avoiding destroying mid intestinal gland), carefully cut whole mid intestinal glands standby.
(B) add distilled water in the mid intestinal gland sample, add methanol-acetone-ether mixing solutions, homogeneous, obtain homogenate after mixing.
Preferred version, add equal-volume distilled water.Methanol-acetone-ether volume ratio is 3～6:2～5:1, optimum volume ratio 3:2:1, and the volume that methanol-acetone-the ether mixing solutions adds is distilled water 1/10～1/5.
(C) obtain study by after described homogenate precooling, carrying out lyophilize.
Preferred method, carry out lyophilize after homogenate being placed in to-196 ℃～-20 ℃ corresponding precooling 30min～24h, obtains the positive study of DSP.
(D) the positive study of the DSP after freeze-drying ground, crossed 30～60 mesh sieves after precomminution, fine grinding, obtained the mussel lyophilized powder.
(E) add water in described shellfish freeze-drying powder, add the mixing solutions of methanol-acetone-ether, after mixing, again carry out lyophilize after precooling and get standard samples.
Preferred version, add pentaploid to amass distilled water, adds the mixing solutions of the long-pending methanol-acetone-ether of monoploid; Methyl alcohol in methanol-acetone in described step e-ether mixing solutions: acetone: the ether volume ratio is 3～6:2～5:1, optimum volume ratio 3:2:1.In addition, precooling temperature is-196 ℃～-20 ℃, and corresponding pre-cool time is 10min～6h.
(F), between fixed temperature and humidity, aseptic technique, the standard model that aforesaid way is obtained is sub-packed in capping sealing in aseptic light-shading bottle.
(G) be the main method of verification sample preparation process validity to homogeneity and the stability test of sample, the sample after encapsulation is carried out to homogeneity and stability test.
(H) the DSP content of standard model carried out to definite value.
In another preferred embodiment, the preparation method of research of diarrhetic shellfish poisons standard model of the present invention comprises the following steps:
(A) get respectively the mussel of the DSP positive, feminine gender, with clear water, thoroughly clean the shellfish shell, cut off closed shell flesh, open shell, with clear water drip washing inner removal silt and other foreign matters, take out shellfish meat (avoiding destroying mid intestinal gland), carefully cut whole mid intestinal glands standby;
(B) the shellfish mid intestinal gland cut is added to distilled water, and add the mixing solutions of methanol-acetone-ether (volume ratio is 3:2:1), homogeneous, mix and obtain homogenate;
(C) carry out lyophilize after homogenate being placed in to-196 ℃～-20 ℃ corresponding precooling 30min～24h, obtain DSP positive or negative study;
(D) DSP after freeze-drying is positive, negative study is ground, cross 30～60 mesh sieves after precomminution, fine grinding;
(E) mix the positive lyophilized powder of DSP, negative lyophilized powder, wherein said DSP is positive, and lyophilized powder accounts for 1%～99% of lyophilized powder gross weight, the mixing solutions that adds the long-pending distilled water of pentaploid and 1 times of volumes methanol-acetone-ether, stir, mix, again carry out lyophilize after being placed in-196 ℃～-20 ℃ corresponding precooling 10min～6h, thoroughly remove moisture and obtain the DSP standard model;
(F), between fixed temperature and humidity, aseptic technique, the standard model that aforesaid way is obtained is sub-packed in capping sealing in aseptic light-shading bottle;
(G) be the main method of verification sample preparation process validity to homogeneity and the stability test of sample, the sample after encapsulation is carried out to homogeneity and stability test;
(H) the DSP content of standard model carried out to definite value.
In addition, the mid intestinal gland of mussel of the mussel of DSP feminine gender and the DSP positive of take is raw material, adopts preparation method of the present invention also can obtain research of diarrhetic shellfish poisons standard model (now can omit in step e mix DSP is positive, the step of negative lyophilized powder).
In the present invention, press the research of diarrhetic shellfish poisons method of inspection in the SN/T1996-2007 shellfish, enzyme-linked immunosorbent assay, DSP in mussel is determined to experiment, the DSP content of the fresh mussel adopted > 0.16 μ g/g, explanation is the sample of the DSP positive, the DSP of the fresh mussel of employing<0.16 μ g/g, and explanation is the sample of DSP feminine gender.
Below in conjunction with specific embodiment, the present invention will be further described.Should be understood that following examples are only for the present invention is described but not for limiting the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1: the mussel of getting the DSP positive, its DSP content is the new scallop meat of 0.56 μ g/g(), with clear water, the shell appearance is thoroughly cleaned, cut off closed shell flesh, open shell, with the inner impurity such as silt and other exotics of removing of distilled water drip washing, closed shell flesh and the tissue that is connected to gummed section are separated, take out whole shellfish meat, carefully cut whole mid intestinal glands.Add isopyknic distilled water to cutting in mid intestinal gland, and account for the methanol-acetone of distilled water volume 1/10 volume-ether mixing solutions (volume ratio is 3:2:1), mix and be placed on-80 ℃ of pre-freezes at least 4 hours, adopt vacuum freeze drier (CHRiST Epsilon2-6D) to carry out lyophilize, organize afterwards grinding, precomminution, adopt the fine grinding of alumina porcelain ball mill, cross 40 mesh sieves, obtain the positive meal of DSP, the mixing solutions that adds distilled water and methanol-acetone-ether, stir, mix, again carry out lyophilize after being placed in-196 ℃～-20 ℃ corresponding precooling 10min～6h, thoroughly remove moisture and obtain the DSP standard model.In between fixed temperature and humidity, aseptic technique, the mussel lyophilized powder that aforesaid way is obtained is sub-packed in capping sealing in the brown cillin bottle of 5ml of 160 ℃ of dry heat treatment cleaning of 2 hours.
Embodiment 2-5 repeats the preparation process in embodiment 1, the DSP content, methanol-acetone-ether mixed liquor volume that difference only is raw material is consumption (with the distilled water volume ratio), precooling temperature, pre-cool time and cross grit number when, and concrete experiment condition is as shown in table 1.
The experiment condition of table 1 embodiment 2-5
Adopt LC-MS to detect sample prepared by embodiment 1-5, result shows: contained toxin component is identical.Concrete, standard model prepared by the embodiment 1 of take is example, through LC-MS, detects, as shown in Figure 4, the toxin component of research of diarrhetic shellfish poisons standard model comprises okadaic acid (OA), pectenotoxins (PTX2).
Reference examples 1～5, be intended to outstanding beneficial effect of the present invention.
Reference examples 1～3 is identical with the preparation process of embodiment 1, and difference is to adopt single agents methyl alcohol, acetone or substituted ether methanol-acetone-ether mixing solutions.Reference examples 4～5 is identical with the preparation process of embodiment 1, and difference is methanol-acetone-ether mixed liquor volume than being 2:6:1 and 7:1:1.
Embodiment 6 homogeneities and stability test
6.1 the uniformity test of sample
Randomly draw the sample of 15 bottles of examples, 1 preparation, every bottle of sample is done 2 repeatability and is detected.Adopt the enzyme-linked immunosorbent assay test in SN/T1996-2007, the DSP content of analytical standard sample, mean with μ g/g.All test portions are tested under repeated condition with random order, i.e. identical testing method and instrument test within a short period of time by identical librarian use in same laboratory.
Detected result is carried out Evaluation for Uniformity with the multilevel variance analysis method of single factor, adopts with the multilevel variance analysis statistical formulas of the factor of placing an order, and result is as shown in table 2 and table 3.
For the homogeneity of check sample, extract i sample, each sample is tested j time under repeat condition.
The testing mean of each sample
The population mean of whole sample tests
The test total degree
The sample room sum of squares
All square
Sum of squares in sample
All square
Degree of freedom f
1=m-1
f
2=N-m
Statistic
The sample homogeneity detected result of table 2 example 1 preparation
| Sample number | Test result μ g/g | Test result μ g/g | |
| 1 | 9.35 | 8.97 | 9.16 |
| 2 | 8.78 | 8.48 | 8.63 |
| 3 | 9.99 | 8.89 | 9.44 |
| 4 | 8.85 | 9.69 | 9.27 |
| 5 | 8.69 | 7.98 | 8.34 |
| 6 | 9.50 | 8.49 | 8.99 |
| 7 | 8.75 | 8.67 | 8.71 |
| 8 | 8.27 | 7.99 | 8.13 |
| 9 | 8.87 | 7.79 | 8.33 |
| 10 | 9.32 | 10.01 | 9.67 |
| 11 | 7.91 | 8.76 | 8.34 |
| 12 | 8.79 | 9.34 | 9.07 |
| 13 | 9.14 | 7.59 | 8.37 |
| 14 | 8.68 | 8.77 | 8.73 |
| 15 | 9.68 | 8.86 | 9.27 |
Table 3 sample homogeneity test the results of analysis of variance
| Soruces of variation | Sum of squares | Degree of freedom | All square | The F ratio | The F threshold value | Fiducial probability | The sample room standard deviation |
| Between group | 6.319834 | 14 | 0.451417 | 1.42 | 2.42 | 0.95 | 0.2593 |
| In group | 4.754157 | 15 | 0.316944 | ? | ? | ? | ? |
| Summation | 11.073991 | ? | ? | ? | ? | ? | ? |
Above result is known: as α=0.05, f
1=14, f
2=15, look into the F-distribution table, the F threshold value is 2.42, because of F, than 1.42<F threshold value 2.42, shows in sample and the sample room there was no significant difference, interpret sample is uniformly, can meet the requirement of experiment such as Quality Control.
Adopt same procedure, the homogeneity of the standard model that checking embodiment 2-5 makes, the F ratio is respectively 1.25,1.68,1.47 1.56 all are less than F threshold value 2.42, show the interior sample room there was no significant difference that reaches of standard model sample that embodiment 2-5 makes, interpret sample is uniformly, can meet the requirement of experiment such as Quality Control.
Adopt above-mentioned same procedure, the homogeneity of the standard model that checking reference examples 1-5 makes, the F ratio is respectively 3.07,3.48,4.02,2.99,2.77 all be greater than F threshold value 2.42, show that the interior sample room that reaches of standard model sample that reference examples 1-5 makes has significant difference, interpret sample is inhomogeneous, can not meet the requirement of experiment such as Quality Control.
6.2 the stability test of sample
According to close rear thin sampling principle before the timed interval, choose at random the sample for packaged embodiment 1 preparation of stability test by the following time respectively: 0th, 2,4,6,9,12,18,24 months.Choose 3 samples is tested at every turn.In whole stability tests, personnel used, instrument, testing method and laboratory are all identical with uniformity test.
The employing straight line model is estimated detected result, uses following statistical formulas, and result and evaluation are as table 4, shown in 5.
The estimated value of slope:
The estimated value of intercept:
Estimate b
1standard deviation:
In formula:
Table 4 sample stability check result
Table 5 sample stability test statistics analytical results
Result shows, degree of freedom is n-2 and p=0.95(95% confidence level) the distribution t-factor equal 2.45.
Due to | b
1|<t
0.95, n-2s (b
1)
Therefore slope is inapparent, do not observe unstable, the having good stability of the checking standard model that makes of embodiment 1.
Adopt above-mentioned same procedure, the stability of the standard model that checking embodiment 2-5 makes, slope is all not remarkable, does not observe the standard model unstable that embodiment 2-5 makes, and result shows that the standard model that embodiment 2-5 makes has good stability.
Adopt above-mentioned same procedure, the stability of the standard model that checking reference examples 1-5 makes, slope is remarkable, and result shows that standard model stability that reference examples 1-5 makes can not reach the requirement of standard model.
The definite value of embodiment 7DSP content
In the research and production of metallurgical standard sample process, adopt research of diarrhetic shellfish poisons method of inspection enzyme-linked immunosorbent assay in the SN/T1996-2007 shellfish to carry out the detection by quantitative of sample.
Adopt 8 laboratories to the DSP content of the standard model of embodiment 1 definite value of cooperating.To process homogeneity and the qualified sample of stability test, randomly draw 40 bottles, be distributed to respectively 8 laboratories of participating in the definite value test, select and appoint and there is the laboratory technician who enriches operating experience by each laboratory respectively, according to unified testing sequence, sample is carried out the detection test of DSP content, result is as shown in table 6.The value data in 8 laboratories adopts Xia Piluo-Weir gram (Shapiro-Wilk) method of inspection to investigate its normality.Approximate, meet under the prerequisite of normality, confirm that through the check of Grubb ' s method all there is not outlier in each laboratory again, with Cochran (Cochran), check each laboratory value data whether to wait precision, result shows that all data are all by check, and each lab investigation precision is consistent.The DSP standard model prepared in the final embodiment 1 of determining is 8.83 μ g/g by the average of 8 experimental determinations, adopts the mode of standard value ± expanded uncertainty to mean the definite value result: DSP content 8.83 ± 0.60 μ g/g.
Table 6 cooperation definite value laboratory test results
Adopt above-mentioned same procedure, to the DSP content of the standard model of the embodiment 2-5 definite value of cooperating, adopt the mode of standard value ± expanded uncertainty to mean, be respectively 4.31 ± 0.29 μ g/g, 7.08 ± 0.47 μ g/g, 12.18 ± 0.89 μ g/g, 15.23 ± 1.02 μ g/g.
Adopt above-mentioned same procedure, to the DSP content of the reference examples 1-5 sample definite value of cooperating, adopt the mode of standard value ± expanded uncertainty to mean, be respectively 5.87 ± 0.78 μ g/g, 5.58 ± 0.72 μ g/g, 5.39 ± 0.69 μ g/g, 6.03 ± 0.63 μ g/g, 6.23 ± 0.71 μ g/g.Reference examples 1-5 is identical with the starting material of example 1, but in the sample of preparing, DSP content, well below example 1, is respectively 66%, 63%, 61%, 68%, 71% of example 1, and the DSP yield is well below embodiment 1.
Further, be the purpose of saving, and produce more standard models, adopt the DSP negative sample to carry out " dilution ".Concrete, get the mussel of DSP feminine gender, the DSP negative sample that will obtain after step (A)～(D) with sieve after the DSP positive mix after, again carry out lyophilize after being placed in-196 ℃～-20 ℃ corresponding precooling 10min～6h, thoroughly remove after moisture gets standard samples to encapsulate and obtain the research of diarrhetic shellfish poisons standard model.
Embodiment 8 research of diarrhetic shellfish poisons standard model application
8.1 the research of diarrhetic shellfish poisons standard model is for detection by quantitative
Accurately take standard model prepared by a certain amount of the present invention, move in beaker, with 30ml90% methanol solution suspendible sample, make the sample suspension, according to the SN/T1996-2007 standard method, detected, that is: mix 2 minutes, centrifugal 10 minutes of 3000g, get supernatant liquor, with distilled water, diluted, as liquid to be measured, ELISA detects, and is calculated as follows result:
X=liquid concentration * 30mL to be measured * extension rate/standard model weight g
In formula: the content of DSP in X-every g sample, unit: μ g/g;
Adopt aforesaid method, the DSP content of the standard model of detection by quantitative embodiment 1-5, result is respectively 4.15 μ g/g, 6.89 μ g/g, 8.97 μ g/g, 11.5 μ g/g, 15.03 μ g/g.
8.2 the research of diarrhetic shellfish poisons standard model is for the quality control activity of result
Research of diarrhetic shellfish poisons standard model prepared by employing preparation method of the present invention, for the quality control activity of quality results, is investigated laboratory and personnel's detectivity.
At first the research of diarrhetic shellfish poisons standard model is distributed to corresponding laboratory 1-10 and personnel thereof, provide standard operation to instruct, then tested, report detected result (μ g/g means), by the robust statistics method, each experimental result is estimated, reflect than mark whether each laboratory test data peels off with Z, be used for discriminating experiment chamber ability, result is as shown in table 7.Wherein, Z than mark calculation formula is: the flat – median that all is worth in Z=(laboratory)/standard interquartile-range IQR
Judging criterion: | Z|≤2, satisfactory result; 2<| Z|<3, suspicious result; | Z| >=3, the result that is unsatisfied with or peels off.
As shown in table 7, laboratory 1,2,3,4,7,8 and 10 | Z|≤2, for satisfactory result, illustrative experiment chamber 1,2,3,4,7,8 and 10 possesses corresponding detectivity, laboratory 9 | Z| >=3 are result dissatisfied or that peel off, and illustrative experiment chamber 9 does not possess detectivity, and laboratory 5 and 6 | Z|<3, | Z| > 2 be suspicious result.For the laboratory of reporting suspicious or the result that peels off, should search reason, corresponding Abarbeitungsmassnahme are proposed, improve detection level.Result shows that the DSP standard model of preparation has reached test requirements document, can effectively guarantee research of diarrhetic shellfish poisons Intercomprison test mass in kind control activity in laboratory, for the ability verification of research of diarrhetic shellfish poisons test event between laboratory, but the accuracy that the effective guarantee shellfish poison detects.
Table 7 sample is for the quality control action result
The research of diarrhetic shellfish poisons standard model that further can also be prepared by employing preparation method of the present invention is for detection of the checking of method, the calibration of testing tool, the quality examination of test result etc.
The inventor is through extensive and deep research, do matrix with the mussel containing research of diarrhetic shellfish poisons, adopt preparation method's preparation standard sample of the present invention, survivable and degraded research of diarrhetic shellfish poisons constituent structure and content, fill up domestic and international blank, raw materials cost is low, and the preparation method is simple, do not relate to expensive processing unit, the standard model obtained is the material standard sample, uniform and stable, easily preserve, for diarrhetic shellfish poison power of test checking between laboratory, quantitatively or the qualitative detection research of diarrhetic shellfish poisons, guarantee the validity that shellfish poison detects, be conducive to shellfish poison is effectively monitored.Research of diarrhetic shellfish poisons standard model of the present invention is suitable for each department such as food, fishing prison, environmental protection, quality inspection, fishery, health to be used, can promote aquaculture, processing and Trade Development, guarantee food safety, will bring significant Social benefit and economic benefit.
The above; it is only preferably embodiment of the present invention; but protection scope of the present invention is not limited to this; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to replacement or changed according to technical scheme of the present invention and inventive concept thereof, within all should being encompassed in protection scope of the present invention.
Claims (6)
1. the preparation method of a research of diarrhetic shellfish poisons standard model, is characterized in that, comprises the following steps:
A) shellfish sample decon is got to mid intestinal gland;
B) add distilled water in described mid intestinal gland, add methanol-acetone-ether mixing solutions, homogeneous, obtain homogenate after mixing;
C) obtain study by after described homogenate precooling, carrying out lyophilize;
D) sieve after grinding described study, obtain shellfish freeze-drying powder;
E) add distilled water in described shellfish freeze-drying powder, add the mixing solutions of methanol-acetone-ether, mix, again carry out lyophilize after precooling and get standard samples;
F) encapsulate described standard model;
G) check homogeneity and the stability of described standard model;
H) the DSP content of described standard model carried out to definite value;
Wherein, the shellfish sample that described shellfish sample comprises the DSP positive or negative;
Methyl alcohol in the mixing solutions of methanol-acetone-ether in described step B: acetone: the ether volume ratio is 3～6:2～5:1, the volume of described mixing solutions be add distilled water volume 1/10～1/5; The add-on of distilled water and described mid intestinal gland equal-volume;
In described step e, in described shellfish freeze-drying powder, add pentaploid to amass distilled water, add the mixing solutions of the long-pending methanol-acetone-ether of monoploid; Methyl alcohol in the mixing solutions of methanol-acetone-ether in described step e: acetone: the ether volume ratio is 3～6:2～5:1.
2. preparation method as claimed in claim 1, is characterized in that, methyl alcohol in the mixing solutions of methanol-acetone-ether in described step e: acetone: the ether volume ratio is 3:2:1.
3. preparation method as claimed in claim 2, is characterized in that, the precooling temperature in described step C is-196 ℃～-20 ℃, and corresponding pre-cool time is 30min～24h; Precooling temperature in described step e is-196 ℃～-20 ℃, and corresponding pre-cool time is 10min～6h.
4. preparation method as claimed in claim 1, is characterized in that, in described step D, the order number sieved is 30～60 orders.
5. according to the described preparation method of any one in claim 1～4, it is characterized in that, the toxin component of prepared research of diarrhetic shellfish poisons standard model is OA, PTX2.
6. preparation method as claimed in claim 5, is characterized in that, the DSP content of described standard model is 1.0～15 μ g/g.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992009891A1 (en) * | 1990-11-21 | 1992-06-11 | National Research Council Of Canada | Method for quantitatively assaying the presence fo diarrhetic shellfish poisoning toxins in marine samples. |
| US20100105928A1 (en) * | 2008-09-25 | 2010-04-29 | Yih Wonho | Methods for massive culture of dinophysis acuminata and isolation of pectenotoxin-2 |
| CN101858833A (en) * | 2010-05-21 | 2010-10-13 | 王秋艳 | Paralytic shellfish poisoning (PSP) standard sample and preparation method and application thereof |
| 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 |
-
2012
- 2012-01-13 CN CN2012100098034A patent/CN102584861B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992009891A1 (en) * | 1990-11-21 | 1992-06-11 | National Research Council Of Canada | Method for quantitatively assaying the presence fo diarrhetic shellfish poisoning toxins in marine samples. |
| US20100105928A1 (en) * | 2008-09-25 | 2010-04-29 | Yih Wonho | Methods for massive culture of dinophysis acuminata and isolation of pectenotoxin-2 |
| CN101858833A (en) * | 2010-05-21 | 2010-10-13 | 王秋艳 | Paralytic shellfish poisoning (PSP) standard sample and preparation method and application thereof |
| 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 |
Non-Patent Citations (10)
| Title |
|---|
| acute * |
| Blanca Sua´ |
| Blanca Sua& * |
| mez,等.New Targets in Diarrhetic Shellfish Poisoning Control.《J.Nat.Prod.》.2005,第68卷(第4期),第596-599页. * |
| PATROKLOS VARELTZIS，等.Removal of Lipids and Diarrhetic Shellfish Poisoning Toxins from Blue Mussels (Mytilus edulis) during Acid and Alkaline Isolation of Proteins.《J.Agric.Food Chem.》.2008,第56卷(第10期),第3675-3680页. * |
| rez-Go´ |
| rez-Go& * |
| 傅博强,等.腹泻性贝类毒素基体标准物质的研制.《计量学报》.2010,第31卷(第5A期),实验方法部分2.2.1节及2.2.3-2.2.5节,第169页左栏最后一段. * |
| 唐守亭.腹泻性贝类毒素及其检测.《卫生研究》.1998,第27卷第88-89页. |
| 腹泻性贝类毒素及其检测;唐守亭;《卫生研究》;19980831;第27卷;全文 * |
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