CN101858833A - Paralytic shellfish poisoning (PSP) standard sample and preparation method and application thereof - Google Patents
Paralytic shellfish poisoning (PSP) standard sample and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a paralytic shellfish poisoning (PSP) standard sample and a preparation method and an application thereof. Positive shellfish raw materials of PSP are initially frozen and dried after being acidized and homogenized to obtain a crude sample, and then the crude sample is frozen and dried once again after being ground and sieved to obtain the product in the invention with good uniformity and stability. The standard sample can be used for capability assurance activities of PSP test items in labs, for PSP qualitative and quantitative detection and for verification of a detection method, correction of a tester and quality control and examination of test results. The invention has low raw material cost and simple preparation method, and the obtained standard sample is a material standard sample, is even and stable and easy for storage, and is beneficial for effective monitoring on shellfish poisoning.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of saxitoxin standard model and its production and application, specifically, relate to a kind of paralytic shellfish poisoning (PSP) standard sample and its production and application.
Background technology
Saxitoxin has become an important public health problem in recent years, and existing a plurality of countries limit the quantity of to shellfish poison in the food and control, 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 about 1,100 ten thousand tons of shellfish per year, accounts for world's total amount 70%.During the nearly last ten years, take place frequently, had a strong impact on the marine aquaculture development and the export trade owing to reasons such as industrial pollution cause China's coastal waters red tide.European Union has taked close the border indefinite duration to the shellfish import of China after nineteen ninety-five, edible Chinese import mussel poisoning incident took place; States such as Korea S, Japan, the U.S. have also formulated strict restriction to China import shellfish and Related product.Saxitoxin has become big public hazards that hinder China's inshore fishing economic development, and the safety of shellfish food is great public health problem, is a key factor of restriction China sea fishery economic development.
The kind of saxitoxin is a lot, find at present mainly contain paralytic shellfish poisoning (PSP) (paralytic shellfishpoisoning, PSP), diarrhoea property saxitoxin (DSP), nerve saxitoxin (NSP) and Toxin-Domoic Acid (ASP) four big classes.It is that the poisoning effect of initial symptoms is gained the name that paralytic shellfish poisoning (PSP) PSP system causes behind the shellfish that contains this toxin because of the people eats by mistake with the peripheral neverous system paralysis, it is the very strong toxoid of toxicity, the mechanism of action is similar to tetraodotoxin with toxicity, it is a kind of neuromuscular paralysis agent, receive ion channel by the blocking-up cell, cause nervous system transmission obstacle and produce the paralysis effect.Dosis toxica to the people is 300-3000MU, and lethal quantity is 600～5000MU, and be 20 minutes latent period, and wherein saxitoxin is 80 times of cobratoxin toxicity, and 0.54mg～0.90mg is enough to make the people to cause death, and does not still have the antidote of suiting the medicine to the illness at present.Since the mussel poisoning incident that takes place from Whangarei port, Irish northern island in 1992 was confirmed to be paralytic shellfish neurotoxin first, the whole world all had this toxoid to cause the report of poisoning every year.At present, paralytic shellfish poisoning (PSP) has become the toxoid the widest, that the accident occurrence frequency is the highest, the extent of injury is maximum that distributes in the world.This toxoid mainly is a series of carbamic acid lipid derivants that wait some kinds that form red tide to produce by the Alexandrium in the dinoflagellate class (Alexandrium) and Goniaulax (Gonyaularx), Prorocentrum (Prorocentrum), is noncrystalline, water-soluble, high polarity, nonvolatile small-molecule substance.They are stable under acid condition, and alkali condition down oxidation takes place, and toxicity disappears, and Heat stability is good can not be destroyed by people's digestive ferment.PSP is a class severe toxicity heterocyclic organic compounds, being separated at present has kind more than 20 approximately, similarity according to group is divided three classes, be respectively (1) carbamyl toxoid (carbamoyl toxin), as saxitoxin (STX), new saxitoxin (neo-STX) and gonyatoxin (GTX); (2) carbamyl-N-sulfo group toxoid (N-sulfo carbamoyl toxin) is as C1, C2, C3, C4, GTX-5, GTX-6; (3) deammoniation formoxyl toxoid (decarbamoyl toxin is called for short dcSTX); they be with saxitoxin (Saxitoxin is called for short STX) be skeleton, the general designation of the multiple compounds that derive because of substituting group is different; as shown in Figure 1; because the difference of group on indivedual positions that many folded basic structures of hexatomic ring derive, the toxicity of each component is difference slightly.
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 at water source or results zone.In each link that shellfish produces, the variation of monitoring saxitoxin at any time is vital.Set up the method for a lot of detection PSP, as bioassay method, immunoassay, cytotoxicity detection method and chemical analysis etc.Be subjected to the restriction of various factors, the most effective now, also be still the mouse bioanalysis by the universally recognized shellfish poison detection method of various countries official.Also be to adopt the mouse bioanalysis in the national standard of China and inspection and quarantine industry standard, this method need use a large amount of multi-component PSP standard items that contain to satisfy Quality Control and quantitative requirement.But the PSP standard items all are the pure product of the standard of single component, no matrix, and prices are rather stiff, and each component is up to about 6000 yuan/100 μ g, and in addition, PSP is an extremely toxic substance, and all there are strict administrative provisions countries in the world to the entry and exit and the use of saxitoxin.Development paralytic shellfish poisoning (PSP) material standard sample can satisfy the market demand that the saxitoxin standard model is constantly enlarged day by day, solve the demand of the famine saxitoxin standard model of domestic and international seashell products test experience chamber, to improving food safety detection technology, technical solution barrier, ensuring food safety and play great function, also improve China's influence power in the world simultaneously.
Summary of the invention
First purpose of the present invention is to provide a kind of preparation method of paralytic shellfish poisoning (PSP) standard sample.
Second purpose of the present invention is to provide the paralytic shellfish poisoning (PSP) standard sample by this method preparation.
The 3rd purpose of the present invention is to provide the application of this paralytic shellfish poisoning (PSP) standard sample.
The preparation method of paralytic shellfish poisoning (PSP) standard sample of the present invention may further comprise the steps:
A), shellfish sample, decon are got shellfish meat;
B), in described shellfish meat, add distilled water and hydrochloric acid solution, regulating pH is 1.5～5.0, obtains homogenate behind homogeneous, the mixing;
C), obtain study with carrying out first freeze drying after the described homogenate precooling;
D), sieve behind the described study of grinding;
E), carrying out freeze drying once more after the sample precooling after will sieving gets standard samples;
F), encapsulate described standard model;
G), the homogeneity and the stability of the described standard model of check;
H), the PSP content to described standard model carries out definite value;
Wherein, described shellfish sample comprises the shellfish sample of the PSP positive.
Preferably, the concentration of described hydrochloric acid solution is 0.1～0.3mol/L; Precooling temperature among the described step C is-196 ℃～-20 ℃, and corresponding pre-cool time is 30min～24h; Precooling temperature in the described step e is-196 ℃～-20 ℃, and corresponding pre-cool time is 10min～6h; The described order number that sieves is 30～60 orders.
Further, preparation method of the present invention, get the scallop of PSP feminine gender, PSP negative sample that will be through obtaining after the step (A)～(D) with sieve after the PSP positive mix in proportion after, carry out freeze drying once more after placing-196 ℃～-20 ℃ corresponding precooling 10min～6h, thoroughly remove moisture, encapsulate and obtain paralytic shellfish poisoning (PSP) standard sample.
In addition, be raw material with the shellfish meat of the scallop of the scallop of PSP feminine gender and the PSP positive, adopt preparation method of the present invention also can obtain paralytic shellfish poisoning (PSP) standard sample.
According to the present invention, provide paralytic shellfish poisoning (PSP) standard sample by above-mentioned preparation method's preparation.
The paralytic shellfish poisoning (PSP) standard sample of preparation in accordance with the present invention preparation, its toxin component comprises C1, C2, C3, C4, dcGTX1, dcGTX2, dcGTX3, dcGTX4, dcSTX, GTX1, GTX2, GTX3, GTX4, GTX5, STX, NEO; PSP content is 40-1000MU/g.
The paralytic shellfish poisoning (PSP) standard sample of preparation in accordance with the present invention preparation, the ability verification that is used for paralytic shellfish poisoning (PSP) test event between the laboratory, also be used for quantitatively or the qualitative detection paralytic shellfish poisoning (PSP), and be used for the quality control of calibration, test result of checking, the testing tool of detection method and examination etc.
Paralytic shellfish poisoning (PSP) standard sample 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 process equipment, the standard model that obtains is the material standard sample, uniform and stable, easily preserve, not only solved the difficult problem that the PSP composition is very easily degraded, and the price that certainly will make the paralytic shellfish poisoning (PSP) standard sample that obtains is far smaller than in the market the price of the pure product of selling of paralytic shellfish poisoning (PSP) standard, guarantees the validity that shellfish poison detects, and helps shellfish poison is effectively monitored.Paralytic shellfish poisoning (PSP) standard sample of the present invention can be used for paralytic shellfish poison's power of test checking between the laboratory, be used 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 security, will bring remarkable social benefit and economic benefit.
Description of drawings
Fig. 1 is the chemical structural formula of paralytic shellfish poisoning (PSP).
Fig. 2 is a paralytic shellfish poisoning (PSP) standard sample preparation technology process flow diagram of the present invention.
Fig. 3 (a)～(v) be the LC-MS spectrogram of paralytic shellfish poisoning (PSP) standard sample of the present invention.
Embodiment
As shown in Figure 2, the preparation method of paralytic shellfish poisoning (PSP) standard sample of the present invention may further comprise the steps: (A) sampling, decon; (B) acidifying, homogenate; (C) first freeze drying; (D) grind, sieve; (E) freeze drying once more; (F) encapsulation; (G) homogeneity and stability test; (H) determine PSP content.
Concrete, the preparation method of paralytic shellfish poisoning (PSP) standard sample of the present invention may further comprise the steps:
(A) get the scallop of the PSP positive, the shell appearance is thoroughly cleaned, cut off closed shell flesh, open shell,, with closed shell flesh and the tissue that is connected gummed portion separately, carefully take out whole shellfish meat with the inner impurity such as silt and other exotics of removing of double distilled water drip washing with clear water;
(B) shellfish meat is added distilled water, reach 0.1～0.3mol/L hydrochloric acid solution and make pH 1.5～5.0, homogeneous, mixing obtains homogenate;
(C) carry out first freeze drying after homogenate being placed-196 ℃～-20 ℃ corresponding precooling 30min～24h, obtain study;
(D) study after the freeze-drying ground, cross 30～60 mesh sieves after the precomminution, fine grinding;
(E) will sieve and carry out freeze drying once more after the sample that obtains places-196 ℃～-20 ℃ corresponding precooling 10min～6h, and thoroughly remove moisture and get standard samples;
(F) between constant temperature and humidity, sterile working, the standard model that aforesaid way is obtained is sub-packed in capping sealing in the aseptic light-shading bottle;
(G) homogeneity and the stability test to sample is the main method of verification sample preparation process validity, and the standard model after the encapsulation is carried out homogeneity and stability checking;
(H) the PSP content to standard model carries out definite value.
Further, preparation method of the present invention, get the scallop of PSP feminine gender, PSP negative sample that will be through obtaining after the step (A)～(D) with sieve after the PSP positive mix in proportion after, carry out freeze drying once more after placing-196 ℃～-20 ℃ corresponding precooling 10min～6h, encapsulate the paralytic shellfish poisoning (PSP) standard sample that obtains different PSP content after thoroughly removing moisture.
In addition, be raw material with the shellfish meat of the scallop of the scallop of PSP feminine gender and the PSP positive, adopt preparation method of the present invention also can obtain paralytic shellfish poisoning (PSP) standard sample.
In the present invention, press the assay method of paralytic shellfish poisoning (PSP) in the GB/T 5009.213-2008 shellfish, PSP content in the scallop is determined experiment, PSP content＞the 4MU/g of the new fresh scallops that adopts, explanation is the sample of the PSP positive, PSP content＜the 4MU/g of the new fresh scallops that adopts, explanation is the sample of PSP feminine gender.
Below in conjunction with specific embodiment, the present invention will be further described.Should be understood that following examples only are used to the present invention to be described but not to be used to limits 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 scallop of getting the PSP positive, its PSP content is 46.2MU/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 gummed portion are separated, carefully take out whole shellfish meat, add distilled water, and the 0.18mol/L hydrochloric acid solution to make pH be 3, homogeneous, mixing were placed on-80 ℃ of pre-freezes at least 4 hours, adopted vacuum freeze drier (CHRiST Epsilon 2-6D) to carry out first freeze drying and obtained the PSP positive, organize grinding afterwards, the fine grinding of alumina porcelain bowl mill is adopted in precomminution, crosses 40 mesh sieves and is placed on-80 ℃ of pre-freezes at least 4 hours, carry out freeze drying once more, thoroughly remove moisture.In between constant temperature and humidity, sterile working, the scallop freeze-dried powder that aforesaid way is obtained is sub-packed in capping sealing in the brown cillin bottle of 5ml of 2 hours cleaning of 160 ℃ of dry heat treatment.
Embodiment 2-5 repeats the preparation process among the embodiment 1, and difference only is PSP content, concentration of hydrochloric acid, pH, precooling temperature, the pre-cool time of raw material and crosses grit number that concrete experiment condition is as shown in table 1.
The experiment condition of table 1 embodiment 2-5
| Embodiment | ??2 | ??3 | ??4 | ??5 |
| ??PSP | ??4.3 | ??13.9 | ??28.6 | ??60.8 |
| Concentration of hydrochloric acid | ??0.1mol/L | ??0.12mol/L | ??0.3mol/L | ??0.2mol/L |
| ??pH | ??1.5 | ??2 | ??3.5 | ??5 |
| First precooling temperature | ??-20℃ | ??-40℃ | ??-80℃ | -196 ℃ (in liquid nitrogen) |
| First pre-cool time | 24 hours | 10 hours | 6 hours | 30 minutes |
| Cross grit number | 30 orders | 40 orders | 50 orders | 60 orders |
| Precooling temperature once more | ??-20℃ | -196 ℃ (in liquid nitrogen) | ??-40℃ | ??-80℃ |
| Pre-once more cool time | 6 hours | 10 |
4 hours | 2 hours |
Adopt LC-MS to detect the sample of embodiment 1-5 preparation, the result shows: contained toxin component is identical.Concrete, standard model with embodiment 1 preparation is an example, detect through LC-MS, as shown in Figure 3, the toxin component of paralytic shellfish poisoning (PSP) standard sample comprises C1, C2, C3, C4, dcGTX1, dcGTX2, dcGTX3, dcGTX4, dcSTX, GTX1, GTX2, GTX3, GTX4, GTX5, STX, NEO.
Embodiment 6 homogeneitys and stability test
6.1 the uniformity test of sample
Randomly draw 16 bottles of samples of embodiment 1 preparation, get in every bottle of sample and do 3 groups of the samples that 3 repeatability detects.Adopt the mouse bioanalysis test among the GB/T 5009.213-2008, the PSP content of analytical standard sample is represented with allen-Doisy unit.All test portions are tested under repeated condition with random order, promptly use identical method of testing and instrument test within a short period of time by identical personnel in same laboratory.
Testing 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 the result is shown in table 2 and table 3.
Be 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 testing total number of times
The sample room quadratic sum
All square
Quadratic sum in the sample
All square
Degree of freedom f
1=m-1
f
2＝N-m
Statistic
The multilevel The results of analysis of variance of the single factor of table 2
Table 3 sample homogeneity test The results of analysis of variance
| Soruces of variation | Quadratic sum | Degree of freedom | All square MS | The F ratio | The F critical value | Fiducial probability |
| Between group | ??0.0027 | ??15 | ??0.000181 | ??1.70 | ??1.99 | ??0.95 |
| In the group | ??0.0034 | ??32 | ??0.000107 | |||
| Summation | ??0.0061 | ??47 |
Above result is as can be known: as α=0.05, f
1=15, f
2=32, look into the F distribution table, the F critical value is 1.99, because of the F ratio is 1.70＜F critical value 1.99, shows in the sample and the sample room there was no significant difference, illustrates that the standard model that embodiment 1 makes is uniformly, can satisfy requirement of experiment such as Quality Control.
Adopt above-mentioned same procedure, the homogeneity of the standard model that checking embodiment 2-5 makes, the F ratio is respectively 1.75,1.69,1.46 1.65 all less than F critical value 1.99, shows the interior and sample room there was no significant difference of standard model that embodiment 2-5 makes, the description standard sample is uniformly, can satisfy requirement of experiment such as Quality Control.
6.2 stability of sample check
According to the sampling principle that dredge close back before the time interval, be used for the sample of packaged embodiment 1 preparation of stability test respectively by following time picked at random: 0th, 10,20,30,40 days, and 3rd month, 6 months, 9 months.Choose 3 samples tests at every turn.In whole stability tests, used personnel, instrument, method of testing and laboratory are all identical with uniformity test.
The employing straight line model is estimated testing result, uses following statistical formulas, and the result is as shown in table 4.
The estimated value of slope:
The estimated value of intercept:
Estimate b
1Standard deviation:
In the formula:
Table 4 sample stability check result
The result shows that degree of freedom is that the distribution t-factor of n-2 and p=0.95 (95% confidence level) equals 2.45.
Because | b
1|＜t
0.95, n-2S (b
1)
So slope is inapparent, promptly do not observe instability, the having good stability of the standard model that checking embodiment 1 makes.
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 instability that embodiment 2-5 makes, and shows that the standard model that embodiment 2-5 makes has good stability.
The definite value of embodiment 7PSP content
Adopt the assay method of paralytic shellfish poisoning (PSP) in the GB/T 5009.213-2008 shellfish to carry out the detection by quantitative of sample in the standard model development process.
Adopt 8 laboratories to the PSP 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 the laboratory that 8 families participate in the definite value test respectively, select and appoint by each laboratory respectively and have the laboratory technician who enriches operating experience, according to unified test routine sample is carried out the detection test of PSP content, the result is as shown in table 5.8 breadboard value data adopt summer skin sieve one Weir gram (Shapiro-Wilk) method of inspection to investigate its normality.Meet under the prerequisite of normality approximate, confirm that through the check of Grubb ' s method all there is not exceptional value in each laboratory again, check each laboratory value data whether to wait precision with Cochran (Cochran), the result shows that all data are all by check, each lab investigation precision unanimity.The PSP standard model for preparing among the final embodiment 1 of determining is 698MU/g by the average of 8 experimental determinations, adopts the mode of standard value ± expanded uncertainty to represent the definite value result: PSP content 698 ± 13MU/g.
Table 5 cooperation definite value laboratory test results
Adopt above-mentioned same procedure,, adopt the mode of standard value ± expanded uncertainty to represent, be respectively 43 ± 2MU/g, 157 ± 9MU/g, 383 ± 15MU/g, 990 ± 11MU/g the PSP content of the standard model of the embodiment 2-5 definite value of cooperating.
Further, be the purpose of saving, and produce more standard models, adopt the PSP negative sample to carry out " dilution ".Concrete, get the scallop of PSP feminine gender, PSP negative sample that will be through obtaining after the step (A)～(D) with sieve after the PSP positive mix after, carry out freeze drying once more after placing-196 ℃～-20 ℃ corresponding precooling 10min～6h, thoroughly remove to encapsulate after moisture gets standard samples and obtain paralytic shellfish poisoning (PSP) standard sample.
In addition, be raw material with the shellfish meat of the scallop of the scallop of PSP feminine gender and the PSP positive, adopt preparation method of the present invention also can obtain paralytic shellfish poisoning (PSP) standard sample.
Embodiment 8 paralytic shellfish poisoning (PSP) standard samples are used
8.1 paralytic shellfish poisoning (PSP) standard sample is used for ability verification between the laboratory
The paralytic shellfish poisoning (PSP) standard sample that adopts preparation method's preparation of the present invention is used for the ability verification of paralytic shellfish poisoning (PSP) test event between the laboratory, investigates each laboratory and personnel's detectability.
At first paralytic shellfish poisoning (PSP) standard sample is distributed to corresponding laboratory 1-10 and personnel thereof, provide standard operation to instruct, experimentize then, report testing result (allen-Doisy unit is represented), with the robust statistics method each experimental result is estimated, promptly reflect than mark whether each laboratory data peels off, and is used for differentiating the laboratory ability, and the result is as shown in table 6 with Z.
Wherein, Z than mark computing formula is:
Z=(laboratory mean value-median)/standard interquartile-range IQR
Criterion: | Z|≤2, satisfactory result; 2＜| Z|＜3, suspicious result; | Z| 〉=3, the result who is unsatisfied with or peels off.
As shown in table 6, laboratory 2,3,4,6,7 and 8 | Z|≤2, be satisfactory result, illustrative experiment chamber 2,3,4,6,7 and 8 possesses detectability, laboratory 5 and 10 | Z| 〉=3, be result dissatisfied or that peel off, illustrative experiment chamber 5 and 10 does not possess detectability, and laboratory 1 and 9 | Z|＜3 and | Z|＞2 are suspicious result.For the laboratory of reporting suspicious or the result that peels off, should search reason, corresponding measures to rectify and reform are proposed, improve detection level.The result shows that the PSP standard model of preparation has reached testing requirements, can effectively guarantee paralytic shellfish poisoning (PSP) blind sample contrastive test quality control activity in kind in the laboratory, the ability verification that is used for paralytic shellfish poisoning (PSP) test event between the laboratory can effectively ensure the accuracy that shellfish poison detects.
Table 6 ability verification result
| The laboratory numbering | Testing result (MU/g) | Z-compares mark | Evaluation of result |
| ??1 | ??1488 | ??2.10 | Suspicious |
| ??2 | ??653 | ??-0.53 | Satisfied |
| ??3 | ??1192 | ??1.38 | Satisfied |
| ??4 | ??710 | ??-0.27 | Satisfied |
| ??5 | ??278 | ??-3.25 | Peel off |
| ??6 | ??497 | ??-1.41 | Satisfied |
| ??7 | ??780 | ??0.03 | Satisfied |
| ??8 | ??802 | ??0.12 | Satisfied |
| ??9 | ??1756.8 | ??2.62 | Suspicious |
| ??10 | ??2073.6 | ??3.15 | Peel off |
8.2 paralytic shellfish poisoning (PSP) standard sample is used for detection by quantitative
Through embodiment 8.1 checking, laboratory 7 | Z|≤2 and compare with other laboratories, | the Z| minimum possesses detectability most.The standard model of embodiment 2-5 preparation is provided to laboratory 7, and by following operation steps, the standard model that embodiment 2-5 is prepared carries out detection by quantitative:
Accurately take by weighing a certain amount of standard model, move in the beaker, divide with 20mL, 0.18mol/L hydrochloric acid solution and wash cillin bottle several times repeatedly, finally merge in the lump and be prepared into the suspendible sample in the beaker, add 20mL distilled water afterwards again, fully mix, regulating pH is 3.5.This moment, the sample suspension detected according to GB/T 5009.213-2008 standard method as a bulk sample, that is: with mixture heated, and boil 5min slowly, be cooled to room temperature, regulate pH to 3.5, can dropwise add the 5mol/L sodium hydroxide solution in the adjustment process and adjust pH, speed wants slow when adding alkali, need simultaneously constantly to stir, prevent that local alkalization from destroying toxin.This mixed liquor is moved on in the graduated cylinder, and constant volume is to 40mL, and the centrifugal 5min of 4000rpm gets supernatant as liquid to be measured (stoste), and liquid to be measured should be finished the animal contaminated experiment in 24 hours.If the death time of mouse is injected another group mouse (3), again up to the death time that obtains 5min～7min after less than 5min, then will diluting stoste to be measured.
Be calculated as follows the result:
X=median CMU * 40mL * extension rate/standard model weight g
In the formula: the content of PSP in the every g sample of X-, unit: MU/g;
Median CMU-test sample is subjected to the median of examination group mouse to proofread and correct allen-Doisy unit;
Adopt the PSP content of the standard model of said method detection by quantitative embodiment 2-5, the result is respectively 40.3MU/g, 160MU/g, 379MU/g, 998.5MU/g.
Further, the paralytic shellfish poisoning (PSP) standard sample that adopts preparation method's preparation of the present invention can also be used for the quality control of calibration, test result of checking, the testing tool of detection method and examination etc.
The inventor is through extensive and deep research, do matrix with the Patinopecten yessoensis that contains paralytic shellfish poisoning (PSP), adopt preparation method's preparation standard sample of the present invention, survivable and degraded paralytic shellfish poisoning (PSP) 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 process equipment, the standard model that obtains is the material standard sample, and is uniform and stable, easily preserve, be used for paralytic shellfish poison's power of test checking between the laboratory, quantitatively or the qualitative detection paralytic shellfish poisoning (PSP), guarantee the validity that shellfish poison detects, help shellfish poison is effectively monitored.Paralytic shellfish poisoning (PSP) standard sample 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 security, will bring remarkable social benefit and economic benefit.
Claims (9)
1. the preparation method of a paralytic shellfish poisoning (PSP) standard sample is characterized in that, said method comprising the steps of:
A), shellfish sample, decon are got shellfish meat;
B), in described shellfish meat, add distilled water and hydrochloric acid solution, regulating pH is 1.5～5.0, obtains homogenate behind homogeneous, the mixing;
C), obtain study with carrying out first freeze drying after the described homogenate precooling;
D), sieve behind the described study of grinding;
E), carrying out freeze drying once more after the sample precooling after sieving gets standard samples;
F), encapsulate described standard model;
G), the homogeneity and the stability of the described standard model of check;
H), the PSP content to described standard model carries out definite value;
Wherein, described shellfish sample comprises the shellfish sample of the PSP positive.
2. preparation method as claimed in claim 1 is characterized in that, the concentration of described hydrochloric acid solution is 0.1mol/L～0.3mol/L.
3. preparation method as claimed in claim 1 is characterized in that, the precooling temperature among the described step C is-196 ℃～-20 ℃, and corresponding pre-cool time is 30min～24h; Precooling temperature in the 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, the described order number that sieves is 30～60 orders.
5. according to the paralytic shellfish poisoning (PSP) standard sample of each described preparation method preparation in the claim 1～4.
6. paralytic shellfish poisoning (PSP) standard sample as claimed in claim 5, it is characterized in that the toxin component of described standard model comprises C1, C2, C3, C4, dcGTX1, dcGTX2, dcGTX3, dcGTX4, dcSTX, GTX1, GTX2, GTX3, GTX4, GTX5, STX, NEO.
7. paralytic shellfish poisoning (PSP) standard sample as claimed in claim 5 is characterized in that, the PSP content of described standard model is 40～1000MU/g.
8. according to the application of the paralytic shellfish poisoning (PSP) standard sample of each described preparation method's preparation in the claim 1～4, it is characterized in that, be used for the ability verification of paralytic shellfish poisoning (PSP) test event between the laboratory.
9. according to the application of the paralytic shellfish poisoning (PSP) standard sample of each described preparation method's preparation in the claim 1～4, it is characterized in that described standard model is used for quantitatively or the qualitative detection paralytic shellfish poisoning (PSP).
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Cited By (11)
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| CN102584861A (en) * | 2011-09-28 | 2012-07-18 | 王秋艳 | Diarrhetic shellfish poison standard sample as well as preparation method and application thereof |
| CN102607909A (en) * | 2012-03-02 | 2012-07-25 | 黑龙江省农业科学院植物脱毒苗木研究所 | Preparation method of standard substance for potato spindle tuber viroid detection |
| CN102706707A (en) * | 2012-05-30 | 2012-10-03 | 徐君怡 | Standard sample of diarrhetic shellfish toxin and preparation method of standard sample |
| CN103575893A (en) * | 2013-10-14 | 2014-02-12 | 广州市疾病预防控制中心 | Method for rapidly detecting shellfish toxin |
| CN103983495A (en) * | 2014-06-10 | 2014-08-13 | 山东出入境检验检疫局检验检疫技术中心 | Tetrodotoxin positive quality control sample as well as preparation method and application thereof |
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| CN106644642A (en) * | 2016-12-29 | 2017-05-10 | 青岛水务集团有限公司科技中心 | Preparing method of organic principle standard sample in urban sludge and prepared sample |
| CN109142595A (en) * | 2018-08-23 | 2019-01-04 | 中国水产科学研究院黄海水产研究所 | A kind of preparation method of paralytic shellfish poisoning (PSP) standard solution |
| CN112110930A (en) * | 2020-09-16 | 2020-12-22 | 中国水产科学研究院东海水产研究所 | Method for extracting neosaxitoxin from toxic shell |
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| CN102584861A (en) * | 2011-09-28 | 2012-07-18 | 王秋艳 | Diarrhetic shellfish poison standard sample as well as preparation method and application thereof |
| CN102584861B (en) * | 2011-09-28 | 2013-12-11 | 王秋艳 | Diarrhetic shellfish poison standard sample as well as preparation method and application thereof |
| CN102607909A (en) * | 2012-03-02 | 2012-07-25 | 黑龙江省农业科学院植物脱毒苗木研究所 | Preparation method of standard substance for potato spindle tuber viroid detection |
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| CN105571918A (en) * | 2016-01-18 | 2016-05-11 | 中国农业科学院茶叶研究所 | Sample pre-treatment method for precise quantification detection of bioactive substances |
| CN105571918B (en) * | 2016-01-18 | 2018-04-10 | 中国农业科学院茶叶研究所 | A kind of sample-pretreating method for the detection of bioactive substance accurate quantification |
| CN106644642A (en) * | 2016-12-29 | 2017-05-10 | 青岛水务集团有限公司科技中心 | Preparing method of organic principle standard sample in urban sludge and prepared sample |
| CN106644642B (en) * | 2016-12-29 | 2019-07-02 | 青岛水务集团有限公司科技中心 | The preparation method of organic principle standard sample and standard sample obtained in city sludge |
| CN109142595A (en) * | 2018-08-23 | 2019-01-04 | 中国水产科学研究院黄海水产研究所 | A kind of preparation method of paralytic shellfish poisoning (PSP) standard solution |
| CN112110930A (en) * | 2020-09-16 | 2020-12-22 | 中国水产科学研究院东海水产研究所 | Method for extracting neosaxitoxin from toxic shell |
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