CN102180967A - Method for extracting immunoglobulin A (IgA) of Przewalski's horse noninvasively - Google Patents
Method for extracting immunoglobulin A (IgA) of Przewalski's horse noninvasively Download PDFInfo
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Abstract
The invention discloses a method for extracting immunoglobulin A (IgA) of Przewalski's horse noninvasively. The method for extracting animal immunoglobulin comprises the following steps of: mixing animal excrement and extraction buffer to obtain a mixture; and centrifuging the mixture to obtain supernatant, namely immunoglobulin in the animal, wherein the extraction buffer consists of phosphate buffered saline (PBS) and Tween-20. The method for extracting the immunoglobulin of the Przewalski's horse noninvasively is simple and high-efficiency. In the method, the fresh excrement of the Przewalski's horse is collected and dissolved in the extraction buffer added with the Tween-20 at certain concentration, and the supernatant is obtained through two-step centrifugation, namely the high-concentration immunoglobulin of the Przewalski's horse can be extracted. The method is easy to operate and high in extraction efficiency.
Description
Technical field
The present invention relates to the method for a kind of non-damage extraction przhevalski's horse Immunoglobulin IgA in the animal immunology field.
Background technology
Przhevalski's horse (Equus przewalskii) once was widely distributed in area, grassland, Eurasia, open-air extinction of the middle of last century.Existing przhevalski's horse is the Western adventurer caught and transported to European stable breeding in Chinese and Mongolia western part in the 19th-century end offspring.China in 1985 from drawing back these species abroad and having set up the center of breeding.Calendar year 2001, wilderness area in the OK a karaoke club wheat of Xinjiang implemented the Gui Yehua of putting of przhevalski's horse.The survival condition of species is heavily introduced in monitoring and assessment, and implements necessary manually managing and protecting, and be to guarantee to put the key link of returning the species long-term surviving and setting up wild stocks, but the work of these aspects is carried out seldom at present.Trace it to its cause, at first, the tissue samples that obtains the free living animal is very difficult, and regular or irregular collection research sample almost is impossible; Moreover catching and gathering animal blood sample itself is exactly a very strong stimulating factor, can the serious orthobiosis of disturbing animal.Obviously, be subjected to that sample obtains and the restriction of aspect such as mensuration, do not see as yet so far and put the immune physiological research of returning species.Therefore, as the physiological important indicator of monitoring animal body immunity, the extraction of immunoglobulin (Ig) and detection return species research particularly important to putting.
Obviously, for monitoring przhevalski's horse immunity physiological situation, traditional extraction blood sampling approach is no longer suitable, and urgent need will develop the method that immunoglobulin (Ig) was extracted and detected in a kind of simple, efficient, non-damage.Usually the non-damage sampling mainly is to gather target animal urine and ight soil, but its space for activities of przhevalski's horse is wide, and mostly be meadow or sandy soil, the urine of discharging will soon permeate the ground, be not easy to collect, and glomerular filtration membrane can not see through sphaeroprotein, so the immunoglobulin (Ig) that contains in the intact animal urine seldom.The gastrointestinal tract mucosa lamina propria can produce secretory immunoglobulin, enters also can carry part in the GI bile from the immunoglobulin (Ig) in the tissue juice.For these reasons, compare with gathering urine, the ight soil approach is the method that an ideal non-damage detects the przhevalski's horse immunoglobulin (Ig).
Summary of the invention
An object of the present invention is to provide a kind of method of extracting the animal immune sphaeroprotein.
A kind of method of extracting the animal immune sphaeroprotein provided by the present invention may further comprise the steps:
Animal excrement are mixed with the extraction damping fluid, obtain mixture; Described mixture is centrifugal, get supernatant liquor, promptly obtain described animal immune sphaeroprotein; Described extraction damping fluid is made up of PBS damping fluid and tween 20.
In the described extraction damping fluid, the concentration of volume percent of described tween 20 is 0.01%-0.5% or 0.03%-0.07% or 0.03% or 0.04% or 0.05% or 0.06% or 0.07%.
Described centrifugation method may further comprise the steps: it is centrifugal that described mixture is carried out the first step, gets supernatant liquor, and note is made supernatant liquor I; With described supernatant liquor I carry out second the step centrifugal; Described the first step centrifugal rotation speed is that 2000rpm, temperature are that 25 ℃ and time are 20min; The described second step centrifugal rotation speed is that 10000rpm, temperature are that 4 ℃ or 25 ℃ and time are 10min.
Before described mixture is centrifugal, comprise step with described mixture mixing.
The method of described mixing for described mixture with the vortex instrument 10min that under 1200rpm, vibrates.
Described PBS damping fluid is made up of sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic and water, and the concentration of each composition is respectively sodium-chlor 8.5g/L, SODIUM PHOSPHATE, MONOBASIC 0.3g/L and Sodium phosphate dibasic 2.2g/L.
The pH value of described extraction damping fluid is 7.2.
The consumption of described extraction damping fluid is: add the described extraction damping fluid of 10mL in the described animal excrement of every 1g.
Described animal excrement are the animal excrement behind the mixing.
Described animal is a przhevalski's horse;
Described immunoglobulin (Ig) is an Immunoglobulin IgA.
The invention provides simple, high-efficiency method that a kind of non-damage extracts the przhevalski's horse immunoglobulin (Ig).By gathering the przhevalski's horse fresh excreta, fresh excreta is dissolved in the extraction damping fluid that is added with finite concentration Tween-20, carry out two step centrifuging and taking supernatant liquors, can extract the przhevalski's horse immunoglobulin (Ig) that obtains higher concentration.This method is simple to operate and extraction efficiency is higher.
Description of drawings
Fig. 1 contains the influence of the extraction damping fluid of different concns Tween-20 to IgA extracted amount in the przhevalski's horse ight soil.
Fig. 2 is the influence of protamine to IgA extracted amount in the przhevalski's horse ight soil.
Fig. 3 is the influences of different centrifuging temperatures to ight soil IgA extracted amount.
Fig. 4 is the typical curve of the IgA concentration and the OD value in standard substance hole.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
One, tween concentration is to the influence of immunoglobulin (Ig) extracted amount in the przhevalski's horse ight soil
(1) non-damage extracts the przhevalski's horse immunoglobulin (Ig)
1, gathers the excrement sample
(the Xinjiang przhevalski's horse is bred research centre (88 ° of 45 ' E to object observing animal przhevalski's horse of same time period of every day (Equus przewalskii), 44 ° 10 ' N)), discharge fresh excreta in case find it, gather immediately, gather the ight soil of female przhevalski's horse and male przhevalski's horse respectively, sample objects is 10 adult wild horse individualities (5
), gather the fresh excreta of target individual for three days on end, respectively gather in every morning and afternoon 1 time, be respectively charged in the valve bag female przhevalski's horse ight soil and the male przhevalski's horse ight soil of being gathered and numbering, in time put into freezing preservation under-20 ℃ of conditions.
2, damping fluid is extracted in preparation
(1) preparation PBS buffered soln
Take by weighing 8.5g sodium-chlor, 0.3g SODIUM PHOSPHATE, MONOBASIC and 2.2g Sodium phosphate dibasic constant volume in the 1000ml ultrapure water, the mixing that vibrates gently after waiting to dissolve, is mixed with the pH value and is 7.2 PBS buffered soln.The concentration of sodium-chlor, SODIUM PHOSPHATE, MONOBASIC and Sodium phosphate dibasic is respectively in the described PBS buffered soln: sodium-chlor 8.5g/L, SODIUM PHOSPHATE, MONOBASIC 0.3g/L and Sodium phosphate dibasic 2.2g/L.
(2) damping fluid is extracted in preparation
Add the tween 20 (Tween-20) of different amounts (see Table 1 and table 2) in the above-mentioned PBS buffered soln of every 1000ml respectively, vibrate gently behind the mixing, preparation obtains PBST (PBS+Tween-20) aqueous solution of 18 different Tween-20 concentration (see Table 1 and table 2), extracts damping fluid as experimental group.Simultaneously organize the extraction damping fluid in contrast with above-mentioned PBS buffered soln.
Table 1 experimental group is extracted addition and the concentration (0.05%-0.50%) of Tween-20 in the damping fluid
Table 2 experimental group is extracted addition and the concentration (0.01%-0.09%) of Tween-20 in the damping fluid
3, extract the przhevalski's horse immunoglobulin (Ig)
Female przhevalski's horse ight soil and male przhevalski's horse ight soil difference mixing (mixing method: take by weighing each 100g of excrement sample that gather every day in each individual three days with step 1 collection, pulverizer (23000r) is pulverized 10min, it is standby to divide pouch to preserve in the sample of mixing) after, take by weighing female przhevalski's horse ight soil and male przhevalski's horse ight soil 1g respectively in the 15ml plastic centrifuge tube, add the experimental group for preparing in the 10ml step 2 respectively and extract damping fluid and control group extraction damping fluid, at 1200rpm vibration 10min mixing, obtain mixture with the vortex instrument; Is that 2000rpm and temperature be 25 ℃ condition under centrifugal 20min with low speed centrifuge (LDZ5-2 type table-type low-speed whizzer) at rotating speed with described mixture, get supernatant liquor I1ml in the 2ml plastic centrifuge tube, is that 10000rpm and temperature be 4 ℃ down centrifugal 10mins with high speed freezing centrifuge (Heal ForceNeofuge 15R desk type high speed refrigerated centrifuge) at rotating speed with described supernatant liquor I, get supernatant liquor II, promptly obtain immunoglobulin (Ig).Get supernatant liquor II in the 2ml plastic centrifuge tube, to be measured in-20 ℃ of freezing preservations.Each sample is established three repetitions.
(2) detect
Purchase is at the single-minded enzyme-linked immunosorbent assay kit of the immunoglobulin (Ig) that will detect, and the strict test kit specification sheets of pressing is operated.
Used kit is that the Holg IgA enzyme-linked immunosorbent assay kit that bio tech ltd is produced is ground in Shanghai more.The significant parameter of test kit is:
(1) measurement range: 5 μ g/ml-100 μ g/ml;
(2) sensitivity:＜2.5 μ g/ml;
(3) average recovery rate: 96-101%;
(4) variation coefficient: in batch＜6%;
(5) specificity: IgA and IgM, IgG and IgE cross reaction are all＜0.01%.
Contain following reagent in this test kit: coating antigen be horse IgA antibody, the standard substance of purifying be horse IgA, ELIAS secondary antibody be the HRP mark IgA antibody, washings (50mM Tris, 0.14M NaCl, 0.05%Tween-20, pH8.0) and stop buffer (0.18M H
2SO
4) and sample diluting liquid (50mM Tris, 0.14M NaCl, 1%BSA, 0.05%Tween-20).
Enzyme plate is carried by test kit.
Concrete detection step is as follows:
1, the dilution of standard substance and application of sample
On the enzyme mark wraps by plate, establish 10 holes, standard substance hole, in first, second hole, add standard substance 100 μ l respectively, in first, second hole, add standard substance diluent 50 μ l then, mixing; From first hole, second hole, respectively get 100 μ l then and be added to the 3rd hole and the 4th hole respectively, add standard substance diluent 50 μ l, mixing again in the 3rd, the 4th hole respectively; In the 3rd hole and the 4th hole, respectively get 50 μ l earlier then and discard, respectively get 50 μ l again and be added to respectively in the 5th, the 6th hole, in the 5th, the 6th hole, add standard substance diluent 50ul, mixing more respectively; Get respectively from the 5th, the 6th hole behind the mixing that 50 μ l are added to the 7th respectively, in the octal, again the 7th, add standard substance diluent 50 μ l respectively in the octal, behind the mixing from the 7th, get 50 μ l respectively the octal and be added in the 9th, the tenth hole, add standard substance diluent 50 μ l again in the 90 hole respectively, from the 90 hole, respectively get 50 μ l behind the mixing and discard.(each hole application of sample amount of dilution back all is 50 μ l, and concentration is respectively 90 μ g/ml, 60 μ g/ml, 30 μ g/ml, 15 μ g/ml and 7.5 μ g/ml).
2, application of sample
Establish blank well (the blank hole does not add sample and enzyme marking reagent, and all the other each step operations are identical), testing sample hole respectively.On wrapping by plate, the enzyme mark adds sample diluent (50mM Tris earlier in the testing sample hole, 0.14M NaCl, 1%BSA, 0.05%Tween-20) 40 μ l, and then add testing sample (being that above-mentioned steps () is extracted experimental group and the control group immunoglobulin (Ig) sample that obtains) 10 μ l (the final extent of dilution of sample is 5 times).Sample is added on bottom, enzyme plate hole, does not touch hole wall as far as possible, rock mixing gently.
3, incubation
With rearmounted 37 ℃ of incubations of shrouding film shrouding 30 minutes.
4, dosing
With 30 times of concentrated cleaning solutions (0.05%Tween-20, pH 8.0 for 50mM Tris, 0.14M NaCl) with standby after 30 times of dilutions of distilled water.
5, washing
Carefully take the shrouding film off, discard liquid, dry, washings is filled it up with in every hole, leaves standstill after 30 seconds to discard, and so repeats 5 times, pats dry.
6, enzyme-added
Every hole adds enzyme marking reagent 50 μ l, except the blank well.
7, incubation
Operation is with step 3.
8, washing
Operation is with step 5.
9, colour developing
Every hole adds developer A 50 μ l earlier, adds developer B50 μ l again, the light shaking mixing, and 37 ℃ of lucifuges developed the color 15 minutes.
10, stop
Every hole adds stop buffer 50 μ l, termination reaction (this moment blueness yellowing) immediately.
11, measure
With the blank well zeroing, the 450nm wavelength detects the absorbancy (OD value) in each hole in regular turn.Mensuration should be after adding stop buffer be carried out with interior in 15 minutes.
According to the OD value (table 3) that application of sample concentration value (μ g/ml) and each hole in standard substance hole records, production standard curve (Fig. 4) is according to typical curve equation y=-0.45186+0.11139X-0.00079X
2(R
2=0.97963), obtain the concentration (μ g/ml) of IgA in the sample well, again according to formula: concentration (μ g/ml) the * 5*10ml/ excrement sample of IgA heavy (g) in the amount of IgA (μ g/g)=sample well in every gram ight soil, converting obtains the amount (μ g/g) of IgA in every gram ight soil.
The IgA application of sample concentration value in table 3 standard substance hole and the OD value in hole
| IgA concentration (μ g/m1) | The OD value |
| 7.5 | 0.4160 |
| 15 | 0.8065 |
| 30 | 2.4315 |
| 60 | 3.2475 |
| 90 | 3.1985 |
The result is as follows:
The concentration that step () is extracted the experimental group obtain and control group immunoglobulin (Ig) sees Table 4 and Fig. 1.The result gets the mean value that all female przhevalski's horse immunoglobulin (Ig)s extract immunoglobulin (Ig) amount in the product, gets the mean value that all male przhevalski's horse immunoglobulin (Ig)s extract immunoglobulin (Ig) amount in the product.
The extracted amount of IgA in the every gram ight soil of table 4 experimental group and control group przhevalski's horse
From Fig. 1 A as seen, Tween-20 concentration is 0.05% o'clock in the used extraction damping fluid of experimental group, the Immunoglobulin IgA extracted amount is the highest, is significantly higher than the extraction damping fluid of other Tween-20 concentration and the extracted amount that control group extracts damping fluid.0 to 0.1%Tween-20 concentration gradient section is encrypted, and as seen B extracts that Tween-20 concentration is in 0.03%-0.07% (v/v) scope in the damping fluid from Fig. 1, and the extracted amount of IgA is higher.Therefore, definite optimal concentration scope of extracting Tween-20 in the damping fluid is 0.03%-0.07% (v/v).Female przhevalski's horse immunoglobulin (Ig) concentration and male przhevalski's horse immunoglobulin (Ig) concentration all do not have significant difference in each group.
Two, protamine is to the influence of immunoglobulin (Ig) extracted amount in the przhevalski's horse ight soil
(1) non-damage extracts the przhevalski's horse immunoglobulin (Ig)
1, gathers the excrement sample
Identical with the collection excrement quadrat method of step 1.
2, damping fluid is extracted in preparation
(1) preparation PBS buffered soln
Identical with the compound method of step 1.
(2) damping fluid is extracted in preparation
Add 0.5mL Tween-20 in the above-mentioned PBS buffered soln of every 1000ml, behind the mixing that vibrates gently, preparation obtains extracting damping fluid.Tween-20 concentration is 0.05% (v/v) in this extraction damping fluid.
3, preparation protamine solution
Take by weighing 0.8g protamine powder and be dissolved in the 10ml ultrapure water, preparation obtain the protamine aqueous solution.
4, extract the przhevalski's horse immunoglobulin (Ig)
Extracting method is with basic identical described in the experiment one, and different is: (1) adds the protamine aqueous solution before centrifugal, and (2) extract the damping fluid composition, and are specific as follows:
(1) adds the protamine aqueous solution that above-mentioned steps 3 is prepared
The add-on of the protamine aqueous solution is: add 100ul before the first step is centrifugal, add 10ul before second step is centrifugal.Experiment is divided into following 4 groups: the 1st group, promptly control group does not add the protamine aqueous solution before two steps are centrifugal; The 2nd group, add the protamine aqueous solution in the centrifugal forward direction supernatant liquor of second step; The 3rd group, all add the protamine aqueous solution before two steps are centrifugal, promptly add in the centrifugal forward direction mixture of the first step in the protamine aqueous solution and the centrifugal forward direction supernatant liquor of second step and add the protamine aqueous solution; The 4th group, add the protamine aqueous solution in the centrifugal forward direction mixture of the first step.
(2) extracting damping fluid is that the Tween-20 concentration for preparing in the step 2 is the extraction damping fluid of 0.05% (v/v).
Each sample is established three repetitions.
(2) detect
Identical with the detection method of experiment one.
The result is as follows:
Step (one) is extracted the concentration of 4 groups of immunoglobulin (Ig)s that obtain and is seen Fig. 2, and 1,2,3 and 4 represent the 1st group, the 2nd group, the 3rd group and the 4th group of female przhevalski's horse immunoglobulin (Ig) and male przhevalski's horse immunoglobulin (Ig) that extracts respectively among Fig. 2.
From Fig. 2 as seen, the 1st group (being control group), the 2nd group and the 4th group extract the immunoglobulin (Ig) concentration that obtains does not have significant difference, and the immunoglobulin (Ig) concentration that the 3rd group (all adding the protamine aqueous solution before two steps are centrifugal) obtains significantly is lower than other three groups.This result shows, can not increase the extracted amount of immunoglobulin (Ig) at the leaching process adding protamine of ight soil immunoglobulin (Ig).Female przhevalski's horse immunoglobulin (Ig) concentration and male przhevalski's horse immunoglobulin (Ig) concentration all do not have significant difference in each group.
Three, different centrifugation are to the influence of ight soil IgA extracted amount
(1) non-damage extracts the przhevalski's horse immunoglobulin (Ig)
1, gathers the excrement sample
Identical with the collection excrement quadrat method of experiment one.
2, damping fluid is extracted in preparation
(1) preparation PBS buffered soln
Identical with the compound method of experiment one.
(2) damping fluid is extracted in preparation
Identical with the compound method of experiment two, obtaining Tween-20 concentration is the extraction damping fluid of 0.05% (v/v).
3, extract the przhevalski's horse immunoglobulin (Ig)
Extracting method is basic identical with the extracting method in the experiment one, and different is the centrifugation difference, specific as follows:
(1) the female przhevalski's horse immunoglobulin (Ig) of gathering in the extraction step 1.
(2) according to the second step centrifugal centrifugation difference, be divided into two groups: first group: second step is centrifugal to be common high speed centrifugation, uses Hunan instrument TG16-WS table model high speed centrifuge, is room temperature (25 ℃) at centrifuging temperature, rotating speed is under the condition of 10000rpm, centrifugal 10min; Second group: second step is centrifugal to be the high speed frozen centrifugation, uses Heal ForceNeofuge 15R desk type high speed refrigerated centrifuge, is 4 ℃ at centrifuging temperature, and rotating speed is under the condition of 10000rpm, centrifugal 10min.
Every kind of centrifugation is established five repetitions, results averaged.
(2) detect
Identical with the detection method of experiment one.
The result is as follows:
Step (one) is extracted the concentration of two groups of immunoglobulin (Ig)s that obtain and is seen Fig. 3, and A represents the female przhevalski's horse immunoglobulin (Ig) that first group of common high speed centrifugation extracts among Fig. 3; The female przhevalski's horse immunoglobulin (Ig) that on behalf of second group of high speed frozen centrifugation, B extract.
From Fig. 3 as seen, first group and second group extract the immunoglobulin (Ig) concentration that obtains does not have significant difference.This result shows that centrifugal high speed frozen centrifugation of second step and common high speed centrifugation do not have significant difference to the influence of the extraction effect of immunoglobulin (Ig).Therefore, the high speed centrifugation step can be used common supercentrifuge, compares with the cryogenic freezing whizzer, and the former has low price, little, the easy to operate advantage of volume, and mini portable high-speed whizzer is also very general, more convenient experimental implementation.
Claims (10)
1. method of extracting the animal immune sphaeroprotein may further comprise the steps:
Animal excrement are mixed with the extraction damping fluid, obtain mixture; Described mixture is centrifugal, get supernatant liquor, promptly obtain described animal immune sphaeroprotein; Described extraction damping fluid is made up of PBS damping fluid and tween 20.
2. method according to claim 1 is characterized in that: in the described extraction damping fluid, the concentration of volume percent of described tween 20 is 0.01%-0.5% or 0.03%-0.07% or 0.03% or 0.04% or 0.05% or 0.06% or 0.07%.
3. method according to claim 1 and 2 is characterized in that:
Described centrifugation method may further comprise the steps: it is centrifugal that described mixture is carried out the first step, gets supernatant liquor, and note is made supernatant liquor I; With described supernatant liquor I carry out second the step centrifugal; Described the first step centrifugal rotation speed is that 2000rpm, temperature are that 25 ℃ and time are 20min; The described second step centrifugal rotation speed is that 10000rpm, temperature are that 4 ℃ or 25 ℃ and time are 10min.
4. according to arbitrary described method among the claim 1-3, it is characterized in that: before described mixture is centrifugal, comprise step described mixture mixing.
5. according to the method described in the claim 1-4, it is characterized in that: the method for described mixing for described mixture with the vortex instrument 10min that under 1200rpm, vibrates.
6. according to arbitrary described method among the claim 1-5, it is characterized in that: described PBS damping fluid is made up of sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic and water, and the concentration of each composition is respectively sodium-chlor 8.5g/L, SODIUM PHOSPHATE, MONOBASIC 0.3g/L and Sodium phosphate dibasic 2.2g/L.
7. according to arbitrary described method among the claim 1-6, it is characterized in that: the pH value of described extraction damping fluid is 7.2.
8. according to arbitrary described method among the claim 1-7, it is characterized in that: the consumption of described extraction damping fluid is: add the described extraction damping fluid of 10mL in the described animal excrement of every 1g.
9. according to arbitrary described method among the claim 1-8, it is characterized in that: described animal excrement are the animal excrement behind the mixing.
10. according to arbitrary described method among the claim 1-9, it is characterized in that:
Described animal is a przhevalski's horse;
Described immunoglobulin (Ig) is an Immunoglobulin IgA.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103149067A (en) * | 2013-02-28 | 2013-06-12 | 苏州和锐医药科技有限公司 | Method and reagent for extracting fecal protein |
| CN107385054A (en) * | 2017-08-08 | 2017-11-24 | 中国农业科学院兰州兽医研究所 | The quick determination method and its quick detection kit of trichina nucleic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1349559A (en) * | 1999-04-15 | 2002-05-15 | 帕德马纳班·P·奈尔 | Noninvasive detection of colorectal cancer and other gasrointestinal pathology |
| CN1401077A (en) * | 2000-02-16 | 2003-03-05 | 国际试药株式会社 | Method for examination of feces ocoult blood |
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| 《Clinical and diagnostic laboratory immunology》 20040930 IR Peters等 Measurement of immunoglobulin concentrations in the feces of healthy dogs 参见第841页右栏倒数第2段 8-10 第11卷, 第5期 * |
| 《Laboratory Animals》 20011031 J Hau等 Development and validation of a sensitive ELISA for quantification of secretory IgA in rat saliva and faeces 参见摘要及第302页右栏第4段 1-10 第35卷, 第4期 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149067A (en) * | 2013-02-28 | 2013-06-12 | 苏州和锐医药科技有限公司 | Method and reagent for extracting fecal protein |
| CN107385054A (en) * | 2017-08-08 | 2017-11-24 | 中国农业科学院兰州兽医研究所 | The quick determination method and its quick detection kit of trichina nucleic acid |
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| CN102180967B (en) | 2013-03-20 |
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