CN112067811B - Test strip for detecting alternaria alternate and application thereof - Google Patents
Test strip for detecting alternaria alternate and application thereof Download PDFInfo
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- CN112067811B CN112067811B CN202010823305.8A CN202010823305A CN112067811B CN 112067811 B CN112067811 B CN 112067811B CN 202010823305 A CN202010823305 A CN 202010823305A CN 112067811 B CN112067811 B CN 112067811B
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Abstract
The invention discloses a test strip for detecting alternaria alternate and application thereof. The test strip comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), a water absorption pad (4) and a bottom plate (7), wherein the reaction membrane is provided with a detection line (5) coated with a cross-linked streptavidin hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, and the conjugate release pad (2) is sprayed with a cross-linked streptavidin monoclonal antibody-colloidal gold marker. The invention also provides a method for detecting the residue of the alternaria alternata in the grains such as wheat, corn and the like and the feed by using the alternaria alternata test strip. The test strip provided by the invention has the characteristics of simplicity in operation, high sensitivity, high detection speed, low cost and the like, and is suitable for screening and on-site monitoring of a large number of samples.
Description
Technical Field
The invention relates to a test strip for detecting alternaria alternata and application thereof, in particular to a colloidal gold test strip for detecting the alternaria alternata, which is particularly suitable for detecting the residue of the alternaria alternata in grains such as wheat, corn and the like and feeds.
Background
The Henan Linzhou city is a high-incidence area of esophageal cancer, and researches prove that the alternaria alternate and toxins thereof separated from the grain in the city play a certain role in inducing esophageal cancer. Although the content of the toxins in the grain of the market is reduced at present, the toxins are still existing in the beverage such as fruit juice and the like, and therefore, the alternaria alternate and the toxins thereof are closely related to human life.
In order to reduce the effects on humans and animals, it is important to effectively and rapidly detect alternaria alternata in cereals and feeds. Compared with the instrument method, the immunochemistry detection method has the advantages of rapidness, specificity, sensitivity, accuracy, batch monitoring, simple sample treatment, automatic operation and the like for mycotoxin detection. The premise of immunochemistry detection is that antibodies against the streptavidin are required. The invention is a rapid detection method, has short time, simple operation and low cost, and is suitable for detecting samples in a large scale in a basic unit.
Disclosure of Invention
The invention aims to provide a test strip for detecting the alternaria alternate residue, which has the advantages of high sensitivity, simple operation, low cost and short detection time.
The test strip for detecting the inter-compartmental streptavidin residue comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), a water absorption pad (4) and a bottom plate (7); the reaction membrane is provided with a detection line (5) coated with a cross-linked streptavidin hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, and the conjugate release pad (2) is sprayed with a cross-linked streptavidin monoclonal antibody-colloidal gold marker.
The conjugate of the cross-linked streptavidin hapten and the carrier protein is obtained by coupling the cross-linked streptavidin hapten and the carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine and human serum albumin.
The inter-alternaria alternata monoclonal antibody is prepared by taking an inter-alternaria alternata hapten-carrier protein conjugate as an immunogen, and is obtained by secretion of inter-alternaria alternata monoclonal antibody hybridoma cell strains; the goat anti-mouse antibody is obtained by immunizing a goat with a mouse-derived antibody.
The sample absorbing pad (1), the conjugate releasing pad (2), the reaction membrane (3) and the water absorbing pad (4) are sequentially adhered to the bottom plate (7), and the conjugate releasing pad 1/3-1/2 is covered below the sample absorbing pad.
The bottom plate can be a PVC bottom plate or other hard non-water-absorbing materials; the sample absorption pad can be suction filter paper or oil filter paper; the conjugate release pad may be a glass wool or polyester material; the water absorbing pad is water absorbing paper; the reaction membrane may be a nitrocellulose membrane or a cellulose acetate membrane.
Another object of the present invention is to provide a method for preparing the above test strip, comprising the steps of:
1) Preparing a conjugate release pad sprayed with a cross-linked streptavidin monoclonal antibody-colloidal gold marker;
2) Preparing a reaction film with a detection line coated with a cross-linked streptavidin hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps 1) and 2) into the test strip.
Specifically, the method comprises the following steps:
1) Hapten preparation: the method comprises the steps of subjecting substances such as the alternaria alternate and DL-homocysteine to a series of chemical reactions to obtain a alternaria alternate hapten;
2) Coupling the streptavidin hapten and the carrier protein to obtain a streptavidin hapten-carrier protein conjugate;
3) Immunizing a mouse by using a cross-linked streptavidin hapten-carrier protein conjugate, and fusing and screening spleen cells and myeloma cells of the mouse to obtain a cross-linked streptavidin monoclonal hybridoma cell strain;
4) Extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibody;
5) Preparing colloidal gold by the reaction of trisodium citrate and chloroauric acid;
6) Adding the inter-digitalis streptavidin monoclonal antibody prepared in the step 3) into the colloidal gold prepared in the step 5) to obtain an inter-digitalis streptavidin monoclonal antibody-colloidal gold marker;
7) Spraying the inter-isolation streptavidin monoclonal antibody-colloidal gold marker on a conjugate release pad, drying at 37 ℃ for 1h, taking out, and storing in a dry environment for standby;
8) Coating the interjacent streptavidin hapten-carrier protein conjugate on a reaction membrane to form a detection line, and coating the goat anti-mouse anti-antibody on the reaction membrane to form a quality control line;
9) Soaking the sample absorption pad in phosphate buffer solution containing 0.5% bovine serum albumin (volume fraction) with pH of 7.2 and 0.1mol/L for 2h, and drying at 37deg.C for 2h;
10 The sample absorbing pad, the conjugate releasing pad, the reaction membrane and the water absorbing pad are sequentially stuck on the bottom plate, the sample absorbing pad covers the conjugate releasing pad, finally, the sample absorbing pad is cut into small strips with the width of 3mm, a plastic box is added, and the sample absorbing pad can be stored for 12 months at the temperature of 4-30 ℃ after vacuum packaging.
The invention also provides a method for detecting the residue of the alternaria alternata in grains and feeds by using the test strip, which comprises the following steps:
(1) Sample pretreatment;
(2) Detecting by using a test strip;
(3) And analyzing the detection result.
The rapid test strip for the inter-digitalis of the invention adopts a highly specific antibody antigen reaction and competitive inhibition immunochromatography analysis technology, and the inter-digitalis monoclonal antibody-colloidal gold label is fixed on a conjugate release pad, and the inter-digitalis in a sample is combined with the inter-digitalis monoclonal antibody-colloidal gold label on the conjugate release pad in the flowing process to form the drug-antibody-colloidal gold label. The medicine in the sample and the inter-digitalis streptavidin hapten-carrier protein conjugate on the reaction membrane detection line compete for binding with the inter-digitalis streptavidin monoclonal antibody-colloidal gold marker, and whether the inter-digitalis streptavidin residues are contained in the sample liquid to be detected is judged according to the existence or the darkness of the red strip of the detection line.
During detection, a sample is dripped into a test strip hole after treatment, when the concentration of the inter-alternaria alternate in the sample is lower than the detection limit or zero, a monoclonal antibody-colloidal gold marker is combined with the inter-alternaria alternate hapten-carrier protein conjugate fixed on a reaction membrane in the chromatography process, and a red strip appears in a detection line (T) and a quality control line (C) respectively; if the concentration of streptavidin in the sample is at or above the limit of detection, the monoclonal antibody-colloidal gold label will bind to all of the streptavidin, so that no red band will appear at the T-line due to competition reactions with the streptavidin hapten-carrier protein conjugate.
The test strip has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitability for various units, simple storage and long shelf life. The method for detecting the alternaria alternate residue by using the test strip is simple, convenient, quick, visual, accurate, wide in application range, low in cost and easy to popularize and use.
Drawings
FIG. 1 is a diagram showing the synthesis of a streptavidin hapten.
Fig. 2 is a schematic diagram of a cross-sectional structure of the test strip.
Detailed Description
The invention is further illustrated below in conjunction with specific examples. It is to be understood that these examples are for illustration of the invention only and are not intended to limit the scope of the invention.
Example 1 preparation of test strips for detecting Interval Neurospora
The preparation method of the test strip mainly comprises the following steps:
1) Preparing a conjugate release pad sprayed with a cross-linked streptavidin monoclonal antibody-colloidal gold marker;
2) Preparing a reaction film with a detection line coated with a cross-linked streptavidin hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the PVC bottom plate which are prepared in the steps 1) and 2) into the test strip.
The following is a stepwise detailed description:
1. preparation of inter-digitalis streptavidin hapten
Dissolving 30mg of inter-cut streptavidin in 50ml of tetrahydrofuran, adding 5ml of trifluoroacetic acid, fully stirring, adding 0.7g of glycolic acid, heating in an oil bath, refluxing for 12h, stopping the reaction, adding 100ml of water, adding 100ml of ethyl acetate, fully oscillating, standing for layering, removing water phase, washing organic phase with 60ml of water, oscillating for standing, separating water phase, evaporating organic phase to dryness, washing and pulping with 1ml of dichloromethane/n-hexane (v/v, 1/9), standing, separating supernatant, drying obtained precipitate, and obtaining 26mg of glycolic acid inter-cut streptavidin hapten with the yield of 70%.
2. Preparation of immunogens
Taking 14mg of hydroxyethylated alternaria alternate, adding 1ml of DMSO for dissolution, adding 9.7mg of NHS and 19mg of DCC, fully dissolving and uniformly mixing, and reacting for 3 hours to obtain hapten activating solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.1M PB buffer solution for dissolution to obtain solution B, dripping all solution A into solution B, reacting for 4h at room temperature, dialyzing and purifying with 0.02M PBS for 3 days, changing the solution 3 times a day to obtain the inter-alternaria alternata-BSA conjugate, namely the immunogen, subpackaging at-20 ℃, and preserving for later use.
3. Preparation of coating Material
Taking 9mg of hydroxyethylated alternaria alternate, adding 1ml of DMF for dissolution, adding 7.6mg of NHS and 9mg of EDC for full dissolution and uniform mixing, and reacting for 3 hours to obtain hapten activating solution A; taking 50mg of Ovalbumin (OVA), adding 0.1M PB buffer solution for dissolution to obtain solution B, dripping all solution A into solution B, reacting at room temperature for 4h, dialyzing and purifying with 0.02M PBS for 3 days, changing the solution 3 times a day to obtain the inter-alternaria alternate-OVA conjugate, namely the coating source, subpackaging at-20 ℃, and preserving for later use.
4. Preparation of inter-digitated streptavidin monoclonal antibody
(1) Immunization of animals
The immunogen obtained in the step 2 is injected into Balb/c mice, and the immune dose is 150 mug/mouse, so that antisera are generated.
(2) Cell fusion and cloning
Spleen cells of an immunized Balb/c mouse are fused with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative proportion), cell supernatant is measured by adopting an indirect competition ELISA method, and positive holes are screened. Cloning the positive hole by limiting dilution method until obtaining hybridoma cell strain for stably secreting monoclonal antibody.
(3) Cell cryopreservation and resuscitation
Hybridoma cellsIs prepared from frozen stock solution (1×10) 6 Cell suspensions/ml were stored in liquid nitrogen for a long period of time. And (3) taking out the frozen storage tube during recovery, immediately putting into a 37 ℃ water bath for medium-speed thawing, centrifuging to remove frozen storage liquid, and transferring into a culture flask for culture.
(4) Preparation and purification of monoclonal antibodies
Incremental culture method: the hybridoma cells are placed in a cell culture medium, cultured at 37 ℃, and the obtained culture solution is purified by an octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, and the monoclonal antibodies are preserved at-20 ℃.
The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium, so that the final concentration of the calf serum in the cell culture medium is 20% (mass fraction), and the final concentration of the sodium bicarbonate in the cell culture medium is 0.2% (mass fraction); the pH of the cell culture medium was 7.4.
5. Preparation of goat anti-mouse antibody
Sheep is used as immune animals, and a murine antibody is used as immunogen to immunize pathogen-free sheep, so that the goat anti-mouse antibody is obtained.
6. Preparation of inter-alternaria alternata monoclonal antibody-colloidal gold marker
(1) Preparation of colloidal gold
Diluting 1% chloroauric acid into 0.01% (mass fraction) with double distilled deionized water, placing 100ml in a conical flask, heating to boil with a constant temperature electromagnetic stirrer, adding 2.5ml 1% trisodium citrate under continuous high temperature and continuous stirring, stopping stirring and heating until the solution is transparent red, cooling to room temperature, recovering original volume with deionized water, and preserving at 4deg.C. The prepared colloidal gold has pure appearance, is transparent, and has no sediment or floaters.
(2) Preparation of inter-alternaria alternata monoclonal antibody-colloidal gold marker
Under the magnetic stirring, the pH value of the colloidal gold is regulated to 7.0 by using 0.2mol/L potassium carbonate solution, 20-50 mug standard is added into each milliliter of the colloidal gold solution, the inter-alternaria alternate monoclonal antibody is added into the colloidal gold solution, the stirring and the mixing are continued for 30min, 10% BSA is added, the final concentration of the inter-alternaria alternate monoclonal antibody in the colloidal gold solution is 1% (volume fraction), and the mixture is kept stand for 10min. Centrifuging at 12000r/min and 4deg.C for 40min, discarding supernatant, washing the precipitate twice with redissolving buffer, re-suspending the precipitate with redissolving buffer with volume 1/10 of that of initial colloidal gold, and standing at 4deg.C for use.
Reconstitution buffer: 0.02 to 0.1 percent (mass fraction) of casein, 0.05 to 0.2 percent (mass fraction) of tween-80 and 0.02mol/L of phosphate buffer solution with pH of 7.2.
7. Preparation of conjugate release pads
The conjugate release pad was soaked in a phosphate buffer containing bovine serum albumin (the concentration of bovine serum albumin in the buffer is 0.5%), having a pH of 7.2 and 0.5mol/L, and was uniformly soaked for 1h and baked at 37℃for 3 h. Uniformly spraying the prepared inter-isolation streptavidin monoclonal antibody-colloidal gold marker on a conjugate release pad by using an Isoflow film spraying instrument, spraying 0.01ml of inter-isolation streptavidin monoclonal antibody-colloidal gold marker on each 1cm conjugate release pad, placing the conjugate release pad in a 37 ℃ environment (humidity is less than 20%) for 60min, taking out the conjugate release pad, and placing the conjugate release pad in a dry environment (humidity is less than 20%) for storage for standby.
8. Preparation of reaction film
Coating the interjacent streptavidin hapten-ovalbumin conjugate on a reaction membrane to form a detection line, and coating the goat anti-mouse anti-antibody on the reaction membrane to form a quality control line.
The coating process comprises the following steps: diluting the cross-linked streptavidin hapten-ovalbumin conjugate to 10mg/ml by using a phosphate buffer solution, and coating the cross-linked streptavidin hapten-ovalbumin conjugate on a detection line (T line) on a nitrocellulose membrane by using an isolow spot membrane tester, wherein the coating amount is 0.8 mu l/cm; the goat anti-mouse antibody was diluted to 200. Mu.g/ml with 0.01mol/L phosphate buffer at pH7.4, and coated on a quality control line (C line) on a nitrocellulose membrane in an isolow spot film apparatus in an amount of 1.0. Mu.l/cm. And (5) drying the coated reaction film for 2 hours at 37 ℃ for standby.
9. Preparation of sample absorbent pad
The sample absorbing pad is placed in phosphate buffer solution containing 0.5% bovine serum albumin (volume fraction), pH7.2 and 0.1mol/L for 2 hours, and is baked at 37 ℃ for 2 hours for standby.
10. Assembly of test strips
Sequentially adhering a sample absorption pad, a conjugate release pad, a reaction membrane and a water absorption pad on a PVC bottom plate; the 1/3 area of the initial end of the conjugate release pad is covered by the sample absorption pad, the tail end of the conjugate release pad is connected with the initial end of the reaction membrane, the tail end of the reaction membrane is connected with the initial end of the water absorption pad, the initial end of the sample absorption pad is aligned with the initial end of the PVC bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC bottom plate; the reaction film is provided with a detection line and a quality control line, and the detection line (T line) and the quality control line (C line) are strip-shaped strips which are perpendicular to the length of the test strip; the detection line is positioned at one side close to the tail end of the conjugate release pad; the quality control line is positioned at one side far away from the tail end of the conjugate release pad; cutting the test paper strip into small strips with the width of 3mm by a machine, and placing the test paper strip in a special plastic card, wherein the test paper strip can be stored for 12 months at the temperature of 4-30 ℃.
Example 2 detection of inter-digitalis residues in samples
1. Detection is carried out by using a test strip
Sucking the sample solution to be detected by a suction pipe, vertically dripping 3 drops of the sample solution into a sample adding hole, starting timing when the liquid flows, reacting for 5-10 min, and judging the result.
2. Analyzing the detection result
The results were read by a colloidal gold analyzer (abbreviated as "analyzer"):
negative (-). Indicating that the concentration of the substance to be detected in the sample is lower than the detection limit;
positive (+): indicating that the concentration of the substance to be detected in the sample is equal to or higher than the detection limit;
invalidation: indicating that retesting is required.
Example 3 sample detection example
1. Limit of detection test
Blank wheat and corn samples were taken, and streptavidin was added to the samples to a final concentration of 5, 10, 20 μg/kg, and the samples were tested using a test strip, and each sample was repeatedly assayed three times.
When the test strip is used for detecting wheat and corn samples, the analyzer shows negative when the adding concentration of the inter-alternaria alternata is 5 mug/kg; the analyzer showed positive when the concentration of the addition of the inter-digitalis was 10, 20. Mu.g/kg.
2. False positive rate and false negative rate test
Taking 20 parts of wheat and corn positive samples with the known inter-alternaria alternata content of 10 mug/kg and 2 parts of wheat and corn negative samples with the known inter-alternaria alternata content of less than 5 mug/kg, detecting by using three batches of test strips, and calculating the negative-positive rate of the test strips. The results are shown in Table 1 and Table 2.
Table 1 wheat test sample results
TABLE 2 corn test sample results
The results show that: when the test strips produced in 3 batches are used for detecting positive wheat and corn samples, the results are positive, the coincidence rate of the positive samples is 100%, and the false negative rate is 0; when 20 negative wheat and corn samples were tested, the results were all negative, and it was found that the negative coincidence rate was 100% and the false positive rate was 0. The test strip for detecting the alternaria alternate provided by the invention can be used for rapidly detecting the alternaria alternate residue in wheat and corn.
3. Specificity test
500 mug/kg of aflatoxin B1, T-2 toxin, ochratoxin and other medicaments are detected by using a test strip of the alternaria alternate. The result shows that the quality control line and the detection line of the test strip are both developed and negative. The test strip has no cross reaction to 500 mug/kg aflatoxin B1, T-2 toxin, ochratoxin and other medicines.
Claims (7)
1. The test strip for detecting the cross-linked streptavidin comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), a water absorption pad (4) and a bottom plate (7), and is characterized in that the reaction membrane is provided with a detection line (5) coated with a cross-linked streptavidin hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, the conjugate release pad (2) is sprayed with a cross-linked streptavidin monoclonal antibody-colloidal gold marker, and the cross-linked streptavidin hapten is prepared by the following steps:
dissolving 30mg of inter-isolation streptavidin in 50ml of tetrahydrofuran, adding 5ml of trifluoroacetic acid, fully stirring, adding 0.7g of glycolic acid, heating in an oil bath, refluxing for 12h, stopping the reaction, adding 100ml of water, adding 100ml of ethyl acetate, fully oscillating, standing for layering, removing water phase, washing organic phase with 60ml of water, oscillating for standing, separating water phase, evaporating organic phase, washing and pulping with 1/9 volume ratio of dichloromethane/n-hexane, standing, separating supernatant, drying obtained precipitate, and obtaining 26mg of glycolic acid inter-isolation streptavidin hapten with a yield of 70%;
the molecular structural formula of the inter-alternaria alternate hapten is as follows:
2. the test strip according to claim 1, wherein the sample absorbing pad (1), the conjugate releasing pad (2), the reaction membrane (3) and the water absorbing pad (4) are sequentially adhered to the bottom plate (7).
3. The test strip of any one of claims 1-2, wherein the conjugate release pad is 1/3 to 1/2 coated under the sample absorbent pad.
4. The test strip of claim 1, wherein the streptavidin-carrier protein conjugate is obtained by coupling a streptavidin hapten with a carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine, or human serum albumin.
5. The test strip of claim 1, wherein the monoclonal antibody of the alternaria alternata is prepared by taking a conjugate of a hapten and a carrier protein of the alternaria alternata as an immunogen, and the anti-mouse antibody of sheep is obtained by immunizing sheep with a mouse-derived antibody.
6. A method of preparing the test strip of any one of claims 1-5, comprising the steps of:
1) Preparing a conjugate release pad sprayed with a cross-linked streptavidin monoclonal antibody-colloidal gold marker;
2) Preparing a reaction film with a detection line coated with a cross-linked streptavidin hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps 1) and 2) into the test strip.
7. A method for detecting inter-digitalis residue in wheat or corn comprising the steps of:
1) Sample pretreatment;
2) Detecting with the test strip of any one of claims 1-5;
3) And analyzing the detection result.
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| CN113588955A (en) * | 2021-07-08 | 2021-11-02 | 上海市农产品质量安全中心 | Test paper for alternaria toxin |
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| JP2003038173A (en) * | 2001-05-18 | 2003-02-12 | Takeda Chem Ind Ltd | Method for selecting antibody, hybridoma, monoclonal antibody and use thereof |
| CN105712970A (en) * | 2016-01-27 | 2016-06-29 | 华南农业大学 | Hapten, artificial antigen and antibody directly targeted to alternariol and preparation method and application thereof |
| CN108912090A (en) * | 2018-02-12 | 2018-11-30 | 北京农业质量标准与检测技术研究中心 | A kind of test strips of quick detection alternariol and alternariol monomethyl ether total amount |
| CN108982690A (en) * | 2018-07-04 | 2018-12-11 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Fast method that is a kind of while detecting six kinds of mould toxin of rod method in pears |
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| US6528325B1 (en) * | 2000-10-13 | 2003-03-04 | Dexall Biomedical Labs, Inc. | Method for the visual detection of specific antibodies in human serum by the use of lateral flow assays |
| EP2708555A3 (en) * | 2012-09-17 | 2014-05-07 | VLN Biotech Inc. | Polyclonal antibodies to cloned fungal polypeptide targets |
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| JP2003038173A (en) * | 2001-05-18 | 2003-02-12 | Takeda Chem Ind Ltd | Method for selecting antibody, hybridoma, monoclonal antibody and use thereof |
| CN105712970A (en) * | 2016-01-27 | 2016-06-29 | 华南农业大学 | Hapten, artificial antigen and antibody directly targeted to alternariol and preparation method and application thereof |
| CN108912090A (en) * | 2018-02-12 | 2018-11-30 | 北京农业质量标准与检测技术研究中心 | A kind of test strips of quick detection alternariol and alternariol monomethyl ether total amount |
| CN108982690A (en) * | 2018-07-04 | 2018-12-11 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Fast method that is a kind of while detecting six kinds of mould toxin of rod method in pears |
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