CN112684180A - Fluorescence immunoassay quantitative POCT detection method for mycotoxin in agricultural products - Google Patents
Fluorescence immunoassay quantitative POCT detection method for mycotoxin in agricultural products Download PDFInfo
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- 238000012123 point-of-care testing Methods 0.000 title claims abstract description 53
- 238000003018 immunoassay Methods 0.000 title claims abstract description 48
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- 239000000047 product Substances 0.000 claims description 46
- 238000000034 method Methods 0.000 claims description 33
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- 238000000605 extraction Methods 0.000 claims description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 claims description 9
- 229930020125 aflatoxin-B1 Natural products 0.000 claims description 9
- 239000002115 aflatoxin B1 Substances 0.000 claims description 8
- UVBUBMSSQKOIBE-DSLOAKGESA-N fumonisin B1 Chemical compound OC(=O)C[C@@H](C(O)=O)CC(=O)O[C@H]([C@H](C)CCCC)[C@@H](OC(=O)C[C@@H](CC(O)=O)C(O)=O)C[C@@H](C)C[C@H](O)CCCC[C@@H](O)C[C@H](O)[C@H](C)N UVBUBMSSQKOIBE-DSLOAKGESA-N 0.000 claims description 8
- QZIADBYRQILELJ-UHFFFAOYSA-N fumonisin B1 Natural products CCCCC(C)C(OC(=O)CC(CC(=O)O)C(=O)O)C(C)(CC(C)CC(O)CCCCC(O)CC(O)C(C)N)OC(=O)CC(CC(=O)O)C(=O)O QZIADBYRQILELJ-UHFFFAOYSA-N 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 7
- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 claims description 7
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- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 claims description 6
- RWQKHEORZBHNRI-BMIGLBTASA-N ochratoxin A Chemical compound C([C@H](NC(=O)C1=CC(Cl)=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 RWQKHEORZBHNRI-BMIGLBTASA-N 0.000 claims description 6
- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 claims description 6
- VYLQGYLYRQKMFU-UHFFFAOYSA-N Ochratoxin A Natural products CC1Cc2c(Cl)cc(CNC(Cc3ccccc3)C(=O)O)cc2C(=O)O1 VYLQGYLYRQKMFU-UHFFFAOYSA-N 0.000 claims description 5
- DAEYIVCTQUFNTM-UHFFFAOYSA-N ochratoxin B Natural products OC1=C2C(=O)OC(C)CC2=CC=C1C(=O)NC(C(O)=O)CC1=CC=CC=C1 DAEYIVCTQUFNTM-UHFFFAOYSA-N 0.000 claims description 5
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- 241000233866 Fungi Species 0.000 description 1
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention belongs to the technical field of agricultural product detection, and particularly relates to a fluorescence immunoassay quantitative POCT detection method for mycotoxin in agricultural products. The invention provides a fluorescence immunoassay quantitative POCT detection method for mycotoxin in agricultural products, which comprises the following steps: a. taking a certain amount of sample, adding 10 times of the extracting solution, extracting for 5-10min, and preparing into a solution to be detected; b. detecting with a fluorescence immunoassay quantitative POCT analyzer, sucking 100 μ l of the solution to be detected obtained in step a, adding into the sample adding hole of the test strip, and covering with a cover for incubation for 8 min; after 8min, inserting the test strip into a test strip slot of a fluorescence immunoassay quantitative POCT analyzer, and reading; c. and (5) judging the detection result. The detection method has wide application range and high detection sensitivity, and is suitable for popularization and application.
Description
Technical Field
The invention belongs to the technical field of agricultural product detection, and particularly relates to a fluorescence immunoassay quantitative POCT detection method for mycotoxin in agricultural products.
Background
During the storage process of the agricultural products, the agricultural products are easily infected by various moulds, and some mycotoxins can remain in the agricultural products, so that the health of consumers can be seriously harmed when the agricultural products are sold on the market.
Mycotoxins are small molecular secondary metabolites produced by fungi, crops are easily polluted by the mycotoxins in the aspects of growth, harvesting, storage, transportation and the like, more than 400 mycotoxins are found to have carcinogenic, teratogenic and mutagenic effects, and the toxicity is obviously enhanced when mixed pollution is carried out. Mycotoxins can enter the food chain through contaminating cereals and feed or through animal-derived foods, causing serious harm to human and animal health, and have become the focus of research in the field of food safety in recent years. The main mycotoxins in agricultural products are aflatoxin B1(Alfatoxin B1, AFB1), Ochratoxin a (Ochratoxin a, OTA), Fumonisin B1(Fumonisin B1, FB1) and zearalenone (Zeralenone, ZEN).
Currently, methods for detecting mycotoxins mainly include biological assays, chemical assays, instrumental assays, immunoassays, and the like. The biological identification method has low specificity; the accuracy of the chemical analysis method is low; the immunoassay method is rapid, high in sensitivity, good in specificity and low in cost, but the detection sensitivity of the immunoassay method is still to be enhanced. The instrument has high analysis sensitivity and can accurately quantify, but the sample pretreatment is complex and the detection instrument is expensive. The methods for detecting the mycotoxin in the agricultural products have the defects of limited use range, incapability of being used on the agricultural products, high detection cost and complex detection flow. Therefore, it is urgently needed to develop some detection methods for mycotoxin in agricultural products, which have short detection flow, simple operation and low detection cost.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the existing method for detecting the mycotoxin in the agricultural product has the problems of complex detection process, high detection cost and difficult popularization.
The technical scheme for solving the technical problems comprises the following steps: a fluorescence immunoassay quantitative POCT detection method for mycotoxin in agricultural products is provided. The method comprises the following steps:
a. sample pretreatment
Taking an agricultural product sample, crushing, sieving with a 100-mesh sieve, taking a certain amount of sample, adding 10-20 times of extracting solution, and performing vortex oscillation extraction or shaking table extraction for 10 min; after extraction is finished, centrifuging at 5000rpm for 1min, taking 100 mu l of supernatant, adding the supernatant into 500 mu l of sample diluent, and uniformly mixing to prepare a solution to be detected;
b. sample on-machine detection
Taking out the fluorescence immunoassay POCT detection test strip, detecting by adopting a fluorescence immunoassay POCT analyzer, starting detection after the temperature of the constant-temperature incubator is raised to 37 ℃ and stabilized, taking out the fluorescence immunoassay POCT detection test strip, horizontally placing the fluorescence immunoassay POCT detection test strip on a heating plate of the constant-temperature incubator, sucking 100 mu l of the solution to be detected obtained in the step a, adding the solution to be detected into a sample adding hole of the test strip, and covering a cover to incubate for 8 min; after 8min, inserting the test strip into a test strip slot of a fluorescence immunoassay quantitative POCT analyzer, and reading;
c. determination of detection result
If the detection result is less than the cutoff value, the judgment result is negative, and the mycotoxin amount is qualified; if the detection result is not less than the cutoff value, the judgment result is positive, and the mycotoxin amount is not qualified.
In the method for fluorescence immunoassay quantitative POCT detection of mycotoxin in agricultural products, the agricultural products in the step a comprise at least one of corn, wheat, barley, grains or sorghum.
In the method for detecting the fluorescence immunoassay quantitative POCT of the mycotoxin in the agricultural product, the sample in the step a is 5ml, and the extracting solution is 70 ml.
In the method for fluorescence immunoassay quantitative POCT detection of mycotoxin in agricultural products, the extracting solution in the step a is at least one of methanol, ethanol, propylene glycol or isobutanol.
In the fluorescence immunoassay quantitative POCT detection method for the mycotoxins in the agricultural products, the mycotoxins detected in the step B comprise at least one of aflatoxin B1, ochratoxin A, fumonisin B1 or zearalenone.
Wherein, in the fluorescence immunoassay quantitative POCT detection method for the mycotoxin in the agricultural products, the environmental conditions detected in the step b are that the room temperature is 25 +/-3 ℃ and the humidity is 40-60%.
In the method for detecting the fluorescence immunoassay quantitative POCT of the mycotoxin in the agricultural product, the reading of the step c needs to be carried out within 2 min.
The invention has the beneficial effects that:
the invention provides a method for detecting mycotoxin in agricultural products by adopting a fluorescence immunoassay POCT (point of care testing) instrument, which widens the application range of the fluorescence immunoassay POCT instrument, can be applied to the detection of mycotoxin in agricultural products, and has the advantages of accurate detection result, detection sensitivity improved by 2-3 orders of magnitude and accurate quantification by adopting the method for detecting mycotoxin. And the detection flow is very short, the detection of the sample can be completed only by pretreatment for 10min and detection for 8min, and the detection efficiency is greatly improved. In addition, the invention also improves the linear range of the detection result and reduces the detection limit of aflatoxin B1, ochratoxin A, fumonisin B1 and zearalenone, wherein the linear range of aflatoxin B1 can reach 1-300ug/kg, the linear range of ochratoxin A can reach 10-5000ug/kg, the linear range of fumonisin B1 can reach 100 plus 65000ug/kg, and the linear range of zearalenone can reach 20-2000ug/kg, thus having good practical value.
Detailed Description
The invention provides a fluorescence immunoassay quantitative POCT detection method for mycotoxin in agricultural products, which comprises the following steps:
a. sample pretreatment
Taking an agricultural product sample, crushing, sieving with a 100-mesh sieve, taking a certain amount of sample, adding 10-20 times of extracting solution, and performing vortex oscillation extraction or shaking table extraction for 10 min; after extraction is finished, centrifuging at 5000rpm for 1min, taking 100 mu l of supernatant, adding the supernatant into 500 mu l of sample diluent, and uniformly mixing to prepare a solution to be detected;
b. sample on-machine detection
Taking out the fluorescence immunoassay POCT detection test strip, detecting by adopting a fluorescence immunoassay POCT analyzer, starting detection after the temperature of the constant-temperature incubator is raised to 37 ℃ and stabilized, taking out the fluorescence immunoassay POCT detection test strip, horizontally placing the fluorescence immunoassay POCT detection test strip on a heating plate of the constant-temperature incubator, sucking 100 mu l of the solution to be detected obtained in the step a, adding the solution to be detected into a sample adding hole of the test strip, and covering a cover to incubate for 8 min; after 8min, inserting the test strip into a test strip slot of a fluorescence immunoassay quantitative POCT analyzer, and reading;
c. determination of detection result
If the detection result is less than the cutoff value, the judgment result is negative, and the mycotoxin amount is qualified; if the detection result is not less than the cutoff value, the judgment result is positive, and the mycotoxin amount is not qualified.
The agricultural product sample suitable for detection by the method mainly comprises at least one of corn, wheat, barley, grain or sorghum, but the application range of the detection method is not limited to the method, other agricultural products such as potato, sweet potato and the like can be detected, and only when the samples with relatively high moisture content are detected, the proportion of the added extracting solution needs to be adjusted correspondingly.
Preferably, in the method for fluorescence immunoassay quantitative POCT detection of mycotoxin in agricultural products, the sample in the step a is 5ml, and the extracting solution is 70 ml. The inventors have found, after extensive experiments, that the best results are obtained when extracting a 5ml sample with 70ml of extract, probably because the mycotoxins in the sample need to be accumulated to a certain amount to facilitate detection.
In the method for fluorescence immunoassay quantitative POCT detection of mycotoxin in agricultural products, the extracting solution in the step a is at least one of methanol, ethanol, propylene glycol or isobutanol. Ethanol or propylene glycol is preferably used. More preferably, the extract is mixed with ethanol and propylene glycol in a volume ratio of 5: 8. When the mixed solution of ethanol and propylene glycol is adopted for extraction, the effective components and the mycotoxin of agricultural products can be extracted into the extracting solution to the maximum extent, so that the detection result is more accurate.
In the fluorescence immunoassay quantitative POCT detection method for the mycotoxins in the agricultural products, the mycotoxins detected in the step B comprise at least one of aflatoxin B1, ochratoxin A, fumonisin B1 or zearalenone.
Wherein, in the fluorescence immunoassay quantitative POCT detection method for the mycotoxin in the agricultural products, the environmental conditions detected in the step b are that the room temperature is 25 +/-3 ℃ and the humidity is 40-60%. The inventor finds out through a large number of tests that the environmental temperature and humidity during detection also have certain influence on the detection result, and the detection result is more sensitive at the room temperature of 25 +/-3 ℃ and the humidity of 40-60%.
In the method for detecting the fluorescence immunoassay quantitative POCT of the mycotoxin in the agricultural product, the reading in the step c needs to be carried out within 2min, and the result is inaccurate due to overlong time.
In the determination of the detection result, the cutoff value may be set as required, and is generally set as a standard limit value of the mycotoxin. When the value is lower than the above range, the mycotoxin in the sample is qualified, and when the value is higher than or equal to the above range, the mycotoxin is out of limits. The invention can quickly judge the result, and the judging method is simple and effective.
The invention adopts a fluorescence immunoassay quantitative POCT detection method to detect the mycotoxin in agricultural products, and belongs to the first case. Because agricultural products generally contain high starch, detection results can be interfered when mycotoxin is detected, and the detection results are inaccurate. When the liquid chromatography is adopted to detect the mycotoxin in the agricultural products, the pretreatment process of the sample is very complicated, otherwise, not only can an accurate result be obtained, but also the use of the instrument is influenced, and the service life of the instrument is shortened. The invention adopts the fluorescence immunoassay POCT instrument, the pretreatment mode of the sample is simple, the sample can be detected on the computer only by about 10min of extraction, the detection efficiency is greatly improved, and the invention obtains good detection effect and is suitable for rapidly detecting a plurality of samples.
The following examples are intended to illustrate specific embodiments of the present invention without limiting the scope of the invention to the examples.
The fluoroimmunoassay POCT apparatus and reagents used in the examples were all common commercial products.
Example the method of the present invention was used to detect aflatoxin B1 in wheat
The specific operation steps are as follows:
a. sample pretreatment
Taking a wheat sample, crushing, sieving with a 100-mesh sieve, taking a certain amount of the sample, adding 10 times of ethanol, carrying out vortex oscillation extraction for 5min, after the extraction is finished, centrifuging at 5000rpm for 1min, taking 100 mu l of supernatant, adding into 500 mu l of sample diluent, and uniformly mixing to prepare a solution to be detected;
b. sample on-machine detection
Taking out the fluorescence immunoassay POCT detection test strip, detecting by adopting a fluorescence immunoassay POCT analyzer, starting detection after the temperature of the constant-temperature incubator is raised to 37 ℃ and stabilized, taking out the fluorescence immunoassay POCT detection test strip, horizontally placing the fluorescence immunoassay POCT detection test strip on a heating plate of the constant-temperature incubator, sucking 100 mu l of the solution to be detected obtained in the step a, adding the solution to be detected into a sample adding hole of the test strip, and covering a cover to incubate for 8 min; after 8min, inserting the test strip into a test strip slot of a fluorescence immunoassay quantitative POCT analyzer, and reading;
c. determination of detection result
If the detection result is less than the cutoff value, the judgment result is negative, and the mycotoxin amount is qualified; if the detection result is not less than the cutoff value, the judgment result is positive, and the mycotoxin amount is not qualified.
Example a total of 5 wheat samples were tested, each repeated 3 times, and the results are shown in table 1 below.
TABLE 1 Aflatoxin B1 results in various corn samples
The embodiment shows that the detection method has good repeatability and high detection sensitivity, and is suitable for popularization and application.
Claims (7)
1. The fluorescence immunoassay quantitative POCT detection method for mycotoxin in agricultural products is characterized by comprising the following steps:
a. sample pretreatment
Taking an agricultural product sample, crushing, sieving with a 100-mesh sieve, taking a certain amount of sample, adding 10-20 times of extracting solution, and performing vortex oscillation extraction or shaking table extraction for 10 min; after extraction is finished, centrifuging at 5000rpm for 1min, taking 100 mu l of supernatant, adding the supernatant into 500 mu l of sample diluent, and uniformly mixing to prepare a solution to be detected;
b. sample on-machine detection
Taking out the fluorescence immunoassay POCT detection test strip, detecting by adopting a fluorescence immunoassay POCT analyzer, starting detection after the temperature of the constant-temperature incubator is raised to 37 ℃ and stabilized, taking out the fluorescence immunoassay POCT detection test strip, horizontally placing the fluorescence immunoassay POCT detection test strip on a heating plate of the constant-temperature incubator, sucking 100 mu l of the solution to be detected obtained in the step a, adding the solution to be detected into a sample adding hole of the test strip, and covering a cover to incubate for 8 min; after 8min, inserting the test strip into a test strip slot of a fluorescence immunoassay quantitative POCT analyzer, and reading;
c. determination of detection result
If the detection result is less than the cutoff value, the judgment result is negative, and the mycotoxin amount is qualified; if the detection result is not less than the cutoff value, the judgment result is positive, and the mycotoxin amount is not qualified.
2. The method for performing fluoroimmunoassay quantitative POCT detection of mycotoxins in agricultural products according to claim 1, wherein the method comprises the following steps: the agricultural product of step a comprises at least one of corn, wheat, barley, grain or sorghum.
3. The method for performing fluoroimmunoassay quantitative POCT detection of mycotoxins in agricultural products according to claim 1, wherein the method comprises the following steps: the sample in the step a is 5ml, and the extracting solution is 70 ml.
4. The method for performing fluoroimmunoassay quantitative POCT detection of mycotoxins in agricultural products according to claim 1, wherein the method comprises the following steps: the extracting solution in the step a is at least one of methanol, ethanol, propylene glycol or isobutanol.
5. The method for performing fluoroimmunoassay quantitative POCT detection of mycotoxins in agricultural products according to claim 1, wherein the method comprises the following steps: the mycotoxin detected in the step B comprises at least one of aflatoxin B1, ochratoxin A, fumonisin B1 or zearalenone.
6. The method for performing fluoroimmunoassay quantitative POCT detection of mycotoxins in agricultural products according to claim 1, wherein the method comprises the following steps: the environmental conditions detected in the step b are that the room temperature is 25 +/-3 ℃ and the humidity is 40-60%.
7. The method for performing fluoroimmunoassay quantitative POCT detection of mycotoxins in agricultural products according to claim 1, wherein the method comprises the following steps: step c readings need to be taken within 2 min.
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2021
- 2021-01-26 CN CN202110103395.8A patent/CN112684180A/en active Pending
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| CN106198966A (en) * | 2016-09-06 | 2016-12-07 | 中国农业大学 | A kind of near-infrared fluorescent immune chromatography reagent kit detecting aflatoxin and application thereof |
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