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Animal Health

 

Rapid testing of total aflatoxin in agricultural raw materials

Michael Zheng and Johann Binder

 

 

Introduction
 

Aflatoxins are secondary metabolites of, primarily, the fungi Aspergillus flavus and A. parasiticus. There are four principle types of aflatoxins: B1, B2, G1 and G2. Aflatoxins could cause liver disease in animals and may cause other diseases involving other organ systems. Aflatoxin B1 is a potent human carcinogen, and may contribute to human liver cancer. Aflatoxins could be found mainly in cereals, corn, peanuts, other nuts and dried distillers grain, a by-product in the ethanol industry.

 

Regulations of aflatoxins have been set in approximately 100 countries by the end of 2003, although the maximum tolerated levels differ greatly among countries. The US Food and Drug Administration's action levels of total aflatoxins are as follows:

 

Total Aflatoxin in Food and Feed, US-FDA Action Levels

ppb

Corn for immature animals and dairy cattle 20 Cottonseed meal for feed for beef, cattle, swine, poultry

300

Maize and peanut products for breeding beef, cattle, swine or adult poultry

100

Maize and peanut products for finishing swine of 100 pounds or greater

200

Maize and peanuts products for finishing beef

300

All foods 

20

 

The European Community set a maximum level for total aflatoxins in foods (Commission Regulation EC 466/2001 and EC 472/2002):

 

Total Aflatoxins Regulation in Food, EU Levels

ppb

Peanuts, nuts and dried fruits (direct consumption)

4

Peanuts for physical processing before consumption

15

Nuts and dried fruits for physical processing before consumption

10

Cereals (direct consumption or processing before consumption)

4

Spices 

10

 

Hence, analysing aflatoxins in agricultural raw materials and products is a must in order to protect the health of human beings and animals.

 

 

Aflatoxin Testing Methods
 

Commonly used analytical methods for the determination of aflatoxins include:

    • Thin-layer chromatography (TLC)

    • High-performance liquid chromatography (HPLC)

    • Gas chromatography (GC)

    • Immunochemical methods (ELISA) 

Although these methods could give quantitative results, there are some disadvantages:

    • high investment costs for equipments/instruments

    • skilled chemists/analysts required

    • laboratory environment is needed

    • long testing time 

The real needs of the industry are rapid on-site test methods to obtain results within minutes without requiring professional training and high investment. Such rapid tests fit perfectly for analysis of aflatoxins in grains, peanuts, dried distillers grain and feed raw materials. These industries not only need to meet regulatory guideline levels, but also need fast decisions based on screening results because time is so important.

 

 

AgraStripTM Total Aflatoxin - one of the fastest methods for total aflatoxins testing in the world

 

The AgraStripTM test has been developed based on the above-mentioned market needs. The method is a one-step lateral flow immunochromatographic assay based on an inhibition immunoassay format.

 

 

Principle:

 

Antibody-particle complex is dissolved in assay diluent and mixed with sample extract. The mixed content is then wicked onto a membrane, which contains a test zone and a control zone. The test zone captures free antibody-particle complexes, allowing colour particles to concentrate and form a visible line. A positive sample containing aflatoxins above the cutoff level would result in no visual line in the test zone (T). Alternatively, a negative sample containing aflatoxin below the cutoff level would form a visible line in the test zone. A line would always be visible in the control zone (C) regardless of the presence of aflatoxin.

 

Assay Procedures:

 

 

(Negative results appear in 1 min for 20ppb cutoff strip)

 

 

Results Interpretation:

 

     Negative: Aflatoxin less than cutoff level  

     (two color lines); results can be obtained 

     in 1 minute for 20ppb cutoff test kit.         

  

 

     Positive: Aflatoxin greater than or equal

     to cutoff level (one control line); results

     in 5 minutes.

    

 

     Invalid results: no control line.

 

 

The AgraStripTM Total Aflatoxin is a fast, easy to use qualitative test. The test kits are available with cutoff levels of 4ppb, 10ppb or 20ppb respectively. It is the only lateral flow test in the market meeting the 4 ppb regulatory level of the EU. Each kit comes complete with 24 strips, 24 dilution wells and a bottle of assay diluent.

 

 

Product features:

    • very fast: obtains results in 5 minutes (1 minute for negative results for 20ppb cutoff kit)

    • easy to use

    • low cost

    • on-site test

    • no equipment needed for the assay

    • long shelf life over a wide range of climates

    • semi-quantitative

 

Your benefits:

    • the only strip test in the market detecting total aflatoxins

    • three different levels available to meet different regulation requirements

    • distinct and clear lines help in result interpretation

    • immediate results in 5 minutes

    • appealing kit appearance & convenient packing 

 

For Aflatoxin screening on:

    • corn

    • peanuts

    • rice

    • dried distillers grain (ethanol industry)

    • soybean

    • feed raw materials

 

Method Validation

 

The AgraStripTM total aflatoxin test kits have been validated in several spiked and naturally contaminated samples. As examples, validation results for the AgraStripTM 4ppb test kits in spiked and naturally contaminated corn are shown below in Tables 1-2, and validation results for AgraStripTM 10ppb and 20ppb test kits for naturally contaminated peanuts and corn are shown  below in Tables 3 and 4 respectively. Validation results collectively indicate that AgraStripTM test kits could effectively measure total aflatoxins in several commodities. Validated commodities include corn, peanuts, rice, DDGs and soybeans.

 

Table 1. Validation Results of AgraStripTM 4ppb Cutoff Test Kit for Spiked Corn Spiked Total Spiked Total Aflatoxin (ppb)

Spiked Total Aflatoxin (ppb)

Analyst 1

Analyst 2

Analyst 3

Scores* 

Results 

Comments

0

2 Lines

2 Lines

2 Lines

100%

Negative 

Valid

2

2 Lines

2 Lines

2 Lines

100%

Negative 

Valid

6

1 Lines

1 Lines

1 Lines

100%

Positive

Valid

10

1 Lines

1 Lines

1 Lines

100%

Positive

Valid

* Scores are based on all testing results which take into account triplicate samples and three analysts.
 

Table 2. Validation Results of AgraStripTM 4ppb Cutoff Test Kit for Naturally Contaminated Corn Naturally Contaminated Corn (ppb)

Naturally Contaminated Corn (ppb)

Analyst 1

Analyst 2

Analyst 3

Scores* 

Results 

Comments

< 2

2 Lines

2 Lines

2 Lines

100%

Negative 

Valid

6.1

1 Lines

1 Lines

1 Lines

100%

Positive

Valid

 

Table 3. Validation Results of AgraStripTM 20ppb Cutoff Test Kit for Naturally Contaminated Corn Naturally Contaminated Corn (ppb)

Naturally Contaminated Corn (ppb)

Analyst 1

Analyst 2

Analyst 3

Scores* 

Results 

Comments

< 2

2 Lines

2 Lines

2 Lines

100%

Negative 

Valid

24.6

1 Lines

1 Lines

1 Lines

100%

Positive

Valid

 

Table 4. Validation Results of AgraStripTM 10ppb Cutoff Test Kit for Naturally Contaminated Peanuts Naturally Contaminated Corn (ppb)

Naturally Contaminated Corn (ppb)

Analyst 1

Analyst 2

Analyst 3

Scores* 

Results 

Comments

< 2

2 Lines

2 Lines

2 Lines

100%

Negative 

Valid

10

1 Lines

1 Lines

1 Lines

100%

Positive

Valid


 

Conclusions

 

AgraStripTM Total Aflatoxin Test is a validated rapid screening method for total aflatoxins in several agricultural products. The test is designed for the on-site testing of aflatoxins in  commodities such as corn, peanut, rice, DDGs and soybeans. Only minimum training and no equipments/instruments are required for this test. The AgraStripTM is particularly useful for those industries in which the assay speed is important.

 

 

References

 

(1)  Richard, J.L. Mycotoxins, Toxicity and Metabolism in Animals - A Systems Approach Overview IN: Mycotoxins

      and Phycotoxins - Developments in Chemistry, Toxicology and Food Safety, Miraglia, M., van Egmond, H.P.,

      Brera, C., Gilbert, J. (eds.), Alaken, Inc., FortCollins, CO, 1998, pp. 363-397.

 

(2)  Council for Agricultural Science and Technology (CAST). Mycotoxins: Risks in Plant, Animal and Human

      Systems. Task Force Report, no. 139, CAST, Ames, Iowa, 2003.

 

(3)  van Egmond, H.P. and Jonker, M.A. Worldwide regulations for mycotoxins in food and feed in 2003.

 

(4)  Zheng, Z.M. Validation Report of AgraStripTM Total Aflatoxin Test, Romer Labs study number SIN-2005-01,

      2005.

 

 

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