title: Low cost quantitative digital imaging as an alternative to qualitative in vivo bioassays for analysis of active aflatoxin B1.
abstract: Aflatoxin B1 (AFB1) producing fungi contaminate food and feed and are a major health concern. To minimize the sources and incidence of AFB1 illness there is a need to develop affordable, sensitive mobile devices for detection of active AFB1. In the present study we used a low cost fluorescence detector and describe two quantitative assays for detection of detoxified and active AFB1 demonstrating that AFB1 concentration can be measured as intensity of fluorescence. When the assay plate containing increasing concentrations of AFB1 is illuminated with a 366 nm ultraviolet lamp, AFB1 molecules absorb photons and emit blue light with peak wavelength of 432 nm. The fluorescence intensity increased in dose dependent manner. However, this method cannot distinguish between active AFB1 which poses a threat to health, and the detoxified AFB1 which exhibits no toxicity. To measure the toxin activity, we used a cell based assay that makes quantification more robust and is capable of detecting multiple samples simultaneously. It is an alternative to the qualitative duckling bioassay which is the "gold-standard" assay currently being used for quantitative analysis of active AFB1. AFB1 was incubated with transduced Vero cells expressing the green fluorescence protein (GFP) gene. After excitation with blue light at 475 nm, cells emitted green light with emission peak at 509 nm. The result shows that AFB1 inhibits protein expression in a concentration dependent manner resulting in proportionately less GFP fluorescence in cells exposed to AFB1. The result also indicates strong positive linear relationship with R(2)=0.90 between the low cost CCD camera and a fluorometer, which costs 100 times more than a CCD camera. This new analytical method for measuring active AFB1 is low in cost and combined with in vitro assay, is quantitative. It also does not require the use of animals and may be useful especially for laboratories in regions with limited resources.
authors.affiliation: Western Regional Research Center, Foodborne Contaminants Research Unit, Agricultural Research Service, USDA, Albany, CA, United States. Electronic address: reuven.rasooly@ars.usda.gov.
authors.forename: Reuven
authors.initials: R
authors.lastname: Rasooly
authors.affiliation: Western Regional Research Center, Foodborne Contaminants Research Unit, Agricultural Research Service, USDA, Albany, CA, United States.
authors.forename: Paula M
authors.initials: PM
authors.lastname: Do
authors.affiliation: Western Regional Research Center, Foodborne Contaminants Research Unit, Agricultural Research Service, USDA, Albany, CA, United States.
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Hernlem
mesh_terms: D016604:Aflatoxin B1 / Q000032:analysis*; D000818:Animals; D015374:Biosensing Techniques / Q000191:economics / Q000295:instrumentation*; D002522:Chlorocebus aethiops; D005470:Fluorometry / Q000191:economics / Q000295:instrumentation; D005516:Food Microbiology / Q000191:economics / Q000295:instrumentation*; D049452:Green Fluorescent Proteins / Q000032:analysis; D057809:HEK293 Cells; D006801:Humans; D061848:Optical Imaging / Q000191:economics / Q000295:instrumentation; D014709:Vero Cells
chemical_list: D049452:Green Fluorescent Proteins; D016604:Aflatoxin B1
keywords: Aflatoxin; CCD camera; Fluorescence detector