Sensitivity analysis of DNA fingerprinting technique for detecting insect fragments in wheat flour

Balasubramanian, A., Jayas, D.S., Fernando, W.G.D., Li, G. and White, N.D.G. 2007. Sensitivity analysis of DNA fingerprinting technique for detecting insect fragments in wheat flour. Canadian Biosystems Engineering/Le génie des biosystèmes au Canada 49: 4.14.5. Tribolium castaneum (Herbst) and T. confusum (Jaquelin du Val) are the most commonly occurring insect species in flour mills and in damaged and milled grains. Deoxyribonucleic acid (DNA) fingerprinting, a molecular tool which helps to identify the unique DNA pattern of an organism was used to detect the presence of insect fragments of T. castaneum and T. confusum at different concentrations in commercial and lab-milled wheat flour. Insect nuclear primers were screened to identify the insect specific primer for T. castaneum and T. confusum. Two sets of primers were found to be specific for each of the Tribolium sp. The primer pair responsible for amplification of elongation factor 1-alpha gene sequence (520 bp) was found to be specific to Tribolium castaneum and the primer pair responsible for amplification of beta-tubulin intron gene sequence (500 bp) was found to be specific to T. confusum. The primers were verified for their specificity against other commonly occurring stored product pests such as Cryptolestes ferrugineus (Stephens), C. pusillus (Schonherr), Rhyzopertha dominica (Fabricius), and Sitophilus oryzae (Linnaeus) and was confirmed to be specific only for the two insect species mentioned above. The crushed insect parts of red flour beetles and confused flour beetles were added at 1.0, 0.5, 0.25, 0.1, and 0.05% by mass to 200 mg of cleaned commercial and lab-milled flour. The DNA fingerprinting technique was able to identify T. castaneum and T. confusum contamination only at the 1.0% contamination level in both commercial and lab-milled flour. The technique was simple and a large number of samples could be evaluated at once. It helps in the identification of the insect species contaminating the flour.