A Molecular Approach for the Detection and Quantification of Tribolium castaneum (Herbst) Infestation in Stored Wheat Flour

Abstract

Research background. The presence of insect fragments is one of the major constrains in stored food commodities and cause considerable loss in the quality of the produce. The management of the pest is viewed as a huge challenge in food processing industry. Conventionally, the detection of T. castenaum in the food processing industry is carried out by acid hydrolysis and staining methods which are not precise and time consuming. Experimental approach. Considering the importance of a quick and effective method, a qPCR-based approach was developed and elucidated in this study. The mitochondrial cytochrome oxidase I (mtCOI) gene was identified as a target due to the abundance in the pest. Specific primers were designed against the target gene by primer premier software and amplified in a qPCR. Results and conclusions. This developed method is capable of detecting all the ontogenic stages of T. castaneum in stored wheat flour. Earlier experiments had demonstrated that about 20 µg of DNA can be obtained from 2.2 mg of insects, to quantify the infestation levels the Ct values obtained from known samples were subjected to regression analysis and expressed as adult equivalents. In the unknown samples tested, the infestation was calculated as 1.74 and 0.046 adult insects in 5 g of wheat flour. The maximum permissible limit of insect fragments in flour is 75 insect fragments or approximately 3 adults per 50 g of flour as per FDA. Hence, by adopting this new method, it is possible for the warehouse operators to arrive at a decision to proceed with efficient management practices were wheat flour is stored. Also, this method can be ratified by government agencies associated with international business to ascertain whether the wheat flour meets the standards set by the respective country before subjecting to foreign trade. Novelty and scientific contribution. This study is the first of its kind in the detection and quantification for T. castaneum in milled products. So far only conventional methods are employed to assess the presence of the pest and manual counting of fragments are practiced to quantify the infestation levels. The developed qPCR method is faster, reliable and can be employed in milling industries, Bakery industries, food processing plants and foreign trade units for critical detection and quantification T. castaneum pest infestation.