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

Aditi Negi1,2orcid tiny, Arunkumar Anandharaj3*orcid tiny, Sureshkumar Kalakandan3orcid tiny and Meenatchi Rajaman1*orcid tiny

1Department of Primary Processing, Storage and Handling, Indian Institute of Food Processing Technology, Thanjavur-613005, Tamil Nadu, India

2Bharathidasan University, Tiruchirappalli - 620024, Tamil Nadu, India

3Department of Food Safety and Quality Testing, Indian Institute of Food Processing Technology, Thanjavur-613005, Tamil Nadu, India

Article history:

Received: 20 July 2020

Accepted: 4 March 2021

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Key words:

insect detection, wheat flour, T. castaneum, qRT-PCR, DNA, red flour beetle


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 Tribolium castaneum in the food processing industry is carried out by acid hydrolysis and staining methods that are time consuming and lack precision.

Experimental approach. Considering the importance of a quick and effective method, a quantitative real-time polymerase chain reaction (qRT-PCR)-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 for amplification in qPCR.

Results and conclusions. This 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 cycle threshold (Ct) values obtained from known samples were subjected to regression analysis and expressed as adult equivalents. In the unknown samples, 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 approx. 3 adults per 50 g of flour as per the US Food and Drug Administration (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 have been employed to assess the presence of the pest and manual counting of fragments is practiced to quantify the infestation levels. The developed qRT-PCR method is faster, more 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.

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