Insect Contaminants in Foods: Detection Limits of a Qualitative PCR-based Method

Introduction: Macroscopic and microscopic methods are currently used in assessing insect contaminants in foods. Sample analyses with these methods are time consuming and require analysts with high degrees of additional training and skills. Molecular based methods for screening of microbial and chemical food contaminants are being rapidly adopted and may be useful in the detection of insect contaminants in foods.

Purpose: This study determined the detection limits of insect fragments in whole wheat flour using singleplex and multiplex qualitative endpoint PCR.

Methods: Fragments of three known vectors of foodborne pathogens (the housefly, Musca domestica; the American cockroach, Periplaneta americana; and the pharaoh ant, Monomorium pharaonis) and two food storage insect pests (the Indian meal moth, Plodia interpunctella, and the red flour beetle, Tribolium castaneum) were added to whole wheat flour at spiking levels of 1, 0.1, 0.01, and 0.001%. Flour without added insect fragments was used as the control. Using custom primers and endpoint PCR, the extracted gDNA was used as template to amplify fragments of the protein-encoding wingless (wg) gene and the cytochrome oxidase I (COI) gene. The visualization of amplicons of expected sizes was considered positive for the presence of insects.

Results: DNA was successfully isolated from all samples. The COI primers amplified a ~150 to180 bp fragment from M. domestica, P. interpunctella and T. castaneum, whereas the wg primers amplified a ~410 to 430 bp fragment from P. americana and M. pharaonis. The intensity of an amplicon band was positively correlated to the spiking level. Insect fragments were detected at spiked levels as low as 0.001% (10 ppm).

Significance: Additional multiplex results will be discussed, and together, these results suggest that molecular methods can potentially be used for the rapid detection of insect contaminants in foods.