Development of a Multiplex Polymerase Chain Reaction (PCR) Assay for the Potential Detection of Insect Contaminants...

Molecular methods can potentially be used to detect insect contaminants of food products. In this study, we used three sets of group-specific primers, two of them targeting the amplification of two regions of the insect's mitochondrial cytochrome c oxidase subunit I (COI-Fa and COI-Fb) and the other targeting a region of the nuclear protein-coding wingless (wg) gene. Using singleplex and multiplex polymerase chain reaction (PCR), we evaluated the three sets of primers using genomic DNA (gDNA) from 48 insect species including food storage insect pests and known vectors of foodborne pathogens. Seven plant-based food matrices were also evaluated for exclusivity testing. Additionally, we spiked fragments from five insect species in a selected food matrix (whole wheat flour). Singleplex and multiplex PCR amplified single specific bands (401-449 bp), corresponding to the wg gene, from insect species belonging to families Blattidae and Formicidae, and in Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). The COI-Fa primers amplified specific bands (171-188 bp) in all Dipteran species and the COI-Fb primers amplified a specific band (∼140 bp) in DNA from Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and P. interpunctella. However, the presence of specific bands in most Coleopterans was not consistent. No amplicon bands were observed in any of the food matrixes tested and the expected pattern of amplicon bands was seen in multiplex reactions using gDNA from spiked food samples. Our multiplex PCR assay targeted specific groups of insects that commonly contaminate foods without amplifying bands from the food matrixes tested; thus, molecular methods may be suitable for detecting insects or their fragments in foods.