Development of a PCR-based method to discriminate between live and dead insect pests present in stored paddy rice

Abstract

Molecular diagnostics offer a significant advancement over conventional methods for detecting insects in stored grain. However, current approaches cannot discriminate between living and dead individuals, which is key for grain management. This study developed a PCR-based method to differentiate between live and dead individuals of insect pests using two pairs of primers: a short fragment (153 bp) expected to amplify DNA from both live and dead insects, and a long fragment (2034 bp) expected to amplify only in live insects. The method was first tested on major pests of stored rice following exposure to high temperatures. After, the methodology was applied to artificially infested paddy rice to evaluate a potential matrix interference. Three species—Plodia interpunctella, Sitophilus zeamais, and Tribolium castaneum—were subjected to high temperature, elevated CO2 concentration, and deltamethrin—under varying exposure and post-exposure durations. PCR amplification of dead insects with the long-fragment primers declined with increasing exposure and post-treatment time when tested with the matrix of paddy rice. High temperatures caused faster DNA degradation than CO2 or deltamethrin. After 4 h of heat, S. zeamais and T. castaneum were undetectable, whereas P. interpunctella persisted up to 16 h. Under CO2 or deltamethrin treatment, insects remained detectable up to 7 days post-exposure. The short-fragment primer pair consistently amplified DNA from all individuals tested. This PCR-based method shows promise for distinguishing between live and dead insects present in stored rice. However, further refinement is needed to shorten post-mortem interval detections, to be use with other stored pest species, and to address other grains to provide the food industry with a more accurate tool to assess insect infestations.