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Development of Marker-Based Diagnosis for Estimating the Inflow Time of Beer-Contaminating Insects

Insects or insect remnants found in beer are a major consumer complaint. An estimation of the inflow time of contaminant insects is an important factor to manage these claims. To estimate the inflow time of contaminant insects, an efficient diagnosis based on the degradation rate of the 18S ribosomal RNA gene was developed. Genomic DNA (gDNA) was extracted from six species of insects that were soaked in 500 mL of beer in a glass bottle for several days (1, 3, 7, 10, 14, 21, and 30 days) at 25°C. Using these gDNAs as a template, quantitative real-time polymerase chain reaction was conducted and the cycle threshold (Ct) values according to soaking times were obtained. We analyzed correlations between Ct values and soaking times and demonstrated that five species showed high correlations (R2 = 0.800–0.977). In the house fly (Musca domestica), honey bee (Apis mellifera), and fruit fly (Drosophila melanogaster) samples stored in beer up to 30 days, the inflow time positively correlated with the Ct values, enabling statistically significant diagnosis of inflow time of these insects. Other insects, including the common house mosquito, German cockroach, and Indian meal moth, displayed poor correlations, which was attributed to the inconsistent gDNA extraction, likely due to small sample size or disintegration of body parts during storage in beer. With proper improvement in DNA extraction, our results suggest that this 18S rRNA-based diagnostic method would be applicable for estimating the inflow time of beer-contaminating insects.