Detection of silkworm (Bombyx mori) and Lepidoptera DNA in feeding stuff by real-time PCR

Abstract

Two real-time PCR methods were developed for the specific detection of silkworm (Bombyx mori) as an illegal ingredient in feed. The methods, a single- and a multi-copy approach, amplify small sequences of 91 and 77 basepairs (bp) size, enabling silkworm detection in severely processed products. High specificity was demonstrated for the single-copy method, with late signals (>Ct = 36) obtained only for some crustaceans. The probability of detection of the single-copy method, relying on a unique silkworm storage protein gene, was calculated to be 11 copies, respectively 10 pg native B. mori DNA. The multi-copy method, based on the classical cytochrome oxidase (COI) barcode region, revealed sensitivities as low as 0.1 pg B. mori DNA. In addition, a previously published sequencing primer pair for the order Lepidoptera (butterflies and moths) proved to be suited for rapid screening purposes. Although the multi-copy and Lepidoptera methods are less specific, unwanted cross-reactions can be screened out with an appropriate cut-off threshold. In conclusion, appropriate real-time methods are now available for the surveillance of an important by-product from the silk industry which is playing a significant role in the world markets.