Abstract
The production of insects on an industrial scale has attracted the attention of the research and agricultural industry as novel protein sources. To detect the presence of Gryllodes sigillatus (GS) in feed and food, a real-time PCR method based on the mitochondrial cytochrome b (CYB) gene is proposed by this study. Forty DNA samples of animal and plant origin were used to confirm the specificity of the qPCR system. The detection method’s performance was evaluated on different processed GS matrices including native GS (UnGS) and different commercial products: crunchy roasted samples (RoGS), insect meal mixtures (ACGS) and energetic snacks containing GS (GSS). Data on sequencing were aligned with the reference gene to confirm the PCR products. The regression curve (y = −3.394 x + 42.521; R2 = 0.994, d.f. 14) between Ct values and Log DNA concentrations of Gryllodes sigillatus resulted in an efficiency of 96.4%. The severity of the technological processing treatments and the matrix structure affected the intensity of the PCR signal with the same amount of insect DNA as observed by different y-intercepts of the three-regression lines for RoGS, ACGS, and GSS. The real-time PCR method resulted in robust and sensitive outcomes able to detect low amounts of GS DNA (5 g/100 g) in a complex matrix, making it suitable for detecting the presence or absence of labeled Gryllodes sigillatus material both in feed and food.
Highlights
Primers exhibited high sensitivity and accuracy. PCR discrimination among insect species. The qPCR system was successfully tested in complex matrices
Untreated insect samples of Gryllodes sigillatus (UnGS), Hermetia illucens, Acheta domesticus (AC), Gryllus assimilis, Musca domestica, Alphitobius diaperinus, Tenebrio molitor were purchased alive from Agripetgarden S.r.l. (PD)—Italy
The GS PCR system was developed based on the mitochondrial cytochrome b (CYB)
Summary
Primers exhibited high sensitivity and accuracy. PCR discrimination among insect species. The qPCR system was successfully tested in complex matrices. The production and marketing of insects as food in Europe is regulated by the so-called ‘Novel Foods’ legislation—i.e. Regulation [14] This regulation applies to all categories of foods that were not used for human consumption to a significant degree within the European Union before May 15, 1997, which is the case of insects. GS, like other Gryllidae species, is susceptible to Acheta domesticus densovirus (AdDNV) with a variable degree, but without cases of severe mortality which instead occurs in AC, where AdDNV provokes high mortality and serious and irreparable damages to the production. For this reason, interest in rearing GS against AC increased after the bankruptcy of AC facilities [21]. In this context, controlling the composition of both feed and food is an important issue according to European rules, especially after outbreaks such as BSE
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