Recently, European Commission (Reg. 2017/893/EC) allowed the use of processed insects in aquafeed and among the class Insect, only seven species have been allowed. DNA detection and identification of animal materials by means of PCR is now considered the official analytical method, thanks to its high sensitivity, to: (i) determine the species origin of processed animal proteins (PAPs); (ii) give evidence of the absence of not allowed ingredients; (iii) confirm the presence of authorised ingredients in animals feed. Among the detection/identification of PAPs, insect species identification may represent a new challenge and PCR methods the solution. In the present study, 7 specific primers against a conservative mitochondrial gene have been designed and validated for cross reactivity with different biological matrices, giving no cross reactivity. In detail, the most promising species to be used in feed according to availability and market price, T. molitor (TM) and H. illucens (HI), were tested for amplification efficiency and sensitivity by six serial tenfold dilutions of pure HI and TM DNA in three replicates. The limit of detection (LOD5) defined, as the lowest dilution level for which three replicates still gave a positive result, was equal to 0.1µg for both the target species. HI DNA at 10ng/µl in purity gave a reproducible signal at Ct=21,51 and TM DNA at 10ng/µl gave a signal at Ct=20,24. TM and HI were detected also in practical feed with graded insect inclusion and the qPCR analysis output resulted in Ct values correlated to the inclusion level of the target species. The efficiency of PCR system for HI detection was described by a linear regression (y = -0,1665x + 39,879 R² = 0,977, n=10) obtained by plotting Ct-values against log DNA concentrations within the range of 0.03 to 0.5 g/g. Similar results were observed for T. molitor. The selected primers sequence revealed good efficiency and affinity in qPCR method and are promising candidates to be used in a portable biosensor for field analysis based on a fluorescence detection technique as a faster and easier detection system useful at industrial level.
Molecular tools for insect detection in animal feed
Daniso, Enrico
2019-01-01
Abstract
Recently, European Commission (Reg. 2017/893/EC) allowed the use of processed insects in aquafeed and among the class Insect, only seven species have been allowed. DNA detection and identification of animal materials by means of PCR is now considered the official analytical method, thanks to its high sensitivity, to: (i) determine the species origin of processed animal proteins (PAPs); (ii) give evidence of the absence of not allowed ingredients; (iii) confirm the presence of authorised ingredients in animals feed. Among the detection/identification of PAPs, insect species identification may represent a new challenge and PCR methods the solution. In the present study, 7 specific primers against a conservative mitochondrial gene have been designed and validated for cross reactivity with different biological matrices, giving no cross reactivity. In detail, the most promising species to be used in feed according to availability and market price, T. molitor (TM) and H. illucens (HI), were tested for amplification efficiency and sensitivity by six serial tenfold dilutions of pure HI and TM DNA in three replicates. The limit of detection (LOD5) defined, as the lowest dilution level for which three replicates still gave a positive result, was equal to 0.1µg for both the target species. HI DNA at 10ng/µl in purity gave a reproducible signal at Ct=21,51 and TM DNA at 10ng/µl gave a signal at Ct=20,24. TM and HI were detected also in practical feed with graded insect inclusion and the qPCR analysis output resulted in Ct values correlated to the inclusion level of the target species. The efficiency of PCR system for HI detection was described by a linear regression (y = -0,1665x + 39,879 R² = 0,977, n=10) obtained by plotting Ct-values against log DNA concentrations within the range of 0.03 to 0.5 g/g. Similar results were observed for T. molitor. The selected primers sequence revealed good efficiency and affinity in qPCR method and are promising candidates to be used in a portable biosensor for field analysis based on a fluorescence detection technique as a faster and easier detection system useful at industrial level.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
