RESUMO
Loop-mediated isothermal amplification (LAMP) is a DNA-based analytical method that can be used as an isothermal alternative to polymerase chain reaction (PCR). In comparison to PCR, the advantage of LAMP is the possibility to perform the isothermal reaction without any sophisticated technical equipment; only a water bath is needed, and naked eye detection is sufficient. Up to now, an application of LAMP methods for the detection of even closely related plant species in food or feed matrices has not been described, whereas a large number of PCR methods for that topic are cited in the literature. The aim of the study was the evaluation of LAMP-based methods for plant species identification with respect to method parameters such as R(2), LOD, and LOQ. An existing (real-time) PCR method (for the detection of spices) was used for comparison. It could be shown that the developed LAMP methods have potential as alternative strategies to PCR in DNA-based analysis.
Assuntos
Análise de Alimentos/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Plantas/classificação , Apium/genética , Carum/genética , Cuminum/genética , Fragmentação do DNA , DNA de Plantas/análise , Alimentos , Contaminação de Alimentos/análise , Mostardeira/genética , Plantas/genética , Reação em Cadeia da Polimerase , Especiarias/análise , Especiarias/classificaçãoRESUMO
Usually spices are identified morphologically using simple methods like magnifying glasses or microscopic instruments. On the other hand, molecular biological methods like the polymerase chain reaction (PCR) enable an accurate and specific detection also in complex matrices. Generally, the origins of spices are plants with diverse genetic backgrounds and relationships. The processing methods used for the production of spices are complex and individual. Consequently, the development of a reliable DNA-based method for spice analysis is a challenging intention. However, once established, this method will be easily adapted to less difficult food matrices. In the current study, several alternative methods for the isolation of DNA from spices have been developed and evaluated in detail with regard to (i) its purity (photometric), (ii) yield (fluorimetric methods), and (iii) its amplifiability (PCR). Whole genome amplification methods were used to preamplify isolates to improve the ratio between amplifiable DNA and inhibiting substances. Specific primer sets were designed, and the PCR conditions were optimized to detect 18 spices selectively. Assays of self-made spice mixtures were performed to proof the applicability of the developed methods.
