Development of a SYBR Green Real-Time PCR method for the detection of and using chloroplast genes.

Unlabelled: (Arabica) and (Robusta) are valuable agricultural products traded worldwide. In this study, we designed specific primer pairs for Arabica and Robusta using chloroplast genes to distinguish and quantify the two types of coffee beans. We assessed the specificity, sensitivity, and applicability of the qRT-PCR assay using all the primer pairs. The six designed primer pairs (three for Arabica and three for Robusta) exhibited a correlation coefficient higher than 0.99 and a slope of approximately - 3.21 to - 3.52. The efficiency ranged from 92.09 to 104.79%. The Real-Time quantitative PCR (qPCR) assay had a detection limit of 0.001 ng DNA and a quantitative detection limit of 0.01% (w/w). Additionally, the specificity of the primer pairs was confirmed by analyzing 12 non-target plant species and verifying their practicality using 10 commercials. This study highlights the effectiveness of the SYBR-based qPCR assay in detecting adulteration in commercial coffee products.

Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-024-01636-7.