Evaluation of a polymerase chain reaction-based heteroduplex assay for detecting the adulteration of processed orange juice with mandarin juice

A polymerase chain reaction (PCR)-based heteroduplex assay was evaluated for the detection of mandarin juice in processed orange juice. PCR amplification of a fragment of the chloroplast trnT-trnL intergenic spacer derived from mixtures of DNA extracted from orange and mandarin juice resulted in heteroduplex formation. The heteroduplex resulted from the co-amplification of a fragment containing an 8 base-pair indel that distinguished mixtures of orange and mandarin juice from orange juice and mandarin juice alone. The heteroduplex assay was evaluated against authentic juices obtained from different citrus species and confirmed that the marker was homogeneous within Citrus. The data obtained demonstrated maternal inheritance of chloroplast type in Citrus sp. and allowed the identification and confirmation of the maternal parentage of unknown and known citrus hybrids. Analysis of the quantitative potential of the PCR and polyacrylamide gel electrophoresis (PAGE) analysis demonstrated good repeatability with a coefficient of variation of 7.5%. Greatest sources of variance in experimental results were attributable to species and varietal differences in the levels of the PCR target. Mandarin juice contained approximately 18% (w/v) less PCR target sequence than did orange juice. The assay was tested in a blind trial using processed juices and correctly identified 20/22 samples with no false-positive results.