title: Detection of mandarin in orange juice by single-nucleotide polymorphism qPCR assay.
abstract: A dual-probe real time PCR (qPCR) DNA-based analysis was devised for the identification of mandarin in orange juice. A single nucleotide polymorphism at the trnL-trnF intergenic region of the chloroplast chromosome was confirmed in nine orange (Citrus sinensis) and thirteen commercial varieties of mandarin, including Citrus reticulata and Citrus unshiu species and a mandarin × tangelo hybrid. Two short minor-groove binding fluorescent probes targeting the polymorphic sequence were used in the dual-probe qPCR, which allowed the detection of both species in single-tube reactions. The similarity of PCR efficiencies allowed a simple estimation of the ratio mandarin/orange in the juice samples, which correlated to the measured difference of threshold cycle values for both probes. The limit of detection of the assay was 5% of mandarin in orange juice, both when the juice was freshly prepared (not from concentrate) or reconstituted from concentrate, which would allow the detection of fraudulently added mandarin juice. The possible use of the dual-probe system for quantitative measurements was also tested on fruit juice mixtures. qPCR data obtained from samples containing equal amounts of mandarin and orange juice revealed that the mandarin target copy number was approximately 2.6-fold higher than in orange juice. The use of a matrix-adapted control as calibrator to compensate the resulting C(T) bias allowed accurate quantitative measurements to be obtained.
authors.affiliation: Centro Tecnológico Nacional de la Conserva, Molina de Segura, Murcia, Spain; Departamento de Ciencia y Tecnología de Alimentos, Universidad Católica San Antonio de Murcia (UCAM), Murcia, Spain.
authors.forename: Miriam
authors.initials: M
authors.lastname: Aldeguer
authors.forename: María
authors.initials: M
authors.lastname: López-Andreo
authors.forename: José A
authors.initials: JA
authors.lastname: Gabaldón
authors.forename: Antonio
authors.initials: A
authors.lastname: Puyet
mesh_terms: D001628:Beverages / Q000032:analysis*; D002957:Citrus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D032084:Citrus sinensis / Q000737:chemistry; D003433:Crosses, Genetic; D056915:DNA Copy Number Variations; D015342:DNA Probes / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D021901:DNA, Intergenic / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D018744:DNA, Plant / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D005506:Food Contamination; D005511:Food Handling; D005513:Food Inspection / Q000379:methods; D005515:Food Labeling; D063427:Food Quality; D005638:Fruit / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057230:Limit of Detection; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction; D012235:Rhodamines / Q000737:chemistry; D013030:Spain; D013045:Species Specificity
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D015342:DNA Probes; D021901:DNA, Intergenic; D018744:DNA, Plant; D005456:Fluorescent Dyes; D012235:Rhodamines
keywords: Adulteration; Citrus; FC; Food quality; MGB; MGB probe; NFC; Real-time PCR; Species identification; Tm; juice from concentrate; juice not from concentrate; melting temperature; minor groove binding