Real-time and conventional polymerase chain reaction systems based on the metallo-carboxypeptidase inhibitor gene for specific detection and quantification of potato and tomato in processed food.
In this paper, a method for the specific detection and quantification of potato and tomato DNA in food samples with the use of conventional and real-time polymerase chain reaction (PCR) is described. This method is adequate for use in food quality routine assays involving highly processed samples for which very tiny amounts of DNA are expected. Detection was achieved by amplifying a region of the metallo-carboxypeptidase inhibitor gene from either the potato (PCI) or the tomato (MCPI) and by using specific primers complementary to the propeptide regions of these inhibitors, which were found to differ for the potato and tomato proproteins. Conventional and real-time PCR systems were based on the same potato- or tomato-specific primer pairs, and quantification was carried out with a TaqMan chemistry-based probe. The methods developed proved to be very specific and sensitive and highly reliable for the identification and quantification of DNA from both plant species. In addition, the construction of plasmids pPAT and pTOM, suitable for use as external calibration standards for the elaboration of comparative amplification profiles, is reported. Limits of detection and quantification with the use of these plasmid standards are given. Specificity and copy number conservation among different cultivars were analyzed, and the reliability of these systems was tested through their application to the analysis of commercial food samples including potato and/or tomato as components.
Hernández
Marta
M
Departamento de Genètica Molecular, Institut de Biologia Molecular de Barcelona, Centro de Investigación y Desarrollo-Consejo Superior de Investigaciones Científicas, Jordi Girona 18-26, 08034 Barcelona, Spain.
Ferrando
Alejandro
A
Esteve
Teresa
T
Puigdomènech
Pere
P
Prat
Salomé
S
Pla
Maria
M
D001483:Base Sequence; D002138:Calibration; D018744:DNA, Plant / Q000032:analysis*; D005506:Food Contamination / Q000032:analysis; D005511:Food Handling / Q000592:standards; D018551:Solanum lycopersicum / Q000201:enzymology / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D011480:Protease Inhibitors; D012015:Reference Standards; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum / Q000201:enzymology / Q000235:genetics*
D018744:DNA, Plant; D010940:Plant Proteins; D011480:Protease Inhibitors