|Home | About | Journals | Submit | Contact Us | Français|
This article is referred to research article entitled “Whole genome sequence analysis of unidentified genetically modified papaya for development of a specific detection method” (Nakamura et al., 2016) .
Real-time polymerase chain reaction (PCR) detection method for unauthorized genetically modified (GM) papaya (Carica papaya L.) line PRSV-YK (PRSV-YK detection method) was developed using whole genome sequence data (DDBJ Sequenced Read Archive under accession No. PRJDB3976). Interlaboratory validation datasets for PRSV-YK detection method were provided. Data indicating homogeneity of samples prepared for interlaboratory validation were included. Specificity and sensitivity test data for PRSV-YK detection method were also provided.
Datasets provided in this article represent reliability of unauthorized genetically modified (GM) papaya (Carica papaya L.) line PRSV-YK detection method (PRSV-YK detection method), including papaya endogenous gene, Chymopapain (Chy), detection method, using real-time polymerase chain reaction (PCR). Table 1 presents specificity of PRSV-YK and Chy detection methods. Fig. 1 shows that Chy detection method amplified papaya DNA, but both PRSV-YK and Chy detection methods did not amplify rice, soybean, maize, potato, rapeseed, pineapple, peach or passion fruit DNA. Fig. 2 presents sensitivity of PRSV-YK detection method. Cycle threshold (Ct) values obtained from real-time PCR amplification plot were quantitative (R2=0.99) in the range of 0.01–100% line PRSV-YK DNA concentrations. Table 2 presents results of homogeneity test on prepared samples. Table 3 presents statistical data obtained from homogeneity test on prepared samples. Table 4 summarizes interlaboratory validation data. Data were statistically analyzed to determine mean, relative standard deviation (RSD), repeatability RSD (RSDr) and reproducibility RSD (RSDR) from Ct values obtained .
DNA purified from fresh papaya fruit was used as sample. DNA purified from GM papaya was mixed with DNA from non-GM papaya to prepare a dilution series of GM papaya DNA. Samples were prepared at three different levels of GM papaya DNA concentrations (0%, 0.05% and 0.10% [w/w]). Aliquots of the diluted DNA were placed in individual tubes. Each tube was then labeled with a randomized number. Six randomly selected tubes of each analyte concentration were tested as blind samples at each participating laboratory owning an ABI PRISM 7900HT Sequence Detection System (Thermo Fisher Scientific Inc.). Samples and real-time PCR primer and probe solutions were stored frozen at −20 °C until use.
According to a procedure described by Thompson et al. , homogeneity of samples was verified before dispatching them to participating laboratories. Ten test samples of each GM papaya DNA concentration (0%, 0.05% and 0.10% [w/w]) were labeled with a randomized number, and randomly selected samples were used. Each blind sample was tested to determine Ct values at threshold value 0.2 from exponential amplification plots obtained using developed real-time PCR method . Data were analyzed by Cochran׳s test and one-way analysis of variance.
Method for interlaboratory validation was followed as described previously , . Twelve laboratory participants were organized to evaluate repeatability and reproducibility of developed real-time PCR method. Reagents and accessories necessary for real-time PCR and experimental protocol were provided to each participating laboratory. ABI PRISM 7900HT Sequence Detection System, owned by each lab, was used for analyses. Real-time PCR was conducted within three months. All data were collected from 12 laboratories. Presence of line PRSV-YK in samples was judged by Ct values at threshold value 0.2 (present, Ct<48.00; absent, Ct≥48.00) obtained using PRSV-YK and Chy detection methods. To statistically analyze interlaboratory validation data, Ct values from all laboratories were used after eliminating outliers by a 1-tailed Cochran׳s test at a probability value of 2.5%.
This work was supported by a Grant from the Ministry of Health, Labour and Welfare of Japan. We would like to thank following laboratories for participating in interlaboratory validation: Tokyo Metropolitan Institute of Public Health (Tokyo, Japan), Food and Agricultural Materials Inspection Centers (Saitama, Japan and Kobe, Japan), Saitama Prefectural Institute of Public Health (Saitama, Japan), Yokohama City Institute of Health (Yokohama, Japan), Kanagawa Prefectural Institute of Public Health (Chigasaki, Japan), Kawasaki City Institute of Health (Kawasaki, Japan), Japan Frozen Foods Inspection Corporation (Yokohama, Japan), Japan Food Research Laboratories (Tama, Japan) and Japan Inspection Association of Food and Food Industry Environment (Tokyo, Japan).
Appendix ASupplementary data associated with this article can be found in the online version at doi:10.1016/j.dib.2016.03.095.