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Quantitative analysis of food and feed samples with droplet digital PCR.

ABSTRACT: In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.

SUBMITTER: Morisset D

PROVIDER: S-EPMC3642186 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Quantitative analysis of food and feed samples with droplet digital PCR.

Morisset Dany D Štebih Dejan D Milavec Mojca M Gruden Kristina K Žel Jana J

PloS one 20130502 5

In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay ...[more]

PMID: 23658750

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Project description:Tuberculosis is a rising global public health emergency. Then, it is a priority to undertake innovations in preventive, diagnostic, and therapeutic methods. Improved diagnostic methods for tuberculosis are urgently needed to address this global epidemic. These methods should be rapid, accurate, affordable, and able to detect drug-resistant tuberculosis. The benefits of these new diagnostic technics include earlier diagnosis and treatment, improved patient outcomes, and reduced economic burden. Therefore, we aimed to systematically review the diagnostic performance of droplet digital PCR (ddPCR)-a third-generation PCR-compared with quantitative Real Time-PCR (qPCR) for diagnosing pulmonary and extrapulmonary tuberculosis. We included 14 diagnostic accuracy test studies performed in Asia, Europe, and Latin America, 1,672 participants or biological samples, and 975 events (pulmonary or extrapulmonary tuberculosis). Most of the included studies had a low risk of bias (QUADAS-C tool). Sensitivity and specificity were lower for ddPCR [0.56 (95% CI 0.53-0.58) and 0.97 (95% CI 0.96-0.98), respectively] than for qPCR [0.66 (95% CI 0.60-0.71) and 0.98 (95% CI 0.97-0.99), respectively]. However, the area under the ROC curve (AUC) was higher for ddPCR than for qPCR (0.97 and 0.94, respectively). Comparing both AUCs using the Hanley & McNeil method, we found statistically significant differences (AUC difference of 4.40%, p = 0.0020). In the heterogeneity analysis, we found significant differences between both techniques according to the continent of origin of the study and the location of tuberculosis (pulmonary or extrapulmonary disease). The AUCs of both methods were similar in pulmonary tuberculosis. However, for extrapulmonary tuberculosis, the AUC was higher for ddPCR. We found some limitations: (1) significant heterogeneity of the studies, and (2) we could not perform subgroup analyses according to other relevant variables, such as the age and sex of the participants. Nonetheless, this study is the first meta-analysis that shows that ddPCR has a comparable diagnostic performance than qPCR for pulmonary tuberculosis. However, for extrapulmonary tuberculosis, ddPCR has a better discriminant capacity to differentiate between patients with and without extrapulmonary tuberculosis. We conclude that ddPCR is likely the best diagnostic technic for tuberculosis diagnosis, especially for extrapulmonary tuberculosis. More studies are still needed yet.Systematic review registrationhttps://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022382768, CRD42022382768.

Dataset Information

Quantitative analysis of food and feed samples with droplet digital PCR.

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Quantitative analysis of food and feed samples with droplet digital PCR.

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