Dataset Information

Establishment of a Screening Method for Epstein-Barr Virus-Associated Gastric Carcinoma by Droplet Digital PCR.

ABSTRACT:

Background

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is classified as one of the molecular subtypes of gastric cancer. We used droplet digital polymerase chain reaction (ddPCR) to enable highly sensitive and quantitative detection of EBV.

Methods

EBV-DNA load was calculated based on the copy number of the BamH1-W fragment of EBV by ddPCR, and the cut-off value of EBV-DNA load was set. We conducted both ddPCR and EBER1 ISH to examine whether their results coincided in 158 gastric cancer specimens of unknown EBV status. We prepared 26 biopsy specimens and 49 serum samples including EBVaGC and assayed them by ddPCR.

Results

The median values of EBV-DNA load for EBVaGC and EBV-negative control were 17.0 and 0.00308, respectively. A cut-off value of 0.032 was determined for which the sensitivity was 1. Among the 158 gastric cancer specimens, 14 lesions were judged as EBV-positive by the 0.032 cut-off value determined by ddPCR. The results of ddPCR and EBER1 ISH were in complete agreement. Even when using a biopsy specimen as a sample for ddPCR, the EBV-DNA load of all EBVaGCs was larger than the cut-off value.

Conclusions

We established a new method of diagnosing EBVaGC from tissue samples by ddPCR.

SUBMITTER: Shuto T

PROVIDER: S-EPMC6956032 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Publications

Establishment of a Screening Method for Epstein-Barr Virus-Associated Gastric Carcinoma by Droplet Digital PCR.

Shuto Takuya T Nishikawa Jun J Shimokuri Kanami K Yanagi Ayaka A Takagi Tatsuya T Takagi Fumiya F Miura Osamu O Iida Michihisa M Nagano Hiroaki H Takemoto Yoshihiro Y Harada Eijiro E Suehiro Yutaka Y Yamasaki Takahiro T Okamoto Takeshi T Sakaida Isao I

Microorganisms 20191129 12

<h4>Background</h4>Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is classified as one of the molecular subtypes of gastric cancer. We used droplet digital polymerase chain reaction (ddPCR) to enable highly sensitive and quantitative detection of EBV.<h4>Methods</h4>EBV-DNA load was calculated based on the copy number of the BamH1-W fragment of EBV by ddPCR, and the cut-off value of EBV-DNA load was set. We conducted both ddPCR and EBER1 ISH to examine whether their results coincided i ...[more]

PMID: 31795435

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Project description:The authorisation of genetically modified food and feed in the EU is subject to the provision of evidence of safety and of the availability of reliable analytical methods. These methods represent an essential tool for official laboratories to enforce a harmonised market control. Here the validation of droplet digital PCR (dPCR) methods has been performed for studying if the performance and acceptance parameters set by EU and other international guidelines for the analysis of genetically modified organisms (GMO) in food and feed are suitable and achievable also with such methods. The single-laboratory validation study showed that performance requirements set for GMO analysis by real time PCR can also be used to assess dPCR-based methods. Moreover, trueness and precision were assessed for both simplex and duplex formats in a multi-laboratory validation study organised according to international standards. Overall, the data on trueness, repeatability and reproducibility precision resulting from the collaborative study are satisfying the acceptance criteria for the respective parameters as stipulated in the EU and other international guidance such as the Codex Committee on Methods of Analysis and Sampling (CCMAS). For instance, the duplex droplet dPCR method for MON810 showed relative repeatability standard deviations from 1.8% to 15.7%, while the relative reproducibility standard deviation was found to be between 2.1% and 16.5% over the dynamic range studied. Moreover, the relative bias of the dPCR methods was well below 25% across the entire dynamic range. In addition, other aspects supporting the application of digital PCR for the control of GMOs on the market were experimentally assessed such as the conversion of the measurement results from copy number ratio to mass fraction, the influence of the DNA extraction step and of the ingredient content. It was found that the DNA extraction step added only a limited contribution to the variability of the measurement results under the studied conditions. The decreasing amount of the target ingredient content may decrease the level of precision of the method, although within the acceptance range of GMO performance parameters. Highlights • In-house and collaborative validation studies of a droplet digital PCR method.• Trueness and precision assessed and compared for simplex and duplex formats in a collaborative study.• Evaluation of the influence of DNA extraction and ingredient content.• Description of compliance to EU and other international performance requirements for GMO analysis methods.• Application of conversion of measurement results from copy number ratios to mass fractions.

Dataset Information

Establishment of a Screening Method for Epstein-Barr Virus-Associated Gastric Carcinoma by Droplet Digital PCR.

Background

Methods

Results

Conclusions

Publications

Establishment of a Screening Method for Epstein-Barr Virus-Associated Gastric Carcinoma by Droplet Digital PCR.

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