AOAC 133rd Annual Meeting - Final Program

Poster Abstracts | Tuesday

Presenter: Reona Takabatake, National Agriculture and Food Research Organization, Tsukuba, Japan, Email: reona@affrc.go.jp P-T-074 Hirotaka Unno , Satoshi Nakazawa , Yuuki Yonekawa , Michie Hashimoto , Ricoh Co., Ltd, Kawasaki, Japan; Reona Takabatake , Kazumi Kitta , National Agriculture and Food Research Organization, Tsukuba, Japan; Satoshi Futo , FASMAC Co., Ltd, Atsugi, Japan Novel DNA Reference Material by Bioprinting III: Quality Control of Real-Time PCR Measurement at Low Range of DNA Copy Number Today, detection of genetically modified organism (GMO) in food industry and virus in the field of regenerative medicine, including detection of an extremely low amount of GMO and virus contaminations in foods and medicines, have been carried out using nucleotide detection and quantification at a consider- ably low concentration (low number of copy). Methods for these detections are using the known real-time PCR methods. However, judgments on whether the nucleotide is existed, true or false positive, and true or false negative are difficult in measurement and testing of a very low nucleotide concentration, leaving the reliability of the testing in question. Moreover, there is no proper technology applicable in quality management for testing instru- ments as well as testing methods i.e., quantification of a very low nucleotide concentration in human and animal clinical testing. In this product, combination of genetically modification technology, bioprinting technology, and high accuracy cell counting injection technology has made it possible to control nucleotide in a unit of one molecule and could provide standard material required for analysis of low concentration nucleotide and evaluation of analysis instruments. We will introduce the application examples of the customized DNA in a unit of one molecule for quality management of analysis instruments and reagents in poster presentation. Presenter: Hirotaka Unno, Ricoh Co., Ltd, Kawasaki, Japan, Email: hirotaka.ax.unno@jp.ricoh.com P-T-075 Akane Kobayashi , Yasutaka Minegishi , Nippon Gene Co., Ltd, Toyama, Japan; Hirotaka Unno , Michie Hashimoto , Ricoh Co., Ltd, Kawasaki, Japan; Ian Riztyan , Mari Onishi , Satoshi Futo , FASMAC Co., Ltd, Atsugi, Japan; Reona Takabatake , Kazumi Kitta , National Agriculture and Food Research Organization, Tsukuba, Japan Novel DNA Reference Material by Bioprinting IV: Evaluation of DNA Amplification Reagents Using a Novel Reference Material To evaluate DNA amplification reagents in a low copy number of target DNA near the limit of detection (LOD), variations in template dispensing and template dilution errors caused by the Poisson distribution may have a significant impact on the results. In this research, we developed two DNA ampli- fication reagents for real-time PCR and LAMP analyses and evaluated the performance of the developed reagents using a novel reference material. First, the real-time PCR reagent was

features, and it is supposed to detect a single copy of DNA. However, there are no measures to assure its performance. For example, the sensitivity of qualitative real-time PCR methods can be expressed as the limit of detection (LOD). To determine the LOD, a series of standard solutions are prepared by diluting DNA solutions of known concentrations. But the copy number of DNA prepared by dilution is not constant because the dilution process is governed by the Poisson distribution. In addition, the relative variation across replicates caused by this distribution tends to become larger at low copy numbers. Thus, no matter how accurately the dilution process is performed, it is impossible to control the copy number of target DNA at the single molecule level. For resolving such problems, we have developed a novel reference material containing a defined copy number of target DNA. In this study, we demonstrate the development of a novel reference material containing as low as 1 to 5 copies of target DNA by means of a new inkjet-based bioprinting system. The newly developed reference material can be used for evaluating the quality of analytical factors such as instrument and reagents as well as for generating an accurate standard curve, which will serve for more reliable data production. Presenter: Kazumi Kitta, National Agriculture and Food Research Organization, Tsukuba, Japan, Email: kaz@affrc.go.jp P-T-073 Reona Takabatake , Masahiro Kishine , Kazumi Kitta , National Agriculture and Food Research Organization, Tsukuba, Japan; Hirotaka Unno , Satoshi Nakazawa , Yuuki Yonekawa , Michie Hashimoto , Ricoh Co., Ltd, Kawasaki, Japan; Ian Riztyan , Mari Onishi , Satoshi Futo , FASMAC Co. Ltd, Atsugi, Japan Novel DNA Reference Material by Bioprinting II: Development and Evaluation of a Novel Reference Material Nucleic acid amplification methods, such as polymerase chain reaction (PCR), have been widely used in many applications including genetic testing to detect target DNA because of its high sensitivity, good reproducibility, and wide dynamic range for quantification. However, analytical quality control for detecting low copy number target DNA is often missing due to a lack of appropriate reference material. In this study, we have developed a novel reference material containing a defined copy number of target DNA using bioprinting technology. In this method, a suspension of cells carrying a single target DNA sequence was ejected by inkjet head and the number of cells in each droplet was counted by highly sensitive cameras. The resulting solutions contain user-defined copy number of target DNA and can be used as reference material. The developed reference material was compared with that of manual serially diluted solutions of target DNA by using quantitative real-time PCR, and it was revealed that the developed reference material showed high detection rate, low variance among replicates, and high linearity for standard curves than those of diluted solutions. The reference material developed in this study will be a promising tool for evaluating the performance of assays as well as for generating an accurate standard curve, particularly focusing on the quantifi- cation of low copy number target DNA.

78 SEPTEMBER 6–12, 2019 SHERATON DENVER DOWNTOWN HOTEL