AOAC 133rd Annual Meeting - Final Program

Poster Abstracts | Tuesday

Japan; Reona Takabatake , Kazumi Kitta , National Agriculture and Food Research Organization, Tsukuba, Japan Novel DNA Reference Material by Bioprinting VI: Determining PCR Primer Concentration for Real-Time PCR Concentration of primer set for real-time PCR applications has been optimized using diluted DNA as templates containing varied copy number of DNA. This dilution step can now be replaced by a novel reference material, resulting in more accu- rate PCR amplification plots. In this experiment, we attempted to determine final concentration of PCR primer set to amplify defined low copy number of DNA. We synthesized a target sequence in pig mitochondrial DNA, and designed sets of PCR primers in the target region. For the evaluation, a 96-well PCR plate which contains 0, 1, 2, 4, 8, 16 copies of target DNA in each well were prepared by the bioprinting technology. We prepared final concentration of the selected primer set ranging from 8 µM to 0.004 µM. Real-time analyses were performed with GenCheck qPCR Probe Master (dUTP) reagent (Fasmac Co., Ltd) and the Applied Biosystems™ 7500 (ThermoFisher Scientific). One selected primer set showed distinctly higher Ct values (>38) than other primer sets when lower concentrations at 0.008 and 0.016 µM. When the concentrations of the primer set were adjusted at ≥0.1 µM, Ct values were clustered nearly with lower (<34). Further, using 0.4, 0.8, 2, and 4 µM of the primer set, higher ΔRn were observed (above >2) with 16 copies of target DNA. For 1 copy of target DNA detection, only 2 and 0.8 µM of the primer set remained higher (ΔRn >2) than its adjacent concentrations. Hence, data across number of DNA copy were compared and discussed. Presenter: Ian Riztyan, FASMAC Co., Ltd, Atsugi, Japan, Email: ian@fasmac.co.jp P-T-078 Mari Onishi , Ian Riztyan , Satoshi Futo , FASMAC Co., Ltd, Atsugi, Japan; Michie Hashimoto , Ricoh Co., Ltd, Kawasaki, Japan; Reona Takabatake , Kazumi Kitta , National Agriculture and Food Research Organization, Tsukuba, Japan Novel DNA Reference Material by Bioprinting VII: Characteristics of Multiplex Real-Time PCR Detection at Low Copy Number of Target DNA Multiplex real-time PCR method has an advantage to detect more than one target DNA simultaneously. In the VI, we devel- oped sets of real-time PCR primer by which 1 copy of a pig DNA standard could be detected. Here, we would perform multiplex real-time PCR using the sets of primer to detect two targets at the same time using a novel reference material. We prepared 10 copies of synthetic DNA as internal positive control (IPC) into all wells of a 96-well PCR plate by the bioprinting technology and, then, added 50 or 100 ng of porcine genomic DNA to the IPC dispensed wells. We further prepared other test samples using the standard DNA instead of porcine genomic DNA in a similar manner. Real-time PCR analyses were performed with GenCheck qPCR Probe Master (dUTP) (Fasmac Co., Ltd) and the QuantStudio™ 5 (Thermo Fisher Scientific). PCR amplifications were observed from both IPC and genomic DNA by the duplex PCR, and Ct values derived from the pig sequence showed lower

evaluated with the freeze-thaw durability and stability. For the evaluation, a 96-well PCR plate which contains 0, 5, 10, 20, 40, and 80 copies of target DNA in each well were prepared by the bioprinting technology. It was revealed that the devel- oped reagent showed high durability and stability compared to other real-time PCR reagents, and highly accurate standard curves were obtained after 40 times of freeze-thaw cycles. Next, we evaluated another reagent for LAMP analysis in low copy number range. It was shown that the LAMP reagent was useful in evaluating the sensitivity and the designed LAMP primers. Based on these results, we concluded that the novel reference material is very effective for the development of nucleic acid amplification reagents because, the sensitivity and stability required for the reagent, can be precisely available in low copy number range. Presenter: Akane Kobayashi, Nippon Gene Co., Ltd, Toyama, Japan, Email: a-moyori@nippongene.com P-T-076 Yuuki Yonekawa , Satoshi Nakazawa , Michie Hashimoto , Hirotaka Unno , 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 V: Quality Control of Real-Time PCR Measurement Using Intercalating System The novel reference material is a result of combined technolo- gies such as genetically modification, bioprinting, and highly accuracy cell counting injection system to control a copy number of target DNA at single molecule level as shown in II. As a result, accurate quality management for instruments, reagents, and analyzing methods are possible at a low copy number range, leading resolution of problems in which have not been observed using standard DNAs containing high copy number of target DNA. DNA-intercalators are widely used for detection of DNA amplification techniques like real-time PCR and several kinds of intercalators such as SYBR Green and EvaGreen have been developed. Reagents for real-time PCR using intercalat- ing systems seems to be no defect for high copy number (> a few dozen of copies) of target DNA. However, it has not been evaluated the sensitivity and stability of intercalator-mediated DNA detections in low copy number range because of lack of reference materials containing defined low copy number of target DNA. Therefore, quality management of the reagents for DNA amplification methods is required within the range. In this study, intercalator-mediated DNA reactions and detections were evaluated using a novel reference material. Difficulties to be evaluate using the customized DNA in a unit of one molecule, and performance of the reagents will be discussed. Presenter: Yuuki Yonekawa, Ricoh Co., Ltd, Kawasaki, Japan, Email: yuuki.yonekawa@jp.ricoh.com P-T-077 Ian Riztyan , Mari Onishi , Satoshi Futo , FASMAC Co., Ltd, Atsugi, Japan; Michie Hashimoto , Ricoh Co., Ltd, Kawasaki,

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