title: Development of direct real-time PCR system applicable to a wide range of foods and agricultural products.
abstract: To improve the efficiency of DNA analysis of foods and agricultural products, we investigated a direct real-time PCR based on the real-time monitoring of DNA amplification directly from crude cell lysates of analytical samples. We established a direct real-time PCR system comprising sample pretreatment with a specified lysis buffer and real-time PCR using the developed master mix reagent. No PCR inhibition was observed in the analysis of crude cell lysates from 50 types of samples, indicating that the direct real-time PCR system is applicable to a wide range of materials. The specificity of the direct real-time PCR was evaluated by means of a model assay system for single nucleotide discrimination. Even when crude cell lysates coexisted in the reaction mixtures, the primer selectivity was not affected, suggesting that the sequence specificity of the direct real-time PCR was equivalent to that of PCR from purified DNA templates. We evaluated the sensitivity and quantitative performance of the direct real-time PCR using soybean flour samples including various amounts of genetically modified organisms. The results clearly showed that the direct real-time PCR system provides sensitive detection and precise quantitation.
authors.affiliation: National Food Research Institute, National Agriculture and Food Research Organization.
authors.forename: Junichi
authors.initials: J
authors.lastname: Mano
authors.forename: Shuko
authors.initials: S
authors.lastname: Hatano
authors.forename: Satoshi
authors.initials: S
authors.lastname: Futo
authors.forename: Yasutaka
authors.initials: Y
authors.lastname: Minegishi
authors.forename: Kenji
authors.initials: K
authors.lastname: Ninomiya
authors.forename: Kosuke
authors.initials: K
authors.lastname: Nakamura
authors.forename: Kazunari
authors.initials: K
authors.lastname: Kondo
authors.forename: Reiko
authors.initials: R
authors.lastname: Teshima
authors.forename: Reona
authors.initials: R
authors.lastname: Takabatake
authors.forename: Kazumi
authors.initials: K
authors.lastname: Kitta
mesh_terms: D018556:Crops, Agricultural / Q000737:chemistry*; D005504:Food Analysis / Q000379:methods*; D030841:Food, Genetically Modified; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013025:Glycine max / Q000737:chemistry