Development of automated PCR monitoring system

一、插层监控聚合酶链式反应(IM-PCR)系统的开发1.原型的构建--我们通过组装零部件和设备，构建了一个均质定量的聚合酶链式反应监控系统的原型。2.聚合酶链式反应条件的优化--通过对反应条件的广泛调查，建立了统一的IM-PCR标准条件。3.核酸提取方法的发展--结合AP(聚集区)型脱蛋白机和半自动质粒机，我们开发了一种从患者血清中提取DNA/RNA的系统，无需预先分离血细胞。2.在基因诊断中的应用4.丙型肝炎病毒RNA的定量-通过RT(反转录)和PCR相结合，我们使IM-PCR方法不仅适用于DNA，也适用于RNA，并对小至-lt；患者血清中存在10^3拷贝的丙型肝炎病毒核糖核酸。5.检测耐甲氧西林金黄色葡萄球菌(MRSA)--以mecA基因为指标，我们非常敏感地区分了MRSA和MSSA(甲氧西林敏感金黄色葡萄球菌)。这些结果表明，我们成功地开发了一种在完全封闭和均匀的系统中进行超灵敏DNA/RNA定量的自动化聚合酶链式反应设备/方法。

I.Development of Intercalation-Monitoring PCR (IM-PCR) System1.Construction of prototype-We constructed a prototype of homogeneous and quantitative PCR monitoring system by assembling parts and devices. We succeeded in removing incompatibilities between the parts, and collected data necessary for the goverment's approval of manufacturing.2.Optimization of PCR conditions-Through an extensive survey of reaction conditions, we established unified standard conditions for IM-PCR.3.Development of nucleic acid extraction method-By combining an AP (aggregation partition) -type deproteinizer and a semiautomatic plasmid extractor, we developed a system of extracting DNA/RNA from patients' sera with no prior separation of blood cells.II.Application to Gene Diagnosis4.Quantitation of hepatitis C virus (HCV) RNA-By combining RT (reverse transcription) with PCR,we made the IM-PCR method applicable to not only DNA but also RNA,and quantitated as little as<10^3 copies of HCV RNA in patients' sera. This high sensitivity proved to be valuable for estimation of interferon effect and prediction of hepatitis recurrence.5.Detection of MRSA (methicillin-resistant Staphylococcus aureus)-By using mecA gene as an indicator, we differentiated between MRSA and MSSA (methicillin-sensitive S.aureus) very sensitively.These results indicate that we succeeded in developing an automated PCR equipment/method for ultrasensitive DNA/RNA quantitfication in a completely closed and homogeneous system, as planned originally.