Development of automated PCR monitoring system

一、嵌入-监测PCR（Intercalation-Monitoring PCR，IM-PCR）系统的研制1.原型的构建-通过组装各部分和器件，构建了一个均相定量PCR监测系统的原型。2. PCR反应条件的优化--通过对反应条件的广泛考察，建立了IM-PCR的统一标准条件; 3.核酸提取方法的开发--通过与AP相结合的方法，建立了一种新的核酸提取方法（聚集分区）型脱蛋白器和半自动质粒提取器，我们开发了一种从患者血清中提取DNA/RNA的系统，而无需预先分离血细胞。II.基因诊断的应用4.丙型肝炎病毒（HCV）RNA的定量-通过将RT（反转录）与PCR相结合，我们使IM-PCR方法不仅适用于DNA，而且适用于RNA，并定量患者血清中小于10^3拷贝的HCV RNA。这种高敏感性被证明是有价值的估计干扰素的效果和预测肝炎复发。5. MRSA的检测（耐甲氧西林金黄色葡萄球菌）-通过使用mecA基因作为指示物，我们区分了MRSA和MSSA这些结果表明，我们成功地开发了一种自动PCR设备/方法，用于超灵敏的DNA/按照最初计划，在完全封闭和均质系统中进行RNA定量。

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.