Development of automated PCR monitoring system

1.插层监测PCR （IM-PCR）系统的建立原型构建-通过组装部件和设备，构建了均质定量PCR监测系统的原型。我们成功地消除了零件之间的不兼容，并收集了政府批准生产所需的数据。优化PCR条件——通过对反应条件的广泛考察，建立了统一的im -PCR标准条件。核酸提取方法的发展-将AP（聚集分割）型脱蛋白器与半自动质粒提取器相结合，开发了一种无需事先分离血细胞即可从患者血清中提取DNA/RNA的系统。2 .基因诊断中的应用丙型肝炎病毒（HCV） RNA的定量——通过RT（逆转录）和PCR的结合，我们使IM-PCR方法不仅适用于DNA，而且适用于RNA，可以定量到患者血清中小于10^3拷贝的HCV RNA。这种高灵敏度被证明是有价值的估计干扰素的作用和预测肝炎复发。MRSA（甲氧西林耐药金黄色葡萄球菌）的检测——以mecA基因为指标，对MRSA和MSSA（甲氧西林敏感金黄色葡萄球菌）进行了非常敏感的鉴别。这些结果表明，我们成功地开发了一种全自动PCR设备/方法，用于在完全封闭和均匀的系统中进行超灵敏的DNA/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.