Development of automated PCR monitoring system

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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.