Isothermal Amplification Of RNA And Its Application In Clinical Samples

Nowadays, the phenomenon of infecting HIV, HBV, HCV by blood transfusion is prevalent and payed much attention by the society. The most important task we face is to strictly control blood screening. Conventional ELISA still has its limits, eg: low-throughput, time-wasting and low sensitivity. The Real time PCR is expensive and requires special equipments. In this research, isothermal amplification was used, combined with biotin-streptavidin system to capture the target sequence, in order to establish a high sensitivity and specialty method to detect low lever nucleic acid samples.The HBV S gene mRNA was captured to magnetic beads with specific probe, then amplified by isothermal amplification, the amplicon was analyzed by 1.5ï¼…agarose gel electrophoresis. After optimization of reaction system, the isothermal amplification technique amplified target sequence more than 10~8 fold, a detection limit of 2 pg/mL was obtained with this technique, which was much higher than the detection limit of 200 pg/mL in RT-PCR.The amplification results of HBV DNA in clinical serum samples, which was analyzed by real time PCR above 10~3 copy/mL, using capture and isothermal amplification technique, had shown great importance in HBV detection, molecular biology and epidemiology research..The method was successfully applied to analysis of HBV S mRNA and HBV DNA. Isothermal amplification technique is a perfect method for the detection of nucleic acid samples. The high sensitivity and specialty in detection are suitable for even low lever nucleic acid samples. It has great potential to replace PCR to become a main technique of nucleic acid amplification.