Application of modified solid-phase adsorption method in real-time fluorescence quantification of HCV RNA / 国际检验医学杂志

ABSTRACT

Objective To explore the application of adopting the solid phase adsorption method to extract serum hepatitis C virus (HCV) RNA in the real-time fluorescence quantification PCR( RT-qPCR) by the improvement of solid phase adsorption method . Methods Firstly ,the surface of silica nano-particles was modified with Si-OH for increasing their adsorption ability with HCV RNA .Then the concentration of guanidinium thiocyanate (GuSCN) in the viral lysate was optimized .Thirdly ,the HCV RNA of standard serum samples was extracted using the modified solid-phase adsorption method and then performed in the RT-qPCR .Its standard curve was drawn and the linear equation was calculated .At the same time ,the lowest detection limit of RT-qPCR for de-tecting HCVRNA in serum sample was measured and the detection repeatability was verified .Results The adsorption ability of sil-ica nano-particles for HCV RNA in serum samples was remarkably improved after surface modification with Si-OH .The optimal GuSCN concentration in viral lysates was 4 .23 mol/L .Using the modified method to extract HCV RNA in serum samples was ap-plied in RT-qPCR ,the standard curve and the linear equation were obtained ,the linear correlation coefficient(R2) reached to 0 .999 and the detected linear range was (2 .52 -5 .52) IU/mL or 102 .52 -105 .52 pathogen number/mL .The detection limit of HCV RNA in serum samples was (2 .52 ± 0 .50)IU/mL with good detection repeatability .Conclusion The modified solid-phase adsorp-tion method can greatly decrease the lowest detection limit of RT-qPCR and increase the positive detection rate of HCV .This meth-od is simple to operate ,has low cost and can be widely applied in the HCV detection and the nucleic acid detection of other viruses in clinic .