Rapid Membrane-Based Viral RNA Isolation Method for the Polymerase Chain Reaction

摘要

Methods used to prepare RNA for use in PCR generally require the use of hazardouschemicals such as guanidine isothiocyanate, chloroform and phenol. Here we describe a rapid membrane-based method for the isolation of RNA from viruses present in serum. In this system whole virus particles present in serum are immobilized on a membrane by positive pressure filtration. Digestion of the membrane bound particle with a detergent and proteinase K releases nucleic acid from its capsid and destroys ribonucleases present in the sample. Strips of Immobilon-P membrane (polyvinylidene difluoride (PVDF), Millipore Corporation, Bedford, MA), measuring 5/32 inches by 1 1/2 inches, were placed on three thicknesses of Whatman 3 MM filter paper. One hundred microliters of human serum spiked with varying amounts of West Mile virus (Family Flaviviridae, RNA genome), strain 956B, were spotted near the end of the membrane in two spots.