Quant. Of HCV RNA Using a DNA Capture Assay

A simple, inexpensive and reliable format amendable for testing large numbers of clinical samplesfor the presence of HCV RNA will be developed in the course of this contract. The conditions for samplecollection, storage and processing will be carefully controlled to ensure maximum reproducibility andoptimum recovery of viral nucleic acids. Amplification will be carried out with biotin-labeled primers,such that the product of the reaction will be labeled. An internal control consisting of a cloned sequencebe co-amplified with the same set of primers as the RNA to allow quantitative measurement of the endproduct. The PCR products will be captured on microtiter wells by means of internal sequences betweenthe two primer sets. The extent of amplification will be measured by calorimetric detection of the biotinlabel using standard ELISA plate reading equipment. This system will be used to obtain a reliablequantitation over a range of 3 to 5,000,000 copies of HCV RNA in the sample. This approach willprovide a rapid, simple procedure for quantitation of HCV RNA that can be easily automated by use ofstandard ELISA plate handling equipment.