Direct detection of RNA mediated by reverse transcriptase lacking RNAse H function

US6277579A · kind A · utility

Assignee

• Digene Corporation · US

Inventors

• James G. Lazar · Bethesda, US
• Abel de la Rosa · Alpharetta, US
• Clayton D. Collier · Germantown, US

Key dates

• Filing date: Nov 15, 1999
• Grant date: Aug 21, 2001
• Expiry date: Nov 15, 2019

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/6804
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

Disclosed is a method of detecting RNA molecules of interest in which reverse transcription primers unique to the RNA molecule of interest are used for reverse transcribing the RNA with a reverse transcriptase lacking RNAse H function and the resulting RNA/DNA hybrid is detected with an antibody specific for RNA/DNA hybrids. The primers are immobilized on a solid support in order to associate the RNA/DNA hybrid with the solid support. This allows easy separation of hybrids form sample solution and specific detection of RNA molecules based on the position of the hybrid on the solid support. This method can be used to detect the presence of one or many specific RNA molecules which may be present in a sample, including RNA from different organisms (such as viruses, bacteria, fungi, plants, and animals), or RNA indicative of an infection, a disease state, or predisposition to a disease in an animal. The specificity of detection is increased relative to current detection methods involving probe hybridization since the reverse transcription primers are shorter and less subject to non-specific hybridization. Specificity of the disclosed method can also be increased by using a thermostable…