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

摘要

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. 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 reverse transcriptase and performing reverse transcription at a high temperature. The disclosed method can also be used to detect reverse transcriptase activity in a sample and to identify inhibitors of reverse transcriptase. Also disclosed is a method for sequencing target RNA molecules using reverse transcriptase lacking an RNAse H function.