Direct RT-PCR detection of RNA pathogens and mRNA expression in crude samples

DESCRIPTION (provided by applicant): We propose to develop a highly simplified and improved method of detecting RNA for use in clinical tests and for scientific research by enabling the RT-PCR amplification of nucleic acids directly in whole blood, serum, plasma, and cell lysates. We propose a dual approach. We will work with two of our blood inhibition Taq mutants combined with a viral reverse transcriptase enzyme in the presence of a specially developed enhancer. In addition, we will make amino acid substitutions to our blood inhibition mutants to render them competent in reverse transcription. The sensitivity of the new technology will drive the evaluations. The method also includes development and optimization of buffers that are compatible with both the RT and DNA polymerase activities, as well as reaction additives that relieve the inhibition and enhance the RT-PCR performance in crude specimens. The tests will begin with mimic samples composed of RNA mixed with blood, serum, and plasma. Once optimized, the method will be applied to clinical RNA pathogen detection and mRNA expression assays in crude samples. The clinical RNA virus pathogens will include HCV and GBV. Comparisons will be made to the standard RNA detection protocol, which requires the RNA to be purified from the sample prior to detection. The focus of the novel method will be to increase the sensitivity of detection to match or exceed the sensitivity of the standard method. Until now, the diagnosis of infectious diseases and genetic disorders has required costly and time-consuming procedures. The standard protocol requires RNA purification prior to RT-PCR which may reduce the quantity of RNA before cDNA is produced. The proposed novel technology not only introduces a significant reduction in cost, but also solves technical problems irrespective of cost. The proposed method would provide improved accuracy, efficiency, and lower cost of RNA detection directly in whole blood or blood fractions samples and cell and tissue lysates. The benefit to the public is by improved and more reliable detection of RNA pathogens in clinical tests and advanced means of measuring mRNA expression in crude samples at a reduced cost. PUBLIC HEALTH RELEVANCE: Many viruses that are harmful to humans, such as hepatitis, HIV, and influenza, are based in RNA, not DNA. To determine if a patient has a harmful RNA virus, a blood sample is often drawn then sent to a lab to use a process called RT-PCR to find the virus. Until now, for RT- PCR to work, the RNA must first be extracted from the blood which can present problems and is expensive, but our proposed method can find RNA directly in blood.