The objective of this application is to introduce a novel, simplified, low cost technology to address the broad need of detecting RNA pathogens directly in crude samples, such as blood. Traditional RT-PCR requires purifying the RNA which increases the cost, time, and risk of cross-contamination. We intend to reduce or eliminate the purification step by producing bifunctional, thermostable DNA polymerases designed for direct RT-PCR via directed mutagenesis of our existing inhibitor-resistant Omni Klentaq and OmniTaq enzymes. The most promising mutant enzymes will serve as components in clinical kits for detection of HIV, HCV, GBV-C, and dengue virus. We will also explore optimized blends of our inhibition-resistant enzymes with reverse transcriptases. First, we will evolve more robust and sensitive bifunctional enzymes by combining the mutations in our Omni Klentaq and OmniTaq enzymes that confer high resistance to PCR inhibitors with recently published mutations rendering Taq DNA polymerase capable of reverse transcription. As an alternative, if after exhausting all AA substitutions of the published mutations the performance of the bifunctional enzymes is not satisfactory, we will use a novel and highly efficient procedure for functional screening of mutagenized Taq libraries to facilitate engineer the unique bifunctional enzymes. A critical milestone in achieving this aim is to obtain balanced dual performance from the selected mutants in inhibitor-resistance and RT activity. The best mutant enzymes will be tested for their inhibition-resistance, reverse transcriptase activity, sensitivity, thermostability, and fidelity. Finally, the enzyme purification protocol for these enzymes will be optimized for large-scale commercial quality enzyme production. Besides inhibition-resistance and RT features, the selected enzymes should have thermostability and fidelity matching or exceeding that of the parental OmniTaq and Omni Klentaq enzymes. Further, we will apply our novel RT-PCR technology to clinical applications. With bifunctional enzymes or blends of enzymes, we will develop unique, sensitive, and reliable single and multiplex real-time RT-PCR assays for direct detection of HIV, HCV, GBV-C, and dengue virus in crude clinical samples. Our measure of success will be that the sensitivity and specificity of our assays in crude clinical samples match or exceed the detection level of existing top commercial kits on purified RNA. We will work in concert with our collaborators in the final validation and marketing of the kits. By eliminating th RNA extraction steps prior to PCR, the proposed novel technology not only introduces a significant reduction in cost, but also solves technical problems. The proposed method would provide higher speed, improved efficiency, and lower cost of RNA detection in important clinical samples, thereby benefitting public health.
Many viruses that are harmful to humans, such as hepatitis and HIV, are based on RNA, not DNA. To determine if a patient has a disease-related RNA virus, a blood sample is often drawn to be used in 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 technical problems and is expensive, but our proposed method can detect virus RNA directly in blood.
