SBIR-STTR Award

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.

Thesaurus Terms:
"aids Virus; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Amino Acid Substitution; Assay; Bioassay; Biologic Assays; Biological Assay; Blood; Blood Plasma Cell; Blood Sample; Blood Serum; Blood Specimen; Body Tissues; Buffers; Cells; Clinical; Complementary Dna; Cytolysis; Dna; Dna Polymerases; Dna, Complementary; Dna-Dependent Dna Polymerases; Dna-Directed Dna Polymerase; Deoxynucleoside-Triphosphate[{..}]dna Deoxynucleotidyltransferase (Dna-Directed); Deoxynucleotide-Triphosphate[{..}]dna Deoxynucleotidyltransferase (Rna-Directed); Deoxyribonucleic Acid; Detection; Development; Direct Costs; Ec 2.7.7.49; Ec 2.7.7.7; Enhancers; Enzymes; Evaluation; Gene Expression; Gene Products, Rna; Genetic Condition; Genetic Diseases; Goals; Government; Grippe; Hcv; Hiv; Htlv-Iii; Hepatitis; Hepatitis C Virus; Hepatitus C; Hereditary Disease; Human; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type Iii; Human T-Cell Lymphotropic Virus Type Iii; Human T-Lymphotropic Virus Type Iii; Human, General; Influenza; Lav-Htlv-Iii; Lymphadenopathy-Associated Virus; Lysis; Man (Taxonomy); Man, Modern; Measures; Methods; Molecular Disease; Mutagenesis, Site-Directed; Nucleic Acids; Pathogen Detection; Patients; Performance; Persons; Plasma Cells; Plasmacytes; Procedures; Process; Protocol; Protocols Documentation; Rna; Rna Transcriptase; Rna Viruses; Rna Purification; Rna, Non-Polyadenylated; Rna-Dependent Dna Polymerase; Rna-Directed Dna Polymerase; Rt-Pcr; Rtpcr; Reaction; Research; Research Specimen; Resistance; Reticuloendothelial System, Blood; Reverse Transcriptase; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Revertase; Ribonucleic Acid; Sampling; Serum; Site-Directed Mutagenesis; Site-Specific Mutagenesis; Specimen; System; System, Loinc Axis 4; Targeted Dna Modification; Targeted Modification; Technology; Testing; Time; Tissues; Viral; Virus; Virus-Hiv; Viruses, General; Whblood; Whole Blood; Work; Base; Cdna; Clinical Applicability; Clinical Application; Communicable Disease Diagnosis; Cost; Design; Designing; Flu Infection; Genetic Disorder; Hereditary Disorder; Improved; Infectious Disease Diagnosis; Influenza Infection; Inhibitor; Inhibitor/Antagonist; Mrna Expression; Mutant; New Technology; Novel; Pathogen; Plasmocyte; Public Health Relevance; Resistant; Reverse Transcriptase Pcr"

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.

Thesaurus Terms:
Address;Base;Biological Assay;Biological Markers;Blood;Blood Specimen;Catalytic Domain;Centers For Disease Control And Prevention (U.S.);Clinical;Clinical Application;Clinical Research;Codon Nucleotides;Collaborations;Cost;Dengue Virus;Design;Detection;Diagnostic Procedure;Diagnostic Tests;Disease;Dna;Dna-Directed Dna Polymerase;Eligibility Determination;Engineering;Enhancers;Enzymes;Equilibrium;Exhaust;Exhibits;Gb Virus C;Gene Expression;Gene Targeting;Genes;Hepatitis;Hepatitis C Virus;Hiv;Hiv-1;Human;Improved;Infection;Inhibitor/Antagonist;Insight;Interest;Libraries;Marketing;Measures;Methods;Mutagenesis;Mutant;Mutation;New Technology;Novel;Pathogen;Pathogen Detection;Patients;Performance;Performance At Work;Phenotype;Plasma;Procedures;Process;Production;Protocols Documentation;Provider;Public Health Medicine (Field);Public Health Relevance;Publishing;Qualifying;Reaction;Research;Resistance;Reverse Transcriptase Polymerase Chain Reaction;Reverse Transcription;Risk;Rna;Rna Purification;Rna Viruses;Rna-Directed Dna Polymerase;Role;Sampling;Screening;Sensitivity And Specificity;Serum;Site-Directed Mutagenesis;Specificity;Speed (Motion);Structure;Success;System;Taq Polymerase;Technology;Testing;Thermostability;Time;Validation;Viral;Viral Load Result;Viral Rna;Virus;Virus Diseases;Work;