Hepatitis C virus (HCV) infections are major cause of morbidity and mortality worldwide. It is estimated that 3% of world population is infected with HCV, with infection rates being higher in low- and middle-income countries and disconnected populations in developed countries. Most people infected with HCV are not aware of infection because of lack of both symptoms (subclinical infection) and regular screening. Barriers to regular screening for HCV are due to the complexity of testing algorithms, cost of diagnostic tests, and availability of point of care (POC) diagnostic tests. The goal of this application is to demonstrate feasibility of a comprehensive molecular detection system, HCVAmp, suitable for use at POC for detection of HCV. The proposed assay will be based on a novel “Duplex reverse transcription- loop mediated isothermal amplification (Duplex RT-LAMP)” technology and performed on a commercially available benchtop instrument (AmpliFire®). While methods currently used for detection of LAMP amplification products are unable to differentiate between different targets, Duplex RT-LAMP allows detection of two targets in the same reaction. This is feasible because of use of fluorescent labels (FAM, HEX etc.) on target specific primers and a specially formulated 10X Isothermal Master Mix. Another innovation that is the basis of this application is a new thermostable Bst polymerase which has been engineered for reverse transcriptase (RT) activity. Total assay time will be 30 minutes including sample preparation with minimal hands-on time and without need of any additional equipment, such as pipettes, centrifuge etc. Results will be displayed on-screen as positive or negative for HCV, minimizing error’s caused by user interpretation. Successful completion of this project will lead to: • Development of a diagnostic kit (HCVAmp) for the detection of HCV genotypes 1 – 3 (including subtypes) at POC. • Minimal hands-on and total assay time (TAT) of 30 minute including sample preparation. • A simple and easy to use sample preparation method for processing of samples at POC. • Diagnostic sensitivity and specificity comparable to reference method. • Dried reagents for storage at ambient temperature. • Platform technology for at home diagnosis of other pathogens such as HBV, Treponema, SARS-CoV-2, influenza etc.