In this collaborative work we developed a proof-of-concept one-pot NAT for detection of S. Typhi and S. Paratyphi A using RPA and Cas12a and fluorescent readout based on trans cleavage of a ssDNA reporter formed of an oligonucelotide flanked by a fluorophore and quencher (Fig.\ref{279272}). We also developed a low-cost detection device and a protocol for manufacturing of the enzymes for RPA in Cameroon, reducing the total cost of reagents per test to $X. We conducted stakeholder engagement with clinicians, medical laboratory scientists and the Cameroon Ministry of Health to determine the strengths and weaknesses of our design in terms of integration in the clinical pathway for typhoid diagnosis in Cameroon and to determine the next steps for development.
 In step 1 (Figure \ref{279272}) at 37C, RPA based amplification initiates by binding of  recombinase (UvsX from T4 bacteriophage) to the primers to form a DNA-protein complex, which then scans the homologous region on the typhi template. Upon recognition of the binding region, assisted by a loading enzyme (UvsY from T4 bacteriphage), the complex invades the target region locally to form a D-loop of the non-target strand, which is stabilized by single strand binding protein (Gp32 from T4 bacteriophage). A strand displacing polymerase (Bsu from Bacillus subtillis) displaces the recombinase to elongate the 3' end of the primer by adding dNTP. In Step 2 (Figure \ref{279272}), when the mixture is incubated at 55C, gRNA-Cas12b complex recognizes the PAM region on the amplified target and binds to the target-strand leading to a staggered cutting of the dsDNA. Additionally, this binding leads to activation of trans cleavage of ssDNA reporter (FQ reporter) to produce fluorescence signal that is detected using a home-built device.