EWH Project : Determine drug resistance in hiv positive patients

1. EWH Project:Determine drug resistance in hiv positive patients

2. Problem: • The HIV virus can mutate in HIV positive patients taking Anti-Retroviral Therapy (ART) • Their HIV strain has now become drug resistant (DR), and their current medication no longer works • This increases the patient’s viral load, and creates the possibility of passing on a drug resistant strain of HIV

3. Goal: • To create a device which can qualitatively detect if an HIV positive patient’s ART is working or if their strain has mutated

4. Method • Create an automated platform which combines RNA extraction, a PCR Thermocycler, and Gel Electrophoresis to give a qualitative result.

5. Process • Use RNA extraction methods to purify RNA • Send a single strand of RNA to PCR group • PCR group turns single strand of RNA to cDNA (complementary DNA) via RNA transcriptase. • Then use PCR to amplify DNA segment • PCR group sends amplified DNA to electrophoresis group

6. Process • Electrophoresis group runs samples through gel, sample can then be analyzed. • If a band is present on a specific segment of the gel, then HIV is present. • Can conclude the patient’s HIV strain is drug resistant, due to the presence of HIV

7. RNA Extraction • Steps of RNA extraction: • Lyse serum • Adjust binding conditions • Bind to glass mesh • Remove contaminants • Inhibitor Removal Buffer • Wash Buffer • Elute

8. Traditional MEthod: Roche : High Pure Viral RNA Kit

9. Our Design

10. PCR Thermocycler Process • Obtain sample from RNA team • Convert RNA to cDNA • Amplify DNA via PCR • Heat and cool sample according to protocol • Export to Gel Electrophoresis team

11. Our Design

12. Gel electrophoresis Process • Make Agarose Gel with Ethidium Bromide • Load amplified DNA from PCR/Thermocycler into gel • Run voltage (100V) through gel for approximately 20-40 minutes • View gel under UV light to determine presence of HIV • Visible due to ethidium bromide tag

13. Specifics • Building our own box based on open source designs • Looking for inexpensive, safer alternatives to ethidium bromide • Create safe waste disposal protocols if ethidium bromide must be used.

14. Things to consider • Designing for Developing Nations. • This means Resource Limited Settings • Size should be reasonable • If it is too large it will be too expensive to ship • Cost • Must be incredibly reliable • Overly complex designs are more likely to break • Would like to be able to run between 20-100 patient samples in a day • Blood pooling methods