EWH PROJECT:

DETERMINE DRUG RESISTANCE IN HIV POSITIVE PATIENTS

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

GOAL: To create a device which can

qualitatively detect if an HIV positive patient’s ART is working or if their strain has mutated

METHOD Create an automated platform which

combines RNA extraction, a PCR Thermocycler, and Gel Electrophoresis to give a qualitative result.

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

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

RNA EXTRACTION Steps of RNA extraction:

Lyse serumAdjust binding conditionsBind to glass meshRemove contaminants

Inhibitor Removal Buffer Wash Buffer

Elute

TRADITIONAL METHOD:

Roche :High Pure Viral RNA Kit

OUR DESIGN

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

OUR DESIGN

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 HIVVisible due to ethidium bromide tag

SPECIFICS Building our own box based on open

source designs Looking for inexpensive, safer

alternatives to ethidium bromideCreate safe waste disposal protocols if

ethidium bromide must be used.

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