Date post: | 24-Dec-2015 |
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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
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
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