Team:JMU Wuerzburg/Description
Test Tonic – a rapid test system for the malaria-causing parasite Plasmodium
Malaria is an infectious disease that affects more than 200 million people and causes 400.000 deaths every year. The disease is caused by different species of the parasite Plasmodium. For a successful trial of Malaria, a fast and sufficient detection of the species affecting the patient is crucial.
Our aim is to construct a test system that is capable of detecting the DNA of Plasmodium. We venture to create a rapid, easily usable and cheap diagnostic device for large area application. To reach this goal, we design primers and probes by analysing published sequences of the human pathogenic Plasmodium species with bioinformatic tools. We reach out to create one primer-probe pair that can detect Plasmodium in general and specific primer/probe pairs for each species that causes malaria in humans.
After identification of fitting sequences we evaluate and optimize our primers by running qPCR assays with E. coli containing a plasmid with a short, synthetic, non-pathogenic sequence of the Plasmodium genome as a positive control. Additionally we conduct qPCR assays with genomic DNA of Plasmodium parasites. We would also like to use inactivated patient-derived samples to confirm selectivity and specificity of our test. Therefore an ethic application is in processing.
To perform the amplification directions in a user friendly way we designed a hardware model. The reactions steps are conducted in a simple test tube with separated chambers to isolate different reactions. A step motor and an Arduino manage the movement and mixing of the reaction fluids. To avoid the need of expensive thermocyclers, the amplification is conducted isothermally by RPA (Recombinase Polymerase Amplification), which is capable of amplifying very low initial concentrations of DNA within 20 to 30 minutes. The amplified DNA product is later detected in a lateral flow assay. The presence of Plasmodium DNA and specifically Plasmodium falciparum DNA is indicated through a colour reaction.
After creating a fundamental model with a multiplex qPCR we elaborate a way to apply our detection system to Recombinase Polymerase Amplification (RPA). RPA is a promising alternative for qPCR to isothermally amplify our target sequences in a short period of time. RPA makes such a test system cheap and avoids the need of an expensive thermocycler. This gives multiple benefits for the application in travelling situations, in areas without proper infrastructure and energy supply.
Our aim is to construct a test system that is capable of detecting the DNA of Plasmodium. We venture to create a rapid, easily usable and cheap diagnostic device for large area application. To reach this goal, we design primers and probes by analysing published sequences of the human pathogenic Plasmodium species with bioinformatic tools. We reach out to create one primer-probe pair that can detect Plasmodium in general and specific primer/probe pairs for each species that causes malaria in humans.
After identification of fitting sequences we evaluate and optimize our primers by running qPCR assays with E. coli containing a plasmid with a short, synthetic, non-pathogenic sequence of the Plasmodium genome as a positive control. Additionally we conduct qPCR assays with genomic DNA of Plasmodium parasites. We would also like to use inactivated patient-derived samples to confirm selectivity and specificity of our test. Therefore an ethic application is in processing.
To perform the amplification directions in a user friendly way we designed a hardware model. The reactions steps are conducted in a simple test tube with separated chambers to isolate different reactions. A step motor and an Arduino manage the movement and mixing of the reaction fluids. To avoid the need of expensive thermocyclers, the amplification is conducted isothermally by RPA (Recombinase Polymerase Amplification), which is capable of amplifying very low initial concentrations of DNA within 20 to 30 minutes. The amplified DNA product is later detected in a lateral flow assay. The presence of Plasmodium DNA and specifically Plasmodium falciparum DNA is indicated through a colour reaction.
After creating a fundamental model with a multiplex qPCR we elaborate a way to apply our detection system to Recombinase Polymerase Amplification (RPA). RPA is a promising alternative for qPCR to isothermally amplify our target sequences in a short period of time. RPA makes such a test system cheap and avoids the need of an expensive thermocycler. This gives multiple benefits for the application in travelling situations, in areas without proper infrastructure and energy supply.