Team:JMU Wuerzburg/Description
Test Tonic – a rapid test system for the malaria-causing parasite Plasmodium
Malaria is a widely spread infectious disease that causes
400.000 deaths every year and affects more than 200 million
people in total according to the WHO's Worlds Malaria Report 2017.
The disease is caused by different species of the protozoan parasite Plasmodium.
For a successful therapy of Malaria, a fast and sensitive
detection of the species affecting the patient is crucial.
1
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 a specific primer/probe pair for P. falciparum.
For the amplification of characteristic parts of the Plasmodium genome we developed two pairs of primers for each species. One of these indicates if a patient is affected of Plasmodium in general. The other one specifically detects the species Plasmodium falciparum.
After identification of fitting sequences, we evaluate and optimize our primers by running qPCR assays with E. coli containing our BioBrick: 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 cultured Plasmodium parasites.
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.
After creating a fundamental model with a multiplex qPCR we elaborate a way to apply our detection system to Recombinase Polymerase Amplification (RPA)2, 3. RPA is a promising alternative for qPCR to isothermally amplify our target sequences in a short period of time. It has been shown to be a sufficient method to detect Plasmodium DNA by Kersting et al. in 20144. RPA makes such a test system cheap and avoids the need of an expensive thermocycler. This results in many advantages for the application in travelling situations and in areas without proper infrastructure and energy supply.
2 https://www.ncbi.nlm.nih.gov/pubmed/27160000
3 https://reader.elsevier.com/reader/sd/pii/S0165993617302583
4 https://malariajournal.biomedcentral.com/articles/10.1186/1475-2875-13-99
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 a specific primer/probe pair for P. falciparum.
For the amplification of characteristic parts of the Plasmodium genome we developed two pairs of primers for each species. One of these indicates if a patient is affected of Plasmodium in general. The other one specifically detects the species Plasmodium falciparum.
After identification of fitting sequences, we evaluate and optimize our primers by running qPCR assays with E. coli containing our BioBrick: 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 cultured Plasmodium parasites.
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.
After creating a fundamental model with a multiplex qPCR we elaborate a way to apply our detection system to Recombinase Polymerase Amplification (RPA)2, 3. RPA is a promising alternative for qPCR to isothermally amplify our target sequences in a short period of time. It has been shown to be a sufficient method to detect Plasmodium DNA by Kersting et al. in 20144. RPA makes such a test system cheap and avoids the need of an expensive thermocycler. This results in many advantages for the application in travelling situations and in areas without proper infrastructure and energy supply.
List of References
1 http://www.who.int/malaria/areas/treatment/overview/en/2 https://www.ncbi.nlm.nih.gov/pubmed/27160000
3 https://reader.elsevier.com/reader/sd/pii/S0165993617302583
4 https://malariajournal.biomedcentral.com/articles/10.1186/1475-2875-13-99