Difference between revisions of "Team:JMU Wuerzburg/Description"
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<h3>Test Tonic – a rapid test system for the malaria-causing parasite <span style="font-style: italic;">Plasmodium</span></h3> | <h3>Test Tonic – a rapid test system for the malaria-causing parasite <span style="font-style: italic;">Plasmodium</span></h3> | ||
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One of these indicates if a patient is affected of<span style="font-style: italic;"> Plasmodium </span>in general. | One of these indicates if a patient is affected of<span style="font-style: italic;"> Plasmodium </span>in general. | ||
The other one specifically detects the species <span style="font-style: italic;">Plasmodium falciparum</span>. | The other one specifically detects the species <span style="font-style: italic;">Plasmodium falciparum</span>. | ||
| + | |||
<br><br> | <br><br> | ||
After identification of fitting sequences, we evaluate and optimize our primers by running qPCR assays with E. | 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<span style="font-style: italic;"> Plasmodium </span>genome as a positive control. | coli containing a plasmid with a short, synthetic, non-pathogenic sequence of the<span style="font-style: italic;"> Plasmodium </span>genome as a positive control. | ||
Additionally we conduct qPCR assays with genomic DNA of cultured<span style="font-style: italic;"> Plasmodium </span>parasites. | Additionally we conduct qPCR assays with genomic DNA of cultured<span style="font-style: italic;"> Plasmodium </span>parasites. | ||
| − | + | 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 2014 <a href="#desc_refs">4</a></sup>. | ||
| + | RPA makes such a test system cheap and avoids the need of an expensive thermocycler. | ||
| + | |||
<br><br> | <br><br> | ||
| − | To perform the amplification directions in a user-friendly way we designed a hardware model. | + | 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. | 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. | + | A step motor and an Arduino manage the movement and mixing of the reaction fluids. |
| − | + | Fluorescence emission then indicates the presence of <i>Plasmodium</i> DNA and specifically <i>P. falciparum</i> DNA. | |
| − | + | ||
<br><br> | <br><br> | ||
| − | After creating a fundamental model with a multiplex qPCR we elaborate a way to apply our detection system to Recombinase Polymerase Amplification (RPA) <sup><a href="# | + | After creating a fundamental model with a multiplex qPCR, we elaborate |
| − | RPA is a promising alternative for qPCR to isothermally amplify our target sequences in a short period of time. | + | a way to apply our detection system to Recombinase Polymerase Amplification (RPA)<sup><a href="#collab_refs">3</a></sup>. |
| − | + | RPA is a promising alternative for qPCR to isothermally amplify our target sequences in a | |
| − | This results in many advantages for the application in travelling situations and in areas without proper infrastructure and energy supply. | + | short period of time. It makes such a test system cheaper 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. | ||
<hr style="width: 50%; margin-bottom: 0;"> | <hr style="width: 50%; margin-bottom: 0;"> | ||
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<sup>1</sup> <a href="http://www.who.int/malaria/areas/treatment/overview/en/">http://www.who.int/malaria/areas/treatment/overview/en/</a><br> | <sup>1</sup> <a href="http://www.who.int/malaria/areas/treatment/overview/en/">http://www.who.int/malaria/areas/treatment/overview/en/</a><br> | ||
<sup>2</sup> <a href="https://www.ncbi.nlm.nih.gov/pubmed/27160000">https://www.ncbi.nlm.nih.gov/pubmed/27160000</a><br> | <sup>2</sup> <a href="https://www.ncbi.nlm.nih.gov/pubmed/27160000">https://www.ncbi.nlm.nih.gov/pubmed/27160000</a><br> | ||
| − | <sup>3</sup> <a href="https://reader.elsevier.com/reader/sd/pii/S0165993617302583?token=AE0F18E7C2136EF7A427BAE926122837FF1300E42C71ECC9B6E963D576D6DA841786904CC29F16458D3472BC66EF6B7F">https://reader.elsevier.com/reader/sd/pii/S0165993617302583?token=AE0F18E7C2136EF7A427BAE926122837FF1300E42C71ECC9B6E963D576D6DA841786904CC29F16458D3472BC66EF6B7F</a> | + | <sup>3</sup> <a href="https://reader.elsevier.com/reader/sd/pii/S0165993617302583?token=AE0F18E7C2136EF7A427BAE926122837FF1300E42C71ECC9B6E963D576D6DA841786904CC29F16458D3472BC66EF6B7F">https://reader.elsevier.com/reader/sd/pii/S0165993617302583?token=AE0F18E7C2136EF7A427BAE926122837FF1300E42C71ECC9B6E963D576D6DA841786904CC29F16458D3472BC66EF6B7F</a><br> |
| + | <sup>4</sup> <a href="https://malariajournal.biomedcentral.com/articles/10.1186/1475-2875-13-99">https://malariajournal.biomedcentral.com/articles/10.1186/1475-2875-13-99</a> | ||
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Revision as of 08:46, 17 October 2018
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 specific primer-probe pairs for each species that causes malaria in humans.
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 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. 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 2014 4. RPA makes such a test system cheap and avoids the need of an expensive thermocycler.
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. Fluorescence emission then indicates the presence of Plasmodium DNA and specifically P. falciparum DNA.
After creating a fundamental model with a multiplex qPCR, we elaborate a way to apply our detection system to Recombinase Polymerase Amplification (RPA)3. RPA is a promising alternative for qPCR to isothermally amplify our target sequences in a short period of time. It makes such a test system cheaper 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?token=AE0F18E7C2136EF7A427BAE926122837FF1300E42C71ECC9B6E963D576D6DA841786904CC29F16458D3472BC66EF6B7F
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 specific primer-probe pairs for each species that causes malaria in humans.
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 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. 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 2014 4. RPA makes such a test system cheap and avoids the need of an expensive thermocycler.
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. Fluorescence emission then indicates the presence of Plasmodium DNA and specifically P. falciparum DNA.
After creating a fundamental model with a multiplex qPCR, we elaborate a way to apply our detection system to Recombinase Polymerase Amplification (RPA)3. RPA is a promising alternative for qPCR to isothermally amplify our target sequences in a short period of time. It makes such a test system cheaper 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?token=AE0F18E7C2136EF7A427BAE926122837FF1300E42C71ECC9B6E963D576D6DA841786904CC29F16458D3472BC66EF6B7F
4 https://malariajournal.biomedcentral.com/articles/10.1186/1475-2875-13-99