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>
 
             <div>
 
             <div>
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                 Malaria is a widely spread infectious disease that causes 400.000  
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                 Malaria is a widely spread infectious disease that causes  
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                deaths every year and affects more than 200 million people in total according to the WHO`s Worlds Malaria Report 2017.
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                400.000 deaths every year and affects more than 200 million  
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                 The disease is caused by different species of the protozoan parasite Plasmodium.
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                people in total according to the WHO's Worlds Malaria Report 2017.  
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                 For a successful therapy of Malaria, a fast and sensitive detection of the species affecting the patient is crucial.
+
                 The disease is caused by different species of the protozoan parasite <i>Plasmodium</i>.  
 +
                 For a successful therapy of Malaria, a fast and sensitive  
 +
                detection of the species affecting the patient is crucial.
 
                 <sup><a href="#desc_refs">1</a></sup>
 
                 <sup><a href="#desc_refs">1</a></sup>
 +
               
 
                 <br><br>
 
                 <br><br>
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                 Our aim is to construct a test system that is capable of detecting the DNA of Plasmodium.
+
               
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                 Our aim is to construct a test system that is capable of  
 +
                detecting the DNA of <i>Plasmodium</i>.  
 
                 We venture to create a rapid, easily usable and cheap diagnostic  
 
                 We venture to create a rapid, easily usable and cheap diagnostic  
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                 device for large area application.
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                 device for large area application. To reach this goal,
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                To reach this goal, we design primers and probes by analysing published sequences  
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                we design primers and probes by analysing published sequences
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                 of the human pathogenic<span style="font-style: italic;"> Plasmodium species </span>with bioinformatic tools.
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                 of the human pathogenic <i>Plasmodium</i> species with bioinformatic tools.
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                 We reach out to create one primer-probe pair that can detect<span style="font-style: italic;"> Plasmodium </span>in general
+
                 We reach out to create one primer-probe pair that can detect <i>Plasmodium</i>  
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                 and specific primer-probe pairs for each species that causes malaria in humans.
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                 in general and a specific primer/probe pair for <i>P. falciparum</i>.
 +
               
 
                 <br><br>
 
                 <br><br>
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                 For the amplification of characteristic parts of the<span style="font-style: italic;"> Plasmodium </span>genome we developed two pairs of primers for each species.
+
               
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                 One of these indicates if a patient is affected of<span style="font-style: italic;"> Plasmodium </span>in general.
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                 For the amplification of characteristic parts of the <i>Plasmodium</i>  
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                 The other one specifically detects the species <span style="font-style: italic;">Plasmodium falciparum</span>.
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                genome we developed two pairs of primers for each species.  
 +
                 One of these indicates if a patient is affected of <i>Plasmodium</i> in general.  
 +
                 The other one specifically detects the species <i>Plasmodium falciparum</i>.
 +
               
 
                 <br><br>
 
                 <br><br>
−
                 After identification of fitting sequences, we evaluate and optimize our primers by running qPCR assays with E.
+
               
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                 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.
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                 After identification of fitting sequences,  
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                Additionally we conduct qPCR assays with genomic DNA of cultured<span style="font-style: italic;"> Plasmodium </span>parasites.
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                we evaluate and optimize our primers by running qPCR assays with  
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                 We would also like to use inactivated patient-derived samples to confirm selectivity and specificity of our test which requires formal permission by the ethics Committee.
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                 E. coli containing our BioBrick: a plasmid with a short,  
 +
                synthetic, non-pathogenic sequence of the <i>Plasmodium</i> genome  
 +
                as a positive control. Additionally we conduct qPCR assays  
 +
                with genomic DNA of cultured <i>Plasmodium</i> parasites.
 +
                  
 
                 <br><br>
 
                 <br><br>
−
                 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.
+
                 To perform the amplification directions in a user-friendly  
−
                 A step motor and an Arduino manage the movement and mixing of the reaction fluids.
+
                way we designed a hardware model. The reactions steps are conducted
−
                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 around 20 minutes<sup><a href="#desc_refs">2</a></sup>. The amplified DNA product is later detected in a lateral flow assay.
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                in a simple test tube with separated chambers to isolate different reactions.  
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                The presence of<span style="font-style: italic;"> Plasmodium </span>DNA and specifically <span style="font-style: italic;">Plasmodium falciparum</span> DNA is indicated through a change of color.
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                 A step motor and an Arduino manage the movement and mixing of the reaction fluids.  
  
 
                 <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="#desc_refs">3</a></sup>.
+
               
−
                 RPA is a promising alternative for qPCR to isothermally amplify our target sequences in a short period of time.
+
                 After creating a fundamental model with a multiplex qPCR we elaborate  
−
                 It makes such a test system cheap and avoids the need of an expensive thermocycler.
+
                a way to apply our detection system to Recombinase Polymerase Amplification  
−
                This results in many advantages for the application in travelling situations and in areas without proper infrastructure and energy supply.
+
                (RPA)<sup><a href="#desc_refs">2</a></sup>,
 +
                 <sup><a href="#desc_refs">3</a></sup>. 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<sup><a href="#desc_refs">4</a></sup>.
 +
                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.
  
 
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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>
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                 <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</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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Latest revision as of 15:31, 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 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