A 3D Printer based DNA extraction method for molecular diagnosis of Chagas disease.

WEHRENDT D.P ; ALMEIDA I.C ; QUARNSTROM Y . ; GASCON, J ; ALONSO-PADILLA J. ; SCHIJMAN A.G.; WONG SIU B.

Resistencia

Congreso; XXX Reunión Anual de la Sociedad Argentina de Protozoologia y Enfermedades Parasitarias; 2018

Sociedad Argentina de Protozoología y Enfermedades Parasitarias

Chagas disease affects around 7 Million people worldwide and it is estimated that only 1% of infected people is treated, mainly due to a lack of proper diagnosis. A reason for this is the requirement of equipment and qualified personnel often unavailable in many disease endemic areas. It is therefore important to develop simpler diagnostic techniques that can be performed in minimally equipped laboratories. The recently developed loop-isothermal amplification (LAMP) kit for Trypanosoma cruzi (etiological agent of Chagas disease) detection is very promising, because it is very sensitive, easy to use (all reagents are dried in the cap of the tubes), no thermocycler is required for the reaction and the positive result can be visualized by the naked eye. However, for optimal LAMP results, DNA of high quality must be purified. Currently available kits for DNA purification are time consuming and require high cost equipment that is generally unavailable in laboratories at endemic regions. Therefore, our goal was to set up an automated extraction method with an adapted low cost 3D Printer ?PrintrLab? to obtain DNA from whole EDTA/blood samples for further T.cruzi detection through LAMP reaction. Using the reagents of the Nuclisens kit (Biomerieux, France), which is based on magnetic beads, optimal conditions for PrintrLab extraction, such as time of washing steps, release of magnetic beads in the washing and elution buffers and elution temperature, were established. To test the quality of eluted DNA we compared the PrintrLab extraction with a standardized manual extraction method (?High Pure PCR template preparation kit?, Roche) followed by LAMP (index test, Besuschio et al, 2017) and a validated qPCR technique for T.cruzi detection (comparator test, Duffy et al 2013), using EDTA/blood samples spiked with 1000 ? 0.1 parasite equivalents/mL of cultured T.cruzi Y stock (Tc II) epimastigotes. Analytical sensitivities of both LAMP and qPCR tests were 0.1 par.eq /mL for both PrintrLab and standardized manual extraction methods. We conclude that the automated PrintrLab extraction method can be used to obtain high quality DNA for further T.cruzi detection through qPCR and LAMP. In the latter case, the PrintrLab device could be coupled to a thermoblock at 65oC for simultaneous DNA extraction and LAMP amplification, which could be of advantage in point of care settings.