Early detection right at epidemic areas can prevent infectious diseases from propagation. Currently, the most common nucleic acid test—polymerase chain reaction (PCR) is time-consuming, complex, expensive and thermocycler required, thus limiting its utility in poor laboratory conditions or even non-laboratory condition of epidemic areas. Loop-mediated isothermal amplification (LAMP) is quick, cheap, sensitive and isothermal assay could be combined with a simple DNA extraction method to integrate into Lab-on-a-chip (LOC) device. Here, we attempted to improve LAMP method for malaria diagnosis on portable microfluidics chip platform by optimizing DNA extraction using boil and spin method and altering Tris-containing amplification buffer for ascertaining changing in pH of reaction solution. Basically, blood sample was mixed with extraction buffer containing Sodium Dedocyl Sulfate (SDS) concentration and treated under high temperature condition. Four concentrations of SDS (0, 0.4, 0.8 and 1%) were tested along with different temperature (65 and 95 °C) to adapt into LOC platform and avoid denaturation of LAMP reagent. All samples treated at 65 °C showed the presence of DNA after extraction. Furthermore, DNA amplification buffer was minimized Tris concentration to facilitate result read-out step. The releasing of hydrogen ion from amplification reaction causes increasing in pH which could be recognized by color of pH indicator paper or dye, for example, phenolphthalein. Throughout a series of experiments, LAMP is demonstrated that it can also occur in low-Tris buffer with pH indicator dye, efficiently. The positive sample will have a change from pink to transparent in solution color, otherwise, the negative sample will maintain pink. These improvements allowed us to adapt LAMP technique into Point-of-care (POC) devices in which the whole process run under isothermal condition (65 °C) and non-instrument required visual detection. The LAMP microfluidics chip will be potential tool for early detection infectious diseases and several other diseases in non-laboratory condition.
LAMP DNA extraction Colorimetric detection Malaria Microfluidics chip
This is a preview of subscription content, log in to check access.
This research is funded by International University, VNU-HCM under grant number T2017-02-BME.
Conflict of Interest The authors declare that they have no conflict of interest.
World malaria report 2017. Geneva: World Health Organization (2017)Google Scholar
Chinkhumba, J., et al.: Comparative field performance and adherence to test results of four malaria rapid diagnostic tests among febrile patients more than five years of age in Blantyre, Malawi. Malar. J. 9(1), 209 (2010)CrossRefGoogle Scholar
Endeshaw, T., et al.: Performance of local light microscopy and the ParaScreen Pan/Pf rapid diagnostic test to detect malaria in health centers in Northwest Ethiopia. PloS one 7(4), e33014 (2012)CrossRefGoogle Scholar
Mekonnen, S.K., et al.: Return of chloroquine-sensitive Plasmodium falciparum parasites and emergence of chloroquine-resistant Plasmodium vivax in Ethiopia. Malar. J. 13(1), 244 (2014)CrossRefGoogle Scholar
Chou, M., et al.: Performance of “VIKIA Malaria Ag Pf/Pan” (IMACCESS®), a new malaria rapid diagnostic test for detection of symptomatic malaria infections. Malar. J. 11(1), 295 (2012)CrossRefGoogle Scholar
Aydin-Schmidt, B., et al.: Loop mediated isothermal amplification (LAMP) accurately detects malaria DNA from filter paper blood samples of low density parasitaemias. PloS one 9(8), e103905 (2014)CrossRefGoogle Scholar
Cunningham, J., Gatton, M.L., Kolaxzinski, K.: Malaria rapid diagnostic test performance: results of WHO product testing of malaria RDTs: Round 7 (2015–2016) (2017)Google Scholar
Notomi, T., et al.: Loop-mediated isothermal amplification of DNA. Nucl. Acids Res. 28(12), e63–e63 (2000)CrossRefGoogle Scholar
Han, E.-T., et al.: Detection of four Plasmodium species by genus-and species-specific loop-mediated isothermal amplification for clinical diagnosis. J. Clin. Microbiol. 45(8), 2521–2528 (2007)CrossRefGoogle Scholar
Mori, Y., Kanda, H., Notomi, T.: Loop-mediated isothermal amplification (LAMP): recent progress in research and development. J. Infect. Chemother. 19(3), 404–411 (2013)CrossRefGoogle Scholar
Poon, L.L.M., et al.: Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin. Chem. 52(2), 303–306 (2006)CrossRefGoogle Scholar
Pourmand, N., et al.: Direct electrical detection of DNA synthesis. Proc. Natl. Acad. Sci. 103(17), 6466–6470 (2006)CrossRefGoogle Scholar
Purushothaman, S., Toumazou, C., Ou, C.-P.: Protons and single nucleotide polymorphism detection: a simple use for the ion sensitive field effect transistor. Sens. Actuators B: Chem. 114(2), 964–968 (2006)CrossRefGoogle Scholar
Rothberg, J.M., et al.: An integrated semiconductor device enabling non-optical genome sequencing. Nature 475(7356), 348 (2011)CrossRefGoogle Scholar
Toumazou, C., et al.: Simultaneous DNA amplification and detection using a pH-sensing semiconductor system. Nat. Methods 10(7), 641 (2013)CrossRefGoogle Scholar
Hopkins, H., et al.: Highly sensitive detection of malaria parasitemia in a malaria-endemic setting: performance of a new loop-mediated isothermal amplification kit in a remote clinic in Uganda. J. Infect. Dis. 208(4), 645–652 (2013)CrossRefGoogle Scholar
Cook, J., et al.: Loop-mediated isothermal amplification (LAMP) for point-of-care detection of asymptomatic low-density malaria parasite carriers in Zanzibar. Malar. J. 14(1), 43 (2015)CrossRefGoogle Scholar