Improvements in DNA Extraction and Loop-Mediated Isothermal Amplification (LAMP) Assist Application of LAMP on Malaria Point-of-Care Diagnostic Devices

摘要

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 fher-mocycler 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.