A Paper-Based Nucleic Acid Testing Device for Rapid Diagnosis of Mosquito-Borne Viral Diseases

Several tropical fever viruses transmitted by mosquitoes including zika, dengue, and chikungunya, are becoming a serious problem in global public health. The three diseases have similar symptoms, making early diagnosis particularly difficult without complex molecular diagnostic equipment. Thus, simple diagnostic tools are strongly required to monitor and prevent these diseases.

In this regard, investigators at the Gwangju Institute of Science and Technology (South Korea) developed LAMDA (lab-on-paper for all-in-one molecular diagnostics), which is a mini laboratory on a paper strip. LAMDA comprises a complete LAMP system in a paper strip. LAMP (Loop-mediated isothermal amplification) is a single-tube technique for the isothermal amplification of DNA and is a low-cost alternative to detect certain diseases. In contrast to the polymerase chain reaction (PCR) technique, in which the reaction is carried out with a series of alternating temperature steps or cycles, isothermal amplification is carried out at a constant temperature, and does not require a thermal cycler. Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) combines LAMP with a reverse transcription step to allow the detection of RNA.

The novel LAMDA platform concentrates the entire process of nucleic acid testing including sampling, extraction, amplification, and detection into a single paper chip. To use the LAMDA test, a drop of blood serum and some drops of distilled water are applied to two pads. The liquids flow through the paper strip horizontally and reach the base of a small vertical stack of layers that extracts all the RNA from the sample and multiplies any viral RNA of the three diseases that might be present. The top layer of the vertical stack comprises individual "reaction" patches, each designed to detect one of the three diseases. After the RNA is extracted, it flows up to the top layer, where LAMP reactions cause the fluorescent indicators on a patch to become dim if its target viral RNA is present in the sample.

Results obtained with the LAMDA platform revealed that three targets, zika virus, dengue virus, and chikungunya virus, in human serum could be detected simultaneously on the all-in-one paper chip within 60 minutes at 65 degrees Celsius. The all-in-one paper chip could be used as a real-time quantitative assay for five to 5000 copies of zika virus RNA, and LAMDA performance was demonstrated with five clinical specimens of zika and dengue virus.

Senior author Dr. Min-Gon Kim, professor of chemistry at the Gwangju Institute of Science and Technology, said "We believe that with minor modifications, such as a portable system to maintain reaction temperature at 65 degrees Celsius and a means to detect the fluorescence change with a smartphone, the proposed all-in-one paper chip can become a portable, low-cost, user-friendly, sensitive, and specific nucleic acid test platform with great potential in point-of-care diagnostics. We certainly hope that our approach and achievements with LAMDA will be helpful to advance research and development of on-site medical diagnostic tools,"

The LAMDA diagnostic platform was described in the October 1, 2020, online edition of the journal Biosensors and Bioelectronics.

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Gwangju Institute of Science and Technology