Publication Type
Journal Article
Publication Date (Issue Year)
2023
Journal Name
Diagnostics
Abstract
Since its discovery, polymerase chain reaction (PCR) has emerged as an important technol- ogy for the diagnosis and identification of infectious diseases. It is a highly sensitive and reliable nucleic acids (NA) detection tool for various sample types. However, stool, which carries the most abundant micro-organisms and physiological byproducts, remains to be the trickiest clinical specimen for molecular detection of pathogens. Herein, we demonstrate the novel application of hydrogel mi- croparticles as carriers of viral RNA from stool samples without prior RNA purification for real-time polymerase chain reaction (qPCR). In each microparticle of primer-incorporated network (PIN) as a self-sufficient reaction compartment, immobilized reverse transcription (RT) primers capture the viral RNA by hybridization and directly initiate RT of RNA to generate a pool of complementary DNA (PIN-cDNA pool). Through a simple operation with a portable thermostat device, a PIN-cDNA pool for influenza A virus (IAV) was obtained in 20 min. The PIN-cDNA pools can be stored at room temperature, or directly used to deliver cDNA templates for qPCR. The viral cDNA templates were freely released in the subsequent qPCR to allow amplification efficiency of over 91%. The assay displayed good linearity, repeatability, and comparable limit of detection (LoD) with a commercial- ized viral RNA purification kit. As a proof of concept, this technology carries a huge potential for onsite application to improve human and animal infectious disease surveillance activities using stool samples without the need for a laboratory or centrifuge for sample preparation
Keywords
hydrogel microparticles, viral RNA carrier, qPCR, influenza A virus, stool samples
Rsif Scholar Name
Emmanuel Kifaro
Thematic Area
Food security and Agribusiness
Africa Host University (AHU)
Sokoine University of Agriculture (SUA), Tanzania
Funding Statement
The work was partly funded by the R and D Convergence Program of the National Research Council of Science and Technology of the Republic of Korea (grant number CRC-20-02-KIST). EGK is a recipient of a scholarship from the Government of Tanzania through the World Bank (WB SACIDS- ACE II Grant PAD1436, IDA Credit 5799-TZ), and the Regional Scholarship and Innovation Fund (RSIF) of the Partnership for Skills in Applied Sciences, Engineering, and Technology (PASET).
Recommended Citation
Kifaro, E., Kim, M. J., Jung, S., Jang, Y., Moon, S., Lee, D., Song, C., Misinzo, G., & Kim, S. (2023). Microparticles as Viral RNA Carriers from Stool for Stable and Sensitive Surveillance. Diagnostics, 13 (2), 261. https://doi.org/10.3390/diagnostics13020261