| Info. | Vol.16 - No.4 (2022.12.20) |
|---|---|
| Title | Direct Reverse Transcription Real-Time PCR of Viral RNA from Saliva Samples Using Hydrogel Microparticles |
| Authors | Emmanuel George Kifaro1,2,3, Mi Jung Kim1, Seungwon Jung1, Jin-Yong Noh4, Chang-Seon Song4,5, Gerald Misinzo2,3, Sang Kyung Kim1,6
*Sang Kyung Kim sangk@kist.re.kr |
| Institutions | 1Molecular Recognition Research Center, Korea Institute of Science and Technology (KIST), 5, Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea
2Department of Veterinary Microbiology, Parasitology, and Biotechnology, Sokoine University of Agriculture (SUA), PO Box 3019, Morogoro, Tanzania 3Southern African Centre for Infectious Disease Surveillance (SACIDS), Africa Centre of Excellence for Infectious Diseases of Humans and Animals in Eastern and Southern Africa (ACE), Sokoine University of Agriculture (SUA), PO Box 3297, Morogoro, Tanzania 4KCAV Co., Ltd., Seoul, Republic of Korea 5Avian Diseases Laboratory, College of Veterinary Medicine, Konkuk University, Seoul 05029, Republic of Korea 6KHU-KIST Department of Converging Science and Technology, Kyung Hee University, Seoul 02447, Republic of Korea |
| Abstract | In recent decades “saliva” has emerged as an important non-invasive biofluid for diagnostic purposes in both human and animal health sectors. However, with the rapid evolution of molecular detection technologies, the limitation has been the lack of an efficient method for the facile amplification of target RNA from such a complex matrix. Herein, we demonstrate the novel application of hydrogel microparticles of primer-immobilized networks (PIN) for direct quantitative reverse transcription PCR (dirRT-qPCR) of viral RNA from saliva samples without prior RNA purification. Each of these highly porous PIN particles operates as an independent reactor. They filter in micro-volumes of the analyte solution. Viral RNA is captured and converted to complementary DNA (cDNA) through the RT step using covalently incorporated RT primers. The PIN with cDNA of the viral target will be ready for subsequent highly specific qPCR. Preceded by heat-treatment for viral lysis, we were able to conduct PIN dirRT-qPCR with 95% efficiency of the matrix (M) gene for influenza A virus (IAV) and 5’ untranslated region (5’ UTR) for chicken coronavirus spiked into saliva samples. The addition of reverse transcriptase enzyme (RTase) and 10% dilution of the matrix improved the assay sensitivity considerably. PIN particles’ compatibility with microfluidic PCR chip technology has significantly reduced total sample processing time to 50 min, instead of an average of 120 min that are normally used by other assays. We anticipate this technology will be useful for other viral RNA targets by changing the incorporated RT primer sequences and can be adapted for onsite diagnostics. |
| Keyword | Primer-immobilized particles, Direct RT-qPCR, Saliva, Influenza A virus, Chicken coronavirus |
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