Kifaro, Emmanuel George Kim, Mi Jung Jung, Seungwon Noh, Jin-Yong Song, Chang-Seon Misinzo, Gerald Kim, Sang Kyung 2024-01-19T10:33:49Z 2024-01-19T10:33:49Z 2022-08-18 2022-12 1976-0280 https://pubs.kist.re.kr/handle/201004/114274 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. English 한국바이오칩학회 Direct Reverse Transcription Real-Time PCR of Viral RNA from Saliva Samples Using Hydrogel Microparticles Article 10.1007/s13206-022-00065-0 1 BioChip Journal, v.16, no.4, pp.409 - 421 BioChip Journal 16 4 409 421 N scie scopus kci ART002906005 000837495700001 Biochemical Research Methods Chemistry, Analytical Nanoscience & Nanotechnology Biochemistry & Molecular Biology Chemistry Science & Technology - Other Topics Article RT-QPCR DNA EXTRACTION RESPIRATORY-SYNDROME-VIRUS ORAL-FLUID HEAT-TREATMENT Primer-immobilized particles Direct RT-qPCR Saliva Influenza A virus Chicken coronavirus