Diagnostic Potential of Rhinovirus C using Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP)

Abstract

Rhinoviruses (RVs), particularly RV-C, frequently cause acute respiratory infections and asthma exacerbations. However, there is a lack of routine detection methods. Thus, this study aims to develop a rapid molecular and differential diagnostic detection method for RV-C using the reverse transcription (RT) loop-mediated isothermal amplification (LAMP) approach. The RT-LAMP assay targeting the 5′UTR region of RV-C genome was optimized by varying the reaction temperature, magnesium sulfate, betaine concentrations, and reaction time. Compared with conventional RT-PCR with a sensitivity of 10⁶ copies of RNA, RT-LAMP demonstrated a significant increase in efficiency and sensitivity with a quantifiable viral load of at least 10¹ copies of RNA by gel electrophoresis and colour change, and 10⁴ copies of RNA for end-point detection with a turbidimeter for 40 minutes. The assay is also specific without amplifying RV-A16 and RV-B72 genomic RNA. In the proof-of-concept assay using 30 clinical respiratory samples with known etiological agents, it detected all RV-C isolates, of which its accuracy was confirmed by sequencing. The newly developed RT-LAMP assay demonstrated good analytical sensitivity and specificity toward RV-C. The assay provides an alternative for improved RV-C diagnosis.