Development of real‐time fluorescent reverse transcription loop‐mediated isothermal amplification assays for rhinovirus detection

Abstract

Human rhinoviruses (RVs) belong to the genus Enterovirus of the family Picornaviridae, and are classified into RV-A, -B, and -C species. Two assays were developed to detect RVs by a real-time fluorescent reverse transcription loop-mediated isothermal amplification method: one was designed based on the 5′-untranslated regions (UTRs) of RV-A and -B, and the other was designed based on the 5′-UTR of RV-C. The competence of both assays for the diagnosis of RV infection was tested using isolated viruses and compared with real-time reverse transcription polymerase chain reaction assays on clinical specimens. Neither assay demonstrated cross-reactivity with other tested enteroviruses, and they detected 19 out of 21 tested RV-As and seven out of eight tested RV-Cs. The specificity of the assays was 100% for the detection of RVs and their sensitivity for RV-A and RV-C was 86.3% and 77.3%, respectively, on clinical specimens by the combined use of both assays. Considering that both developed assays were highly specific and detected the majority of recently circulating RVs, they are helpful for the diagnosis of RV infection. Consequently, the results generated by these assays will enhance the surveillance of respiratory illness and the study of the roles of RVs associated with clinical features and disease severity. K E Y W O R D S fluorescence, quenching primer, reverse transcription loop-mediated isothermal amplification, rhinovirus

Clinical evaluation of fully automated molecular diagnostic system “Simprova” for influenza virus, respiratory syncytial virus, and human metapneumovirus

“…In this Simprova-RV, multiple reaction wells are mounted on the testing chip, and multiple targets can be tested at the same time. In a previous study, we developed RT-LAMP assays to detect influenza A subtypes of H1pdm09 and H3 viruses and other respiratory viruses 20,27,28 . Therefore, by combining these assays, IVs can be easily and simultaneously identified with respect to type and subtype, and other respiratory viruses can also be detected.…”

Evolution of the Probe-Based Loop-Mediated Isothermal Amplification (LAMP) Assays in Pathogen Detection

“…Takayama et al developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using a Q probe for the detection of the influenza virus (IV), respiratory syncytial virus (RSV), and rhinovirus [44,61]. Shirato et al used the Q probe to develop a real-time LAMP assay for the detection of the Middle East respiratory syndrome coronavirus (MERS-CoV) [43].…”

Propagation of Rhinovirus C in Differentiated Immortalized Human Airway HBEC3-KT Epithelial Cells

“…A total of 23 nasopharyngeal swabs or nasal aspirates obtained from patients enrolled in clinical studies that were approved by the National Institute of Infectious Diseases, Showa General Hospital Ethics Committee, and which screened positive for RV by real-time RT-PCR and partial 5′UTR sequencing [18], were used for the study.…”