Human rhinoviruses (HRV) comprise 3 species representing more than 150 genotypes. As an important human respiratory pathogen, molecular detection is an indispensable tool for diagnosis and surveillance. However, the sequence diversity of HRV genotypes poses challenges for developing robust molecular methods that detect all genotypes with equal efficiencies. This study compares the accuracies of reverse transcription-quantitative PCR (RT-qPCR) and reverse transcription-digital PCR (RT-dPCR) for quantifying HRV RNA using genotype-specific primers and probes and a consensus primer/probe set targeting the 5= noncoding region of HRV. When using consensus primers and probes for the quantification of HRV, RT-dPCR outperformed RT-qPCR by consistently and accurately quantifying HRV RNAs across more genotype groups, despite the presence of up to 2 target-sequence mismatches within the primer or probe binding region. Because it does not rely on amplification efficiency, which can be affected by sequence mismatches in primer/probe binding regions, RT-dPCR may be the optimal molecular method for future HRV quantification studies and for quantitating other viruses with high sequence diversity.KEYWORDS human rhinovirus, digital PCR, qPCR H uman rhinoviruses (HRV) are important human respiratory pathogens that are small positive-sense RNA viruses within the family Picornaviridae. There are more than 150 genotypes of HRV that have been recognized within species A, B, and C of the genus Enterovirus (1). Molecular assays, such as reverse-transcription PCR (RT-PCR), are the most useful methods for detecting HRV in clinical samples (2-4). Most HRV RT-PCR assays target the conserved 5= noncoding region (NCR), which exhibits the greatest sequence homology among the HRV genotypes. However, even in the 5= NCR, consensus primer and probe sets must be designed with degenerate and modified bases or multiple oligonucleotides to amplify all HRV genotypes (5-7).Although qualitative detection of HRV by RT-PCR is currently sufficient for determining HRV infection, accurate quantification of HRV RNA in clinical samples is needed for studies associating HRV viral load with viral transmission and with patient symptoms and outcomes. Viral load studies of other respiratory viruses have shown that a correlation exists between viral load and disease severity (8-10). Accurate quantification of HRV will also be required for evaluating the performances of future antiviral drugs. Real-time RT-PCR assays, when accompanied by the amplification of standard curves (RT-qPCR), can be used to quantify the number of viral copies in a sample. However, RT-qPCR assays using quantification with a consensus HRV primer and probe set may not give accurate results for all genotypes due to amplification inefficiencies caused by base mismatches between the primer and probe sequences and the specific
We describe metagenomic next-generation sequencing (mNGS) of a human coronavirus 229E from a patient with AML and persistent upper respiratory symptoms, who underwent hematopoietic cell transplantation (HCT). mNGS revealed a 548-nucleotide deletion, which comprised the near entirety of the ORF4 gene, and no minor allele variants were detected to suggest a mixed infection. As part of her pre-HCT conditioning regimen, the patient received myeloablative treatment with cyclophosphamide and 12 Gy total body irradiation. Iterative sequencing and RT-PCR confirmation of four respiratory samples over the 4-week peritransplant period revealed that the pre-conditioning strain contained an intact ORF4 gene, while the deletion strain appeared just after conditioning and persisted over a 2.5-week period. This sequence represents one of the largest genomic deletions detected in a human RNA virus and describes large-scale viral mutation associated with myeloablation for HCT.
We describe metagenomic next-generation sequencing (mNGS) of a human coronavirus 229E from a patient with AML and persistent upper respiratory symptoms, who underwent hematopoietic cell transplantation (HCT). mNGS revealed a 548-nucleotide deletion, which comprised the near entirety of the ORF4 gene, and no minor allele variants were detected to suggest a mixed infection. As part of her pre-HCT conditioning regimen, the patient received myeloablative treatment with cyclophosphamide and 12 Gy total body irradiation. Iterative sequencing and RT-PCR confirmation of 4 respiratory samples over the 4-week peritransplant period revealed that the pre-conditioning strain contained an intact ORF4 gene, while the deletion strain appeared just after conditioning and persisted over a 2.5-week period. This sequence represents one of the largest genomic deletions detected in a human RNA virus and describes large-scale viral mutation associated with myeloablation for HCT.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.