Superiority of Digital Reverse Transcription-PCR (RT-PCR) over Real-Time RT-PCR for Quantitation of Highly Divergent Human Rhinoviruses

Abstract

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

An intramolecular catalytic hairpin assembly on a DNA tetrahedron for mRNA imaging in living cells: improving reaction kinetics and signal stability

“…[5][6][7][8] Reverse transcription-polymerase chain reaction (RT-PCR) is a traditional amplication method that has been developed to detect RNA at low concentration levels, but it only operates in cell lysate or buffer, and cannot reect the difference in the expression level between cells. 9,10 Isothermal enzymatic signal amplication-based uorescence in situ hybridization (FISH) has been used to achieve in situ imaging of mRNA in single cells, but because the xation of cells is required for inward transportation of enzymes, it is inapplicable for living cell imaging. 11,12 In recent years, enzyme-free amplication techniques based on dynamic DNA self-assembly (DDSA) have been developed for amplication detection of targets of interest and also imaging of RNA in living cells, such as the hybridization chain reaction (HCR) [13][14][15] and catalyzed hairpin assembly (CHA).…”

Applications of Digital PCR for Clinical Microbiology

“…If amplification is inhibited by impurities in the sample or amplification efficiency is poor due to mismatches between target, primer, and probe sequences, quantification will be underestimated. Because dPCR quantifies using endpoint instead of real-time amplification, quantification is less affected by inhibitors of amplification that may be present in the sample (10,11), and it is also less affected by poor amplification efficiency (4,12,13). In fact, it may be possible to perform dPCR on samples without prior extraction of the nucleic acids (14).…”

“…Quantitation of these viruses by qPCR and RT-qPCR is often performed using consensus primer and probe sets that are intended to detect all genotypes of the virus. However, inefficient amplification of some genotypes due to sequence mismatches between the consensus primers and probe and the target nucleic acid may lead to inaccurate quantitation (12,33). Compared to RT-qPCR, human rhinovirus, which has a highly diverse genome, was more accurately quantified by RT-dPCR, which is less affected by amplification efficiency (12).…”

“…However, inefficient amplification of some genotypes due to sequence mismatches between the consensus primers and probe and the target nucleic acid may lead to inaccurate quantitation (12,33). Compared to RT-qPCR, human rhinovirus, which has a highly diverse genome, was more accurately quantified by RT-dPCR, which is less affected by amplification efficiency (12). Amplification efficiency can also be compromised by impurities in a nucleic acid sample that inhibit the PCR.…”

Development of a Digital RT-PCR Method for Absolute Quantification of Bluetongue Virus in Field Samples

“…Digital PCR overcomes the need for a standard curve, and it is increasingly used for DNA/RNA viral quantification, in human and animal health (11)(12)(13)(14)(15)(16)(17). Bluetongue (BT) is an Office International des Epizooties (OIE)-listed infectious disease of domestic and wild ruminants (18), transmitted mainly through the bites of Culicoides midges.…”