Critical Analysis of Rhinovirus RNA Load Quantification by Real-Time Reverse Transcription-PCR

Abstract

bRhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an in vitro-transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5=-untranslated regions (5=UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5=UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ؎10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible. Human rhinoviruses (HRVs) are small nonenveloped viruses containing a positive-strand RNA genome. They belong to the Enterovirus genus, which is part of the Picornaviridae family. HRVs are important human pathogens and the most frequent cause of respiratory infections. Their tropism is not restricted to the upper respiratory tract, and they can cause complications in the lower respiratory tract, especially in children, the elderly, and immunocompromised patients (5-7, 15, 16). Many HRVs, in particular those belonging to the newly discovered HRV-C species, do not grow under traditional cell culture conditions. Therefore, molecular diagnostic tools, such as real-time reverse transcription-PCR (RT-PCR), are the methods of choice for diagnosing HRVs.Thanks to numerous clinical studies, data have been gathered regarding implication of HRV in the exacerbation of underlying diseases, such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease (1, 3,7,8,10,17,18,20,21,25), as well as concerning the possible impact of a given HRV species on disease severity (9, 11-13, 23). However, there are inconsistent data about whether viral load is correlated with disease severity, and the threshold cycle (C T ) values from real-time RT-PCR are converted too frequently into viral copies per milliliter without any validation. This is particularly important because many published assays include primers with degenerate nucleotides or probe sequences that are biased toward a given genotype or species (4,14,22,24).We validated previously a two-step real-time RT-...

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

“…In comparison, other reports on the quantification of HRV used only consensus RT-qPCR assays, quantified from standard curves generated from a single HRV genotype, and tested a limited number of HRV genotypes (14)(15)(16). Nevertheless, our findings confirm the results from some of these studies and show that assay quantification ability is linked to target variability and that accurate HRV quantification by a single RT-qPCR assay is not feasible for all HRV genotypes (17,18).…”

Growth and characterization of different human rhinovirus C types in three-dimensional human airway epithelia reconstituted in vitro

“…The calculated VP4/VP2 nucleotide sequence homology for each of them with the closest reference type ranges from 92.1% to 97% (Table 1). Each of these HRV-C clinical samples, HRV-A41 clinical sample, and HRV-A16 stock was inoculated on ALI human airway epithelia reconstituted in vitro (named thereafter MucilAir ™ for simplicity), and the HRV RNA load was quantified at the apical surface at different times post-inoculation by real-time RT-PCR (Schibler et al, 2012b;Tapparel et al, 2009a). The RNA load present in the HRV inoculum is indicated for each clinical specimen (Fig.…”

“…RNA quantification was performed by one-step real-time RT-PCR as previously described (Schibler et al, 2012b;Tapparel et al, 2009a). HRV RNA was measured with the Panenterhino/Ge/08 assay using HRV-14 10-fold diluted RNA transcript as reference standard, while HEV RNA was quantified with the Entero/Ge/08 assay using HEV-71 (EU414331) 10-fold diluted RNA transcript as reference standard.…”

Novel human astroviruses in pediatric respiratory samples: A one‐year survey in a Swiss tertiary care hospital

“…Each individual NPS specimen (380 μL) was spiked with 20 μL of a standardized canine distemper virus as previously described 14. Nucleic acids were extracted using the NucliSENS easyMAG (bioMérieux, Geneva, Switzerland) in 100 μL elution volume.…”