Critical analysis of rhinovirus RNA load quantification by real-time reverse transcription-PCR - PubMed (original) (raw)
Critical analysis of rhinovirus RNA load quantification by real-time reverse transcription-PCR
Manuel Schibler et al. J Clin Microbiol. 2012 Sep.
Abstract
Rhinoviruses 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.
Figures
Fig 1
Linearity and variability of the Panenterhino/Ge/08 two-step (A) and one-step (B) real-time RT-PCR on three 10-fold dilution series of _in vitro_-transcribed HRV-A16 RNA (A and B). The standard curves, slopes, and _r_2 values are shown.
Fig 2
One-step Panenterhino/Ge/08 HRV RNA quantification values compared to two-step Panenterhino/Ge/08 HRV RNA quantification values. A Bland-Altman plot shows the distribution of HRV RNA quantification values in log copies/ml, compared to the values obtained with the two-step assay. Two-step HRV quantification values are represented on the x axis. Differences in quantification values between the one-step and two-step assays in log copies/ml are represented on the y axis. The dashed line represents the mean difference of log copies/ml between both assays (two-step assay = one-step assay + 0.15 log), and the dotted lines represent the mean difference of log copies/ml between both assays + 2 SD (1.09) and − 2 SD (−0.79), respectively. Raw data are available in Table S2 in the supplemental material.
Fig 3
Experimental evaluation of HRV nucleic acid quantification variation linked to HRV genetic variability. The plot shows chimeric plasmid quantifications expressed in log copies/ml using the Panenterhino/Ge/08 assay (y axis) compared to the HRV-A16 3D values (x axis), using five 10-fold dilutions of chimeric plasmids, ranging from 4.30 to 8.30 copies/ml. Raw data are available in Table S4 in the supplemental material.
Fig 4
Putative sources of real-time RT-PCR viral load quantification variation. Box plots show the variation of viral load quantification (% CV) related to interassay variability (one-step and two-step assays), HRV genetic variability, HRV/HEV genetic variability, and one-step assay versus two-step assay variability.
References
- Garbino J, et al. 2004. Lower respiratory viral illnesses: improved diagnosis by molecular methods and clinical impact. Am. J. Respir. Crit. Care Med. 170:1197–1203 - PubMed
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