Improvement of HRV Quantification Using cRNA-Based Standards for Real Time RT-PCR

Abstract

Real Time RT-PCR developed in recent years represents an useful tool in the diagnosis of RNA viruses. In order to accurately quantify and normalize a RNA target, efficiency of reverse-transcription must be considered. In this study a cRNA-standard based quantitative Real Time RT-PCR have been developed for HRV quantification on bronchoalveolar lavage (BAL) specimens. Results has been compared to a quantitative plasmid standard-based Real Time RT-PCR previously developed by us.Large amount of pHRV was linearized and purified. Blunt ends were generated and cRNA production was carried out. Dilutions of cRNA were generated and dynamic range, intraand inter-test variability, sensitivity and limit of detection were evaluated. Sixty-seven BAL, previously resulted positive to our plasmid standard-based method, were evaluated using cRNA-standard quantification. cRNA curve showed a broad dynamic range with a good intra-and inter-test variability, with an average of 3.23 threshold cycles more in comparison to plasmid standard-based curve. In terms of specimen quantification, a difference of 1.07log was found, showing a significant underrate using plasmid standard-based quantification.The method for cRNA-standard construction seems more suitable for quantification of RNA viruses, in order to normalize the quantification in reverse-transcription.

Standardized One-Step Real-Time Reverse Transcription-PCR Assay for Universal Detection and Quantification of Hepatitis Delta Virus from Clinical Samples in the Presence of a Heterologous Internal-Control RNA

“…1. We found a 1-log difference when plasmid standards were used instead of RNA standards (data not shown), a difference also described by Terlizzi et al (16). Indeed, the use of in vitro-synthesized RNA standards provides the control for both the RT and PCR steps and is thus more reliable than other methods using DNA-based standards.…”

“…LNA nucleotides, chosen to be directed to highly (7,9,10,11,14). Plasmid-based standard curves usually underrate RNA samples (16). In vitro-transcribed RNAs (HDV-1, -5, -6, -7, and -8) were thus serially diluted in QuantiTect nucleic acid dilution buffer (Qiagen, Courtaboeuf, France) and used as standards.…”

Development of a Tetraplex qPCR for the Molecular Identification and Quantification of Human Enteric Viruses, NoV and HAV, in Fish Samples

“…The existing literature suggests that in the assessment of a qPCR assay several factors should be considered, namely, the effect of using different templates to generate standard curves for the absolute quantification of RNA viruses, which can be made from either a plasmid, a synthetic oligonucleotide, or an in vitro transcribed RNA [11]. The advantage of using in vitro transcribed RNA as a template is that it implicates cDNA synthesis, and the efficiency of the reverse transcription reaction can be considered [11,12]. On the other hand, it involves in vitro RNA and cDNA synthesis steps, which are time consuming and expensive [11,12].…”

Can We Predict Sustained Virologic Response to Interferon α Therapy in Patients With Chronic Hepatitis Delta Virus Infection?

“…There is no robust commercially available assay for HDV RNA detection 14 . The use of a plasmid-standard curve in PCR assays, as it was the case in the HIDIT-1 study 15 , is a recognized cause of decreased sensitivity [16][17][18] . Thus, some samples with very low serum HDV RNA levels in the HIDIT-1 study might have been considered falsely undetectable.…”