Specific detection of hepatitis C virus in clinical samples using a novel simplification strategy
Abstract number: P994
Boulter N., Siah S.P., Vockler C.J., Coulston N.A., Warton K., Rajasekariah P., Melki J.R., Millar D.S.
Objective: We have shown previously that DNA simplification is a novel procedure that is able to specifically detect all high-risk strains of the Human Papilloma Virus in a single reaction. We wished to determine if it was possible to use a modified simplification strategy to detect RNA encoding viruses and used Hepatitis C virus (HCV) as a model organism.
Background: HCV is a major cause of chronic hepatitis, cirrhosis and liver cancer on a global scale. One of the most sensitive methods for the detection of HCV is reverse transcription PCR (RT-PCR), which in addition to improved sensitivity compared to serological methods, can also be used to monitor disease activity in response to anti-viral drugs. We aimed to produce a simplification-based RT-PCR assay for HCV RNA that was as sensitive and specific as current viral-load monitoring assays.
Methods: Treatment of DNA with sodium bisulfite results in the conversion of cytosine residues to uracil, which are subsequently amplified as thymine. Although such methods have been applied to the detection of DNA viruses such as HPV, the treatment of RNA with sodium bisulfite results in total degradation of the RNA and thus was of no clinical utility. We have developed a novel method for the simplification of RNA using sodium bisulfite, which eliminates RNA degradation and shows similar sensitivity and specificity to conventional approaches.
Results: Baseline sensitivity tests were conducted using serial dilutions of HCV (Acrometrix cat# 960203) diluted in normal human serum and purified using the Qiagen QiAmp UltraSens viral kit. Positive signals were generated from as little as 15 IU/mL of virus, indicating negligible RNA degradation. In addition, a number of commercially available panels were obtained including; BBI Diagnostics Genotype Panel (PHW202), Worldwide HCV panel (WWHV301), HCV RNA genotype performance panel (PHW202) and HCV RNA linearity panel (PHW804), all of which produced results indicating that the HGS simplification strategy performed as well as conventional RT-PCR. Furthermore, sequencing of amplicons derived from different genotypes demonstrated that the method could be used for genotyping individual HCV strains. Finally the method was assessed on a blinded panel of clinical samples the results of which shall be discussed in more detail.
Conclusion: RNA simplification is a novel approach for the reliable detection and genotyping of RNA containing pathogens such as HCV or HIV.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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