Comparison of Simple RNA Extraction Methods for Molecular Diagnosis of Hepatitis C Virus in Plasma

Abstract

Nucleic acid extraction from biological samples is an important step for hepatitis C virus (HCV) diagnosis. However, such extractions are mostly based on silica-based column methodologies, which may limit their application for on-site diagnosis. A simple, rapid, and field-deployable method for RNA extraction is still needed. In this study, we evaluated the efficacy of four simple RNA extraction methods for the detection of HCV in plasma samples: a silica-membrane-based method, a magnetic-beads-based method, boiling with diethyl pyrocarbonate (DEPC)-treated distilled water, and using a commercial lysis buffer. HCV RNA was detected using both real-time reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Using real-time RT-PCR, extracted RNA from the silica-membrane-based and magnetic-beads-based methods had a 100% detection rate for RNA extraction from plasma. Using RT-LAMP, extracted RNA from the silica-membrane-based method showed a 66% detection rate, while the magnetic-beads-based method had a 62% detection rate. In summary, magnetic-beads-based extraction can be used as an alternative RNA extraction method for on-site HCV detection. Boiling with DEPC-treated distilled water was not appropriate for low HCV load samples, and boiling with a lysis buffer was not recommended.

A semi-automated and high-throughput approach for the detection of honey bee viruses in bee samples

“…Our data demonstrates that the sensitivity of DWV detection by both RT-qPCR and nanopore sequencing is comparable after total RNA extraction by NucleoSpin® Virus and NucleoMag™ Virus isolation kits. Both methods provided similar analytical results obtained from RT-qPCR showing that both DWV strain loads and C t values from honey bee pooled samples processed by affinity purification columns and magnetic beads-based technology method correlated well, which is in agreement with previous work [ 37 , 38 ]. Differences observed in the strength of amplification signal could be due to higher RNA yields and purity delivered by NucleoSpin®.…”

“…PCR remains one of the most favored and applied techniques for virus detection and disease diagnosis. It is routinely used for virus diagnostics in many research fields and is a standard for evaluating viral loads in biomedical research, agricultural and environmental sectors [33][34][35][36][37][38]. NGS technology is an additive and sometimes alternative method, capable of identifying unknown viruses in the sample, including variants and quasispecies [39][40][41], and is a reliable tool for validation of PCR results [32,39,40,41].…”

“…The magnetic beads-based technology has been widely used in biomedical research [33][34][35][36][37], and has become one of the most used methods to extract viral RNA for screening for SARS-CoV-2 [36], as rapid diagnosis of COVID-19 is essential to restrict its spreading. A same extraction method combined with an automated approach, which is rapid, high-throughput, uniform and bears low cross contamination risk [50][51][52] may be employed for honey bee research, in particular for virus screening surveys to screen multiple colonies in a limited timeframe.…”

Examination and comparison of the RNA extraction methods using mouse serum

“…This was consistent with the current results in terms of RNA yields and ease of use. Silica-based or magnetic beads-based was recommended for RNA extraction of hepatitis C virus in serum, as used in a recent study ( 21 ). The miRNeasy Serum/Plasma Advanced kit and mirVana™ PARIS™ RNA and Native Protein Purification Kit are silica-based kits, and TRIzol-LS is a guanidinium phenol-based kit.…”

Point-of-Care Testing for Hepatitis Viruses: A Growing Need

“…The performance of these methods has typically only been reported using nucleic acids that are purified from serum or plasma samples using commercial nucleic acid extraction kits that are poorly compatible with POC use due to time and equipment needs. In studies that did evaluate simpler nucleic acid extraction methods, heat treatment of plasma samples exhibited poor sensitivity, while magnetic-bead-based purification approaches looked promising [150,151]. Several devices to simplify the amplification and detection of HCV RNA using RT-LAMP have been developed.…”