Automated extraction protocol for quantification of SARS-Coronavirus RNA in serum: an evaluation study

Rossa Wk Chiu,Yongjie Jin,Wing-Bong Lui,Ym Dennis Lo,Grace Ty Chung,Wilina Lim,Anthony Tc Chan

doi:10.1186/1471-2334-6-20

Abstract

BackgroundWe have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (SARS) based on the detection of the SARS-coronavirus RNA in serum by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). In this study, we evaluated the feasibility of automating the serum RNA extraction procedure in order to increase the throughput of the assay.MethodsAn automated nucleic acid extraction platform using the MagNA Pure LC instrument (Roche Diagnostics) was evaluated. We developed a modified protocol in compliance with the recommended biosafety guidelines from the World Health Organization based on the use of the MagNA Pure total nucleic acid large volume isolation kit for the extraction of SARS-coronavirus RNA. The modified protocol was compared with a column-based extraction kit (QIAamp viral RNA mini kit, Qiagen) for quantitative performance, analytical sensitivity and precision.ResultsThe newly developed automated protocol was shown to be free from carry-over contamination and have comparable performance with other standard protocols and kits designed for the MagNA Pure LC instrument. However, the automated method was found to be less sensitive, less precise and led to consistently lower serum SARS-coronavirus concentrations when compared with the column-based extraction method.ConclusionAs the diagnostic efficiency and prognostic value of the serum SARS-CoV RNA RT-PCR test is critically associated with the analytical sensitivity and quantitative performance contributed both by the RNA extraction and RT-PCR components of the test, we recommend the use of the column-based manual RNA extraction method.

Highlights

We have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (SARS) based on the detection of the SARS-coronavirus RNA in serum by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR)
We addressed the possibility of carry-over contamination within the MagNA Pure LC instrument by introducing nine aliquots of transport medium containing inactivated SARS-CoV ranging from 102 to 107 copies/mL alternating with aliquots of plain transport medium on the instrument and using the modified large volume protocol for extraction
When the sample contained 10 copies/mL SARS-CoV, the large volume kit yielded positive detection in four replicates, while viral RNA extractions from the total nucleic acid kit was only positive in one replicate

Summary

Introduction

We have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (SARS) based on the detection of the SARS-coronavirus RNA in serum by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The severe acute respiratory syndrome (SARS), etiologically related to a newly emerged coronavirus (SARS-CoV) [1], caused an epidemic in 2003 with reported cases in 29 countries around the world [2]. We have previously reported the development of a diagnostic test based on the detection of the SARS-CoV RNA in serum/plasma by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) [4,5]. Eighty percent of infected individuals were shown to be positive by the test on the first day of hospital admission with no false-positive results [4,5]. The approach has been demonstrated to be useful for serial monitoring of treatment efficacy [6]

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