Abstract
The molecular detection system has evolved over last two decades and is rapidly replacing the conventional confirmatory techniques in diagnostic virology. However the major limitation in implementation of available molecular detection assays is the non availability of field deployable nucleic acid isolation platform coupled with gene amplification technique. The rapid and early molecular detection is crucial for employing effective measure against many viral infections. The re-emergence of chikungunya virus (CHIKV) has led to epidemics since 2004 in several parts of the world including India. The main association of CHIKV with severe arthritis and long-lasting arthralgia and closely mimics symptoms of Dengue and Zika virus infection requiring laboratory confirmation. In this study, a simple magnetic bead based ribonucleic acid extraction method was optimized, which was coupled with isothermal polymerase spiral reaction (PSR) technique for early and rapid detection. Subsequently, the polymerase spiral reaction reagents were converted to dry down format that led to a rapid user friendly field compatible sample processing to answer method for rapid and onsite detection of Chikungunya virus. Both the methods were evaluated with a panel of clinical samples. The sensitivity of the assays were compared with available commercial viral RNA extraction platform and qRT-PCR. The in-house nucleic acid extraction system based on magnetic bead followed by dry down RT-Polymerase Spiral Reaction assay was found to be highly sensitive with 10 copies of RNA as limit of detection in CHIKV clinical specimens. With respect to other closely related viruses no cross reactivity was observed. This novel methodology has the potential to revolutionize the diagnosis of infectious agents in resource limited settings around the world.
Highlights
The molecular detection system has evolved over last two decades and is rapidly replacing the conventional confirmatory techniques in diagnostic virology
Initial experiments were performed with silica coated magnetic bead nucleic acid extraction protocol along with commercially available QIAamp viral RNA kit in order to study quality and quantity of RNA
The whole set up of in-house magnetic bead based nucleic acid extraction protocol was represented in Supplementary Fig. S1
Summary
The molecular detection system has evolved over last two decades and is rapidly replacing the conventional confirmatory techniques in diagnostic virology. A simple magnetic bead based ribonucleic acid extraction method was optimized, which was coupled with isothermal polymerase spiral reaction (PSR) technique for early and rapid detection. The in-house nucleic acid extraction system based on magnetic bead followed by dry down RT-Polymerase Spiral Reaction assay was found to be highly sensitive with 10 copies of RNA as limit of detection in CHIKV clinical specimens. Qiagen extraction + SYBR Green qRT-PCR (Ct value) 23 25.5 No Ct 22 No Ct acidic and neutral buffers for binding and elution of ribonucleic acid from clinical samples was attempted This method offers a rapid and field compatible nucleic acid extraction employing in-house ingredients, obviating the requirements of any high end equipments. The limitations of other isothermal assays viz., nucleic acid sequence-based amplification (NASBA) and helicase-dependent amplification (HDA) includes requirement of multiple enzymes to initiate a r eaction[6,7,8]
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