A Novel Rna Virus Detection System Based On Duplex Specific Nuclease
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A Novel RNA Virus Detection System Based on Duplex Specific Nuclease
The Norwalk viral agent or Norovirus is the most prevalent cause for non-bacterial gastroenteritis worldwide [1-4] and is named for the northern Ohio town where it was first identified [5]. Every year 21 million cases of acute gastroenteritis are caused by norovirus infection, leading to 70,000 hospitalizations and 800 deaths, mainly young children and the elderly [6]. According to the Centers for Disease Control and Prevention (CDC), 11% of deaths in the United States from food-borne illnesses were caused by norovirus in 2011 and outbreaks frequently occur in semi-closed communities such as schools, hospitals, retirement homes and cruise ships [7, 8 9], producing the nickname "cruise ship virus". A particularly virulent Australian strain has emerged as a global threat in 2012, readily passing between individuals, making Norovirus detection of paramount interest [10-11]. Efforts to study the impact of norovirus infection have been hindered by the difficulty of controlled laboratory cultivation. In addition, norovirus culture techniques are fraught with the risk of exposure to the real virus. However, non-culture based techniques such as Reverse-Transcription Polymerase Chain Reaction (RT-PCR) have confirmed norovirus involvement in epidemics and sporadic outbreaks of gastroenteritis. While RT-PCR has proven useful for detection it requires the essential enzymatic step of reverse transcription which increases the experimental cost, time and complexity. The detection of norovirus is also hindered by factors like inappropriate sample storage (light exposure), low virus concentrations and ineffective viral RNA extraction methods [12], particularly for surface waters. As a result there remains a need for an improved Norovirus assay, one which is faster, more cost effective and more reliable in environmental waters. The main goal of this study was to develop a method which is not limited by the current drawbacks in detection of norovirus. An ideal system would enable quick and accurate detection without increase in cost. We have used the inherent specificity of the Duplex-Specific Nuclease (DSN) for cleaving single stranded DNA in a DNA-RNA duplex since norovirus contains a single stranded RNA genome. DSN, recently isolated from the Kamchatka (King) Crab [13], was combined with FRET (Forsters Resonance Energy Transfer) based DNA probes (TaqmanTM) that have been designed to target the most common norovirus RNA genogroups (I and II). The fundamental idea is to have the FRET probes bind to the viral RNA template to form a DNA: RNA duplex. DSN would then preferentially digest the DNA probe within the duplex, resulting in a fluorescent signal when the quencher and reporter dye are separated. As the RNA template usually endures this procedure, an amplified fluorescent signal will result proportional to the target, as multiple probe molecules are cleaved for individual RNA template. The total reaction time for this strategy is less than 30 minutes, compared to the 4-6 hours required for quantitative RT-PCR [14], and can be achieved on a simple heat block, rather than a more costly thermocycler. The real-world efficacy of our assay was tested using environmental water (Little Miami River) and the results demonstrate a detection limit of 10 norovirus copies per assay, below the minimum concentration required for infection (18 viral copies) [15-17]. Norovirus is included in the EPA Contaminant Candidate List 3 (CCL3) [18], which means that presently it is not subject to any proposed national drinking water regulations but may require regulation in future under the Safe Drinking Water Act (SDWA).
Detection and Management of New and Emerging Mystery Plant Virus Sources
Plant virus infections are estimated to cost billions of dollars in economic losses. Despite intensive efforts to manage and prevent plant viruses and their potential vectors in crop production processes, the threat posed by newly emerging or re-emerging viruses is becoming more common and poses potential threats even in previously uninfected areas. In this new volume, the editors review the progress made and the challenges ahead to address the hidden world of plant viruses, their impact on agriculture and food security, and the need for new detection systems and management strategies.
The Handbook of Plant Genome Mapping
While the complete sequencing of the genomes of model organisms such as a multitude of bacteria and archaea, the yeast Saccharomyces cerevisiae, the worm Caenorhabditis elegans, the fly Drosophila melanogaster, and the mouse and human genomes have received much public attention, the deciphering of plant genomeswas greatly lagging behind. Up to now, only two plant genomes, one of the model plant Arabidopsis thaliana and one of the crop species rice (Oryza sativa) have been sequenced, though a series of other crop genome sequencing projects are underway. Notwithstanding this public bias towards genomics of animals and humans, it is nevertheless of great importance for basic and applied sciences and industries in such diverse fields as agriculture, breeding in particular, evolutionary genetics, biotechnology, and food science to know the composition of crop plant genomes in detail. It is equally crucial for a deeper understanding of the molecular basis of biodiversity and synteny. The Handbook of Genome Mapping: Genetic and Physical Mapping is the first book on the market to cover these hot topics in considerable detail, and is set apart by its combination of genetic and physical mapping. Throughout, each chapter begins with an easy-to-read introduction, also making the book the first reference designed for non-specialists and newcomers, too. In addition to being an outstanding bench work reference, the book is an excellent textbook for learning and teaching genomics, in particular for courses on genome mapping. It also serves as an up-to-date guide for seasoned researchers involved in the genetic and physical mapping of genomes, especially plant genomes.