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      Current Developments and Challenges in Plant Viral Diagnostics: A Systematic Review


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          Plant viral diseases are the foremost threat to sustainable agriculture, leading to several billion dollars in losses every year. Many viruses infecting several crops have been described in the literature; however, new infectious viruses are emerging frequently through outbreaks. For the effective treatment and prevention of viral diseases, there is great demand for new techniques that can provide accurate identification on the causative agents. With the advancements in biochemical and molecular biology techniques, several diagnostic methods with improved sensitivity and specificity for the detection of prevalent and/or unknown plant viruses are being continuously developed. Currently, serological and nucleic acid methods are the most widely used for plant viral diagnosis. Nucleic acid-based techniques that amplify target DNA/RNA have been evolved with many variants. However, there is growing interest in developing techniques that can be based in real-time and thus facilitate in-field diagnosis. Next-generation sequencing (NGS)-based innovative methods have shown great potential to detect multiple viruses simultaneously; however, such techniques are in the preliminary stages in plant viral disease diagnostics. This review discusses the recent progress in the use of NGS-based techniques for the detection, diagnosis, and identification of plant viral diseases. New portable devices and technologies that could provide real-time analyses in a relatively short period of time are prime important for in-field diagnostics. Current development and application of such tools and techniques along with their potential limitations in plant virology are likewise discussed in detail.

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          Most cited references203

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          We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.
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            PacBio Sequencing and Its Applications

            Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing (SGS) technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation (SV) in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with diseases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Additionally, PacBio’s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone.
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              Metagenomics: genomic analysis of microbial communities.

              Uncultured microorganisms comprise the majority of the planet's biological diversity. Microorganisms represent two of the three domains of life and contain vast diversity that is the product of an estimated 3.8 billion years of evolution. In many environments, as many as 99% of the microorganisms cannot be cultured by standard techniques, and the uncultured fraction includes diverse organisms that are only distantly related to the cultured ones. Therefore, culture-independent methods are essential to understand the genetic diversity, population structure, and ecological roles of the majority of microorganisms. Metagenomics, or the culture-independent genomic analysis of an assemblage of microorganisms, has potential to answer fundamental questions in microbial ecology. This review describes progress toward understanding the biology of uncultured Bacteria, Archaea, and viruses through metagenomic analyses.

                Author and article information

                Role: Academic Editor
                05 March 2021
                March 2021
                : 13
                : 3
                [1 ]Department of Biotechnology, Mizoram University, Aizawl, Mizoram 796004, India; gtmehetre@ 123456gmail.com (G.T.M.); vincentvineethleo@ 123456gmail.com (V.V.L.)
                [2 ]Department of Botany, Pachhunga University College, Aizawl, Mizoram 796001, India; garima.singh106@ 123456gmail.com
                [3 ]Institute of Biochemistry and Genetics, Ufa Federal Research Center of the Russian Academy of Sciences, pr. Oktyabrya 71, 450054 Ufa, Russia; fourtyanns@ 123456googlemail.com (A.S.); igor.mak2011@ 123456yandex.ru (I.M.)
                [4 ]Department of Biotechnology, Pachhunga University College, Aizawl, Mizoram 796001, India; mukiyadav@ 123456gmail.com
                [5 ]Department of Forestry, Mizoram University, Aizawl, Mizoram 796004, India
                [6 ]Botany and Microbiology Department, College of Science, King Saud University, P.O. Box. 2460, Riyadh 11451, Saudi Arabia; habeer@ 123456ksu.edu.sa (A.H.); asmalsaleh@ 123456ksu.edu.sa (A.N.A.); dturki@ 123456gmail.com (T.M.D.); Kalmaary@ 123456ksu.edu.sa (K.S.A.)
                [7 ]Mycology and Plant Disease Survey Department, Plant Pathology Research Institute, ARC, Giza 12511, Egypt
                [8 ]Department of Agriculture and Environmental Sciences, National Institute of Food Technology Entrepreneurship & Management (NIFTEM), Industrial Estate, Kundli 131028, India
                Author notes
                [* ]Correspondence: kumzu70@ 123456gmail.com (K.U.); bhimpratap@ 123456gmail.com (B.P.S.); Tel.: +91-9436374242 (K.U.); +91-9436353807 (B.P.S.)
                © 2021 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).


                Microbiology & Virology
                plant viruses,diagnostics,next-generation sequencing,omics technologies,nanopore sequencing,quasi-species,in-field analysis


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