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      Detection of melon necrotic spot virus by one-step reverse transcription loop-mediated isothermal amplification assay

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          Abstract

          Melon necrotic spot virus (MNSV) can cause significant economic losses due to decreased quality in cucurbit crops. The current study is the first to use reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of MNSV. A set of four LAMP primers was designed based on the coat protein gene sequence of MNSV, and a RT-LAMP reaction was successfully performed for 1 h at 62°C. The results of RT-LAMP showed high specificity for MNSV and no cross-reaction with other viruses. Compared to traditional reverse transcription-PCR (RT-PCR), the RT-LAMP assay was 10 3-fold more sensitive in detecting MNSV. Due to its sensitivity, speed and visual assessment, RT-LAMP is appropriate for detecting MNSV in the laboratory.

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          Most cited references38

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          Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.

          The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that uses only one type of enzyme. One of the characteristics of the LAMP method is its ability to synthesize extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced, yielding white precipitate of magnesium pyrophosphate in the reaction mixture. Judging the presence or absence of this white precipitate allows easy distinction of whether nucleic acid was amplified by the LAMP method. Since an increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized, real-time monitoring of the LAMP reaction was achieved by real-time measurement of turbidity. Copyright 2001 Academic Press.
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            Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue.

            Loop-mediated isothermal amplification (LAMP), a novel gene amplification method, enables the synthesis of larger amounts of both DNA and a visible byproduct--namely, magnesium pyrophosphate--without thermal cycling. A positive reaction is indicated by the turbidity of the reaction solution or the color change after adding an intercalating dye to the reaction solution, but the use of such dyes has certain limitations. Hydroxy naphthol blue (HNB), a metal indicator for calcium and a colorimetric reagent for alkaline earth metal ions, was used for a new colorimetric assay of the LAMP reaction. Preaddition of 120 microM HNB to the LAMP reaction solution did not inhibit amplification efficiency. A positive reaction is indicated by a color change from violet to sky blue. The LAMP reaction with HNB could also be carried out in a 96-well microplate, and the reaction could be measured at 650 nm with a microplate reader. The colorimetric LAMP method using HNB would be helpful for high-throughput DNA and RNA detection.
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              Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures.

              Staphylococcus aureus is the most important pathogen in nosocomial infections, including bloodstream infections. Prompt identification of S. aureus from blood cultures and detection of methicillin resistance are essential in cases of suspected sepsis. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63 degrees C) with high specificity, efficiency, and rapidity, was applied to detect methicillin-resistant S. aureus (MRSA) directly from positive blood culture bottles. MRSA-LAMP, which targets the spa gene, encoding S. aureus-specific protein A, and the mecA gene, encoding penicillin-binding protein-2' for methicillin resistance, could detect MRSA within 2 h after the blood culture signal became positive. The diagnostic values of LAMP, compared to a duplex real-time polymerase chain reaction (Drt-PCR) assay, were 92.3% and 96.2% sensitivity, 100% and 100% specificity, 100% and 100% positive predictive value (PPV), and 96.9% and 98.4% negative predictive value (NPV), respectively. These two methods had almost the same results, but the LAMP method is more cost-effective and provides excellent availability for rapid examination in a hospital clinical laboratory. Therefore, the LAMP assay appears to be a sensitive and reliable new method to diagnose MRSA bloodstream infection for appropriate antibiotic therapy.
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                Author and article information

                Contributors
                Role: Data curationRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draft
                Role: Data curationRole: InvestigationRole: MethodologyRole: ResourcesRole: Writing – original draft
                Role: MethodologyRole: Writing – original draft
                Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: Writing – review & editing
                Role: MethodologyRole: ResourcesRole: Visualization
                Role: MethodologyRole: Visualization
                Role: InvestigationRole: Methodology
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                5 March 2020
                2020
                : 15
                : 3
                Affiliations
                [1 ] College of Plant Protection, Shandong Agricultural University, Taian, Shandong, China
                [2 ] Facility Horticulture Laboratory of Universities in Shandong, Weifang University of Science and Technology, Shouguang, Shandong, China
                University of Georgia, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Article
                PONE-D-19-26004
                10.1371/journal.pone.0230023
                7058275
                32134962
                7f6a29e9-1bc9-43a6-bf2f-b2a96b679c45
                © 2020 Qiao et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 5, Tables: 1, Pages: 10
                Product
                Funding
                Funded by: Program for Applied Agricultural Technology Innovation of Shandong
                Award ID: SD2019ZZ004
                Award Recipient :
                Funded by: Shandong Provincial Natural Science Foundation, China
                Award ID: ZR2017CM058
                Award Recipient :
                Funded by: Shandong Key R&D Program (Public Welfare)
                Award ID: 2019GSF109118
                Award Recipient :
                Funded by: the Scientific Research Project of Facility Horticulture Laboratory of Universities in Shandong
                Award ID: 2018YY002
                Award Recipient :
                Funded by: Weifang Science and Technology Planed Project
                Award ID: No. 2019GX074
                Award Recipient :
                This research has been supported by the following grants: Program for Applied Agricultural Technology Innovation of Shandong (No. SD2019ZZ004), Shandong Provincial Natural Science Foundation, China (No. ZR2017CM058), the Science and Technology Development Planning Program of Weifang (No. 2017GX076), and the Scientific Research Project of Facility Horticulture Laboratory of Universities in Shandong (No. 2018YY002).
                Categories
                Research Article
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Reverse Transcriptase-Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Reverse Transcriptase-Polymerase Chain Reaction
                Biology and Life Sciences
                Organisms
                Eukaryota
                Plants
                Fruits
                Melons
                Biology and Life Sciences
                Plant Science
                Plant Anatomy
                Leaves
                Research and analysis methods
                Extraction techniques
                RNA extraction
                Research and Analysis Methods
                Electrophoretic Techniques
                Gel Electrophoresis
                Agarose Gel Electrophoresis
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Engineering and Technology
                Equipment
                Biology and Life Sciences
                Organisms
                Eukaryota
                Plants
                Vegetables
                Cucumber
                Biology and Life Sciences
                Organisms
                Eukaryota
                Plants
                Vines
                Cucumber
                Custom metadata
                All gene sequence files are available from the GenBank database (accession number EU016217, DQ922807, GU480022, KT923150, KY124137, AB044292, AB106106, AF488692, MK604924, KR094068).

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                Uncategorized

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