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      Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination

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          Abstract

          Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples.

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          Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases

          Yasuyoshi Mori, Tsugunori Notomi (2009)
          Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. This technology has been developed into commercially available detection kits for a variety of pathogens including bacteria and viruses. The current focus on LAMP methodology is as a diagnostic system to be employed in resource-limited laboratories in developing countries, where many fatal tropical diseases are endemic. The combination of LAMP and novel microfluidic technologies such as Lab-on-a-chip may facilitate the realization of genetic point-of-care testing systems to be used by both developed and developing countries in the near future. This review will describe the historical, current, and future developments of such technologies.
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            Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions.

            M Longo, M Berninger, J. L. Hartley (1990)
            Polymerase chain reactions (PCRs) synthesize abundant amplification products. Contamination of new PCRs with trace amounts of these products, called carry-over contamination, yields false positive results. Carry-over contamination from some previous PCR can be a significant problem, due both to the abundance of PCR products, and to the ideal structure of the contaminant material for re-amplification. We report that carry-over contamination can be controlled by the following two steps: (i) incorporating dUTP in all PCR products (by substituting dUPT for dTTP, or by incorporating uracil during synthesis of the oligodeoxyribonucleotide primers; and (ii) treating all subsequent fully preassembled starting reactions with uracil DNA glycosylase (UDG), followed by thermal inactivation of UDG. UDG cleaves the uracil base from the phosphodiester backbone of uracil-containing DNA, but has no effect on natural (i.e., thymine-containing) DNA. The resulting apyrimidinic sites block replication by DNA polymerases, and are very labile to acid/base hydrolysis. Because UDG does not react with dUTP, and is also inactivated by heat denaturation prior to the actual PCR, carry-over contamination of PCRs can be controlled effectively if the contaminants contain uracils in place of thymines.
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              Simultaneous elimination of carryover contamination and detection of DNA with uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP).

              Kuangwen Hsieh, Peter L Mage, Andrew Csordas … (2014)
              We report a one-pot, closed-vessel enzymatic assay that eliminates carryover contamination while preserving robust DNA amplification in loop-mediated isothermal amplification (LAMP), providing reliable and rapid detection of target DNA in contaminated samples.
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                Author and article information

                Journal
                Journal ID (nlm-ta): J Vet Sci
                Journal ID (iso-abbrev): J. Vet. Sci
                Journal ID (publisher-id): JVS
                Title: Journal of Veterinary Science
                Publisher: The Korean Society of Veterinary Science
                ISSN (Print): 1229-845X
                ISSN (Electronic): 1976-555X
                Publication date (Print): September 2016
                Publication date (Electronic): 20 September 2016
                Volume: 17
                Issue: 3
                Pages: 421-425
                Affiliations
                [1 ]College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University, Daegu 41566, Korea.
                [2 ]RAD Inc., Daegu 39852, Korea.
                [3 ]M Monitor Inc., Daegu 41914, Korea.
                [4 ]Virology Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Korea.
                [5 ]Avian Disease Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Korea.
                Author notes
                Corresponding author: Tel/Fax: +82-53-950-5973; parkck@ 123456knu.ac.kr
                Article
                DOI: 10.4142/jvs.2016.17.3.421
                PMC ID: 5037312
                PubMed ID: 26726027
                SO-VID: dafb29de-0e26-45c1-80d0-982d6e97f585
                Copyright © © 2016 The Korean Society of Veterinary Science.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 17 September 2015
                Date revision received : 07 December 2015
                Date accepted : 30 December 2015
                Funding
                Funded by: Ministry of Agriculture, Food and Rural Affairs, CrossRef http://dx.doi.org/10.13039/501100003624;
                Award ID: 313060-03-1-HD020
                Award ID: PJ009921
                Award ID: 311007-5
                Award ID: 009410
                Categories
                Subject: Short Communication

                ScienceOpen disciplines: Veterinary medicine
                Keywords: avian influenza virus,contamination,loop-mediated isothermal amplification,uracil-dna glycosylase
                Data availability:
                ScienceOpen disciplines: Veterinary medicine
                Keywords: avian influenza virus, contamination, loop-mediated isothermal amplification, uracil-dna glycosylase

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