| MAPREC assay for quantitation of mutants in a recombinant flavivirus vaccine strain using near-infrared fluorescent dyes. | |
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MedLine Citation:
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PMID: 21514323 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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Mutant analysis by PCR and restriction enzyme cleavage (MAPREC) is a quantitative assay of revertants in batches of live viral vaccines. The assay is highly sensitive and reliable but requires radioactive isotopes, which complicates its use in quality control laboratories. To quantify mutants in the cDNA of the West Nile (WN)/Dengue 4 chimera that was proposed as a new candidate of live vaccine against West Nile disease, alternative MAPREC protocols using non-radioactive dyes were explored. To compare the utility of different fluorescent dyes for MAPREC, the G(2337)→C mutation that was revealed by microarray hybridization in WN/Dengue 4 chimera virus was used as a model. DNA fragments produced by restriction endonuclease digestion were visualized in polyacrylamide gels by visible-range fluorescent dyes including ethidium bromide (EtBr) and SYBR Green I as well as by near-infrared (NIR) dye SYTO 60 and NIR dyes 700 and 800. The MAPREC assay performed with SYTO 60 and SYBR Green I was more sensitive than with EtBr but less sensitive than with NIR dyes 700 or 800. The NIR dyes 700 and 800 exhibited a wide linear range that may enable the detection of 0.05% of mutants in viral stocks. The NIR-based MAPREC assay was validated by using World Health Organization (WHO) international references for poliovirus type 3 with known contents of mutants. Values of mutant content produced by the non-radioactive assay were similar to the values determined in a previous WHO international collaborative study. The modified MAPREC assay could be used as an alternative to the radioisotope-based standard protocol for quality control of live viral vaccines. |
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Authors:
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Bella Bidzhieva; Majid Laassri; Konstantin Chumakov |
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Publication Detail:
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Type: JOURNAL ARTICLE Date: 2011-4-14 |
Journal Detail:
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Title: Journal of virological methods Volume: - ISSN: 1879-0984 ISO Abbreviation: - Publication Date: 2011 Apr |
Date Detail:
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Created Date: 2011-4-25 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 8005839 Medline TA: J Virol Methods Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Copyright Information:
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Copyright © 2011. Published by Elsevier B.V. |
Affiliation:
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Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, Rockville, MD 20852, USA. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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