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(PDF) Detection of Influenza Virus Types A and B and Type A Subtypes (H1, H3, and H5) by Multiplex Polymerase Chain Reaction | Apiradee Theamboonlers - Academia.edu
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RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus negative specimens. Furthermore, the assays could detect and subtype up to 105 dilution of each of the reference viruses that had an original infectivity titer of 106 EID50/ml. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes...</p><div class="ds-related-work--ctas"><button class="ds2-5-text-link ds2-5-text-link--inline js-swp-download-button" data-signup-modal="{"location":"wsj-grid-card-download-pdf-modal","work_title":"Development of multiplex rt-PCR assays for rapid detection and subtyping of influenza type A viruses from clinical specimens","attachmentId":91016025,"attachmentType":"pdf","work_url":"https://www.academia.edu/86601677/Development_of_multiplex_rt_PCR_assays_for_rapid_detection_and_subtyping_of_influenza_type_A_viruses_from_clinical_specimens","alternativeTracking":true}"><span class="material-symbols-outlined" style="font-size: 18px" translate="no">download</span><span class="ds2-5-text-link__content">Download free PDF</span></button><a class="ds2-5-text-link ds2-5-text-link--inline js-wsj-grid-card-view-pdf" href="https://www.academia.edu/86601677/Development_of_multiplex_rt_PCR_assays_for_rapid_detection_and_subtyping_of_influenza_type_A_viruses_from_clinical_specimens"><span class="ds2-5-text-link__content">View PDF</span><span class="material-symbols-outlined" style="font-size: 18px" translate="no">chevron_right</span></a></div></div><div class="ds-related-work--container js-wsj-grid-card" data-collection-position="6" data-entity-id="43086916" data-sort-order="default"><a class="ds-related-work--title js-wsj-grid-card-title ds2-5-body-md ds2-5-body-link" href="https://www.academia.edu/43086916/Identification_of_genetic_markers_of_the_genetic_variability_of_avian_influenza_a_subtypes_h1n1_and_h7n9_Determination_of_primers_for_the_diagnostic_method_lamp_method_">Identification of genetic markers of the genetic variability of avian influenza a subtypes h1n1 and h7n9. Determination of primers for the diagnostic method (lamp method)</a><div class="ds-related-work--metadata"><a class="js-wsj-grid-card-author ds2-5-body-sm ds2-5-body-link" data-author-id="2263402" href="https://kharkov-ua.academia.edu/BuriachenkoSemen">Buriachenko Semen</a></div><p class="ds-related-work--metadata ds2-5-body-xs">Biopolym.cell , 2019</p><p class="ds-related-work--abstract ds2-5-body-sm">Background. HA, NA, and NP genes are potential candidates as markers of genetic variability in avian influenza virus in birds, animals, and humans. Aim. The purpose of this study was to extract the potential of the HA, NA, and NP genes as markers of the polymorphism of avian influenza A viruses in domestic animals and humans with respect to the subtype H7N9 birds. Methods. The study used the MEGA6 and VectorNTI-11 Smith-Waterman algorithm nucleotide sequences of the hemaglutinin (HA), neurominidase (NA) and nucleoprotein (NP) genes taken from the virus database. A common region at the 3’-end of the segments was used to select the reverse primer sequences for amplifying the HA-NA and NP genes. To select direct primers, the multiple alignment of all the HA, NA, and NP gene sequences deposited in the ISD database (www.flu.lanl.gov) was performed. Then, the consensus sequences were formed for the HA and NA-ta NP genes; the common conservative areas (motifs) and selected sequences of direct primers were found for the first and second stages of amplification in compliance with the general principles; the primers were chosen. The following algorithm was used to select oligonucleotide markers capable of specifically detecting of a specific subtype of AIV: all HA, NA and NP sequences in the database were divided into groups according to certain variants of hemagglutinin (H1 and H7), neuraminidase (N1 and N9) and nucleoprotein (NP). 40 resulting groups were divided into subgroups according to the origin of the virus (isolated from humans, birds, etc.), which formed a consensus sequence. Then, the most conservative motifs, limited by the primers of the second amplification stage, of the genomic segment, were determined. When it was impossible to identify a rather conservative motive, phylogenetic analysis of the sequences was performed within the subgroup with its division into smaller subgroups. Then a consensus sequence was formed and a search for conservative areas was conducted. Results. A total of 3500 sequences of hemagglutinin, neuraminidase, and AIV nucleoproein segments were analyzed. The variable structural regions of the nucleotide sequences of genes were determined. In all genes, single nucleotide substitutions are acquired, and the most polymorphic loci are located at the 3 ‘and 5’ ends of the sequences. The number of oligonucleotides selected to determine a specific variant of HA, NA and NP depended on the number of identified conservative regions in the amplified region and on the degree of variability within the region. Based on the analysis, the primers were selected (4 for each gene of two subtypes) for an isothermal loop amplification reaction of viral genes, the set was constructed consisting of 24 discriminating oligonucleotides for specific analysis of AIV subtypes. Conclusions. The results of the analysis showed that the identification of RNA mutations and the most variable gene loci could be used for the genotyping of influenza A viruses.</p><div class="ds-related-work--ctas"><button class="ds2-5-text-link ds2-5-text-link--inline js-swp-download-button" data-signup-modal="{"location":"wsj-grid-card-download-pdf-modal","work_title":"Identification of genetic markers of the genetic variability of avian influenza a subtypes h1n1 and h7n9. 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