Selected article for: "dna polymerase and second round PCR"
Author: Katherine E. Arden; Ristan M. Greer; Claire Y.T. Wang; Ian M. Mackay
Title: Genotypic diversity, circulation patterns, and co-detections among rhinoviruses in Queensland, 2001
  • Document date: 2018_5_30
    • ID: d0w40mkn_7
    Snippet: RV/EV-positive extracts were genotyped using a conventional nested VP4/VP2 RT-PCR assay incorporating previously described primers that include part of the 5' untranslated region (UTR), viral protein coding regions VP4 and partial VP2. [14] [15] [16] first-round RT-PCR used OneStep RT-PCR (QIAGEN, Australia) or SensiFAST Probe No-ROX One-Step (Bioline, Australia) kits and subsequent second-round PCR used Bioline MyTaq HS DNA polymerase kit. In ca.....
    Document: RV/EV-positive extracts were genotyped using a conventional nested VP4/VP2 RT-PCR assay incorporating previously described primers that include part of the 5' untranslated region (UTR), viral protein coding regions VP4 and partial VP2. [14] [15] [16] first-round RT-PCR used OneStep RT-PCR (QIAGEN, Australia) or SensiFAST Probe No-ROX One-Step (Bioline, Australia) kits and subsequent second-round PCR used Bioline MyTaq HS DNA polymerase kit. In cases of failure to amplify a VP4/VP2 amplicon (~540bp), part of the 5' untranslated region (5'UTR; ~400nt) was amplified. 16 Amplicons were sequenced (BigDye sequencing kit v3.1, Applied Biosystems Pty. Ltd.) and, after removal of the primer sequence (Geneious Pro v6) 17 , RV and EV sequences were submitted to GenBank (accession numbers KF499366-KF499501, KF688607-KF688723). Curated and detailed methods are available online. [18] [19] [20] A virus variant was described as an "untypeable RV" when a clean sequence could not be obtained from either genotyping assay, despite the extracts being repeatedly positive by RT-rtPCR assay. RV genotype was determined by best match using the . CC-BY-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/334334 doi: bioRxiv preprint Page 5 of 21 following algorithm: when a query sequence returned BLAST comparison with 97% sequence identity in the 5'UTR or 90% (for RV-B and RV-C) or 91% (for RV-A) in the VP4/VP2 region, with members assigned to a given type, our sequence was assigned as being a variant of that RV genotype.

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