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Image Search Results
Journal: Frontiers in Veterinary Science
Article Title: Rapid Detection and Typing of Actinobacillus pleuropneumoniae Serovars Directly From Clinical Samples: Combining FTA ® Card Technology With Multiplex PCR
doi: 10.3389/fvets.2021.728660
Figure Lengend Snippet: Optimization of FTA ® card processing for direct PCR amplification. FTA ® card discs inoculated with reference strain 7213384-1 at OD 600 = 0.01 producing serovar 19-specific product are shown for different optimization conditions. (A) PCR amplification from FTA ® cards using different polymerases: A = HotStarTaq ® Plus Polymerase, B = HotStarTaq ® Polymerase, and C = DreamTaq Polymerase. (B) The number of washes in “deionized water” required to achieve amplification: 0–4 = no wash to 4 washes, respectively. (C) Length of incubation with deionized water per wash: 1 = 5 min, 2 = 10 min, and 3 = 15 min. (D) Altering the volume of water used to wash in a single step for 5 min: 1 = 100 μl, 2 = 200 μl, 3 = 300 μl, 4 = 400 μl, and 5 = 500 μl. (E) Various PCR reaction volumes: 1 = 30 μl, 2 = 40 μl, and 3 = 50 μl. (F) Limit of detection is shown for amplification of the serovar 19-specific product from discs inoculated with strain 7213384-1. Lanes labelled 1–5 are for OD 600 = 0.01 and four subsequent 10-fold dilutions, respectively, of bacterial culture on discs prepared either with the Whatman FTA ® protocol (left hand lanes) or our developed wash protocol (right hand lanes). For all gels, lane M = 100 bp Plus DNA Ladder (GeneRuler, Thermo Fisher Scientific).
Article Snippet: For optimization of FTA ® card processing conditions, single-plex PCR reactions were prepared with either a PCR kit containing
Techniques: Amplification, Incubation