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. 2015 Dec 8;5:17925. doi: 10.1038/srep17925

Figure 4. Detection specificity of primers and fluorogenic oligonucleotides.

Figure 4

Ability of the assay to detect specific targets was evaluated utilizing nucleic acid extracts of clinical specimens and multiplex master reaction mixture containing all the primer-sets in Table 1 and only one virus-specific fluorogenic oligonucleotide (LRp) per panel. (A) Electrophoretic results in all 4 panels show detection of the targets as demonstrated by virus-specific ladder-like banding-patterns. No banding pattern was observed in the controls, NTC, CMV, and PV. Distinguishing characteristics of the banding patterns were identified by the grouping/pattern-formation of the bands, size and number of the bands in each pattern, and spacing/distance between the patterns (also see Supplementary Figure S4 for target identification). (B) Intense fluorescence and higher RFU demonstrate that: (i) HIV-LRp specifically hybridized only to HIV RNA in Panel-1; (ii) HCV-LRp specifically hybridized only to HCV RNA in Panel-2; (iii) WNV-LRp specifically hybridized only to WNV RNA in Panel-3; and, (iv) HBV-LRp specifically hybridized only to HBV DNA in Panel-4. M = 100 bp marker; NTC = non-template control; CMV = cytomegalovirus; PV = parvovirus; Human Immunodeficiency virus (HIV); Hepatitis-B virus (HBV); Hepatitis-C virus (HCV); Hepatitis-E virus (HEV); Dengue virus (DENV); West Nile virus (WNV); RFU = Relative Fluorescence Units; UV = ultraviolet naked-eye visualization.