HPV Genotype Detection Using Hybrid Capture Sample Preparation Combined with Whole Genome Amplification and Multiplex Detection with Luminex XMAP Brian.

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HPV Genotype Detection Using Hybrid Capture Sample Preparation Combined with Whole Genome Amplification and Multiplex Detection with Luminex XMAP Brian Lowe, Lori Kobayashi, Attila Lorincz, Rick Mallonee, Dominic O'Neil, Ha Thai, Irina Nazarenko The Journal of Molecular Diagnostics Volume 12, Issue 6, Pages 847-853 (November 2010) DOI: 10.2353/jmoldx.2010.100045 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic diagram of the HC-WGA-LMX assay. 1: Hybrid Capture enrichment shows a double-deleted RNA (dotted line) single-stranded target DNA (solid line) hybrid captured by a hybrid-specific antibody (Y-shape) bound to a paramagnetic bead (shaded circle). 2: Isothermal, whole-genome, strand displacement amplification by ϕ-29 shows the DNA template (long black line) with single strands being polymerized (gray lines with arrowheads) at random priming sites (primers are short black lines) and displacing complementary amplicon strands. The arrowheads indicate the polymerization direction. 3: Hybrid Capture detection shows a double-deleted RNA probe (dotted line)-DNA amplicon (solid line) hybrid captured onto a detection oligonucleotide (short black line) of a LMX bead. Two detection oligonucleotide probes in separate genomic regions are labeled a and b. There is one LMX bead set for each HPV type, each with a distinct color and a distinct pair of oligonucleotide capture probes. The hybrid is decorated with hybrid capture antibodies (Y shapes) conjugated with phycoerythrin (PE), which acts as the reporter in the LMX assay (see text). The Journal of Molecular Diagnostics 2010 12, 847-853DOI: (10.2353/jmoldx.2010.100045) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Plot of the average (n = 4) HC-WGA-LMX assay signal (median fluorescence intensity, MFI) for various HPV 16 inputs (clinical HPV DNA diluted in clinical-negative specimen pool, dotted line; HPV plasmids diluted in STM with 5 μg herring DNA, dashed line). Error bars show SD of signal. The no target (0 copies) control is less than 200 MFI. The Journal of Molecular Diagnostics 2010 12, 847-853DOI: (10.2353/jmoldx.2010.100045) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Plots of the HC-WGA-LMX assay signal (average MFI, n = 2) for various structures of HPV 16 DNA targets; HPV 16 plasmid DNA linearized with SalI (gray bars); circular HPV 16 plasmid DNA (cross-hatched bars); and integrated (genomic) HPV 16 DNA from CasKi cells (dark gray bars). Targets were diluted in STM. Error bars indicate SD. The Journal of Molecular Diagnostics 2010 12, 847-853DOI: (10.2353/jmoldx.2010.100045) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Assay signal-to-noise (S/N, average, n = 3) was unequal for each of the 19 HPV plasmid types (500 copies, A; 1,000,000 copies, B). Each HPV plasmid was assayed separately by the multiplex HC-WGA-LMX (19 types) assay. Background signal of controls with no targets was low. The Journal of Molecular Diagnostics 2010 12, 847-853DOI: (10.2353/jmoldx.2010.100045) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Suppression of the HC-WGA-LMX assay (A) and the gp-PCR assay (B) with variable amounts of HPV 16 and HPV 56. The traces represent the assay S/N (y axis; n = 3 replicates) for increasing amounts of HPV 16 plasmid copies (square, 10; triangle, 100; solid star, 1000; open star, 10000; x-shape, 1000,000) amplified with various HPV 56 copies (x axis). The Journal of Molecular Diagnostics 2010 12, 847-853DOI: (10.2353/jmoldx.2010.100045) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 6 Analytical limit-of-detection of the HC-WGA-LMX assay is approximately 2 logs lower than for the HC2 screening assay. The signal for a serial dilution (n = 2 replicates) of HPV 16–positive signal is shown for two assays; dark bars, HC2 assay; light bars, HC-WGA-LMX assay. The Journal of Molecular Diagnostics 2010 12, 847-853DOI: (10.2353/jmoldx.2010.100045) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions