Point-of-Care Extraction-Free Sample Preparation for Recombinase Polymerase Amplification of Nucleic Acid Targets in...

Introduction: Recombinase polymerase amplification (RPA) amplifies nucleic acid targets quickly and with low-complexity instrumentation. Simple sample preparation is needed for RPA testing at the point of care (POC). We describe and evaluate sample preparation workflows that do not require nucleic acid extraction for detecting β-globin DNA via RPA in swabs and blood. Methods: Amplification of targets present in crude sample lysate is suitable for the POC, but inhibitors can pose challenges. We determined the tolerance of RPA to various chemical lytic agents and sample matrices. We compared sample preparation methods determining the relative limit of detection as a function of pooled swab and comparing the performance across the dynamic range of white blood cell (WBC) counts. We selected preferred workflows balancing performance, time, and the number of liquid handling steps required. Finally, we evaluated sample-to-answer performance of an optimized assay to detect β-globin DNA with samples from normal volunteers. Results: The RPA assay tolerates 1 μL of a buccal swab or 0.1 μL of total reaction volume blood. When using pooled representative buccal swab or blood samples, multiple lysis strategies had similar performance. However, performance differences were observed with different concentrations of samples. One-step sodium hydroxide lysis was selected for buccal swabs and blood because consistent performance was achieved with a range of cellular materials, minimal time, and low process complexity. The integrated sample-to-answer assay demonstrated consistent target amplification across all normal volunteer samples. Conclusions: The method to develop a POC extraction-free sample preparation workflow for a sample-to-answer assay for β-globin enabled robust amplification. This method could be employed to maximize the extraction-free POC sample preparation performance for varied isothermal nucleic acid amplification tests.