Comparative Evaluation of Extraction Protocols for Point‐Of‐Need Molecular Diagnostics

ABSTRACT Background In the rapidly evolving field of molecular diagnostics, identifying a suitable extraction method is crucial in determining the applicability of the point‐of‐need device. Extraction, which serves as the initial and important step in the diagnostic process, plays a vital role in the accuracy, reliability, and speed of detecting pathogens. Extraction methods range from traditional approaches like organic extraction to modern advancements such as magnetic bead‐based separation, each offering unique advantages and limitations. Methods This study explored the comparative effectiveness of 10 commercially available protocols (denoted as I–X), focusing on their practicality and efficiency in point‐of‐need scenarios. By considering criteria such as ease of use, turnaround time, and robustness in handling different sample matrices, we aimed to highlight the critical factors that influence the selection of an appropriate extraction method for immediate and reliable diagnostic outcomes in diverse settings. The effectiveness of each protocol was evaluated by comparing the time threshold and fluorescence signal using isothermal amplification (namely reverse transcription‐recombinase‐aided amplification). For comparison, samples were also extracted with Qiagen spin column extraction. Results The performance of each protocol in extracting feline Coronavirus (FCoV) RNA differed depending on the sample type, such as nasal swab, serum, and feces. Overall, protocol VIII proved to be flexible and reliable for point‐of‐need diagnostics owing to its consistent extraction efficiency across different sample types and its excellent sensitivity (2 × 10 1 RNA copies/μL from supernatant and nasal swab and 2 × 10 2 RNA copies/μL from serum). Conclusion This study emphasizes the significance of considering the specific sample type and diagnostic goal in selecting the right extraction protocol.