Limited-resource preparable chitosan magnetic particles for extracting amplification-ready nucleic acid from complex biofluids.

Abstract

Extraction and concentration of pure nucleic acid from complex biofluids are the prerequisite for nucleic acid amplification test (NAAT) applications in pathogen detection, biowarfare prevention, and genetic diseases. However, conventional spin-column mediated nucleic acid extraction is constricted by the requirement for costly power-intensive centralized lab infrastructure, making it unsuitable for limited-resource settings. Significant progress in lab-on-a-chip devices or cartridges (e.g., Cepheid GeneXpert®) that integrate nucleic acid extraction and amplification has been made, but these approaches either require additional equipment or are costly. Similarly, their complexities make them difficult to fabricate in low-resource settings by the end-user themselves. The application of magnetic particles such as silica-coated iron oxide beads for nucleic acid extraction is relatively instrument-free, rapid, user-friendly, and amenable to automation. But, they rely on hazardous chaotropic salt chemistry and ethanol desalting that could limit their efficacy for downstream NAATs. Recent advances in several types of novel material (e.g., polyamine) coated magnetic bead-based chaotropic salt-free extraction methods offer a possible solution to this problem. However, these materials also involve multistep synthesis impermissible in limited-resource settings. To offer a possible instrument-free magnetic particle-based nucleic acid extraction doable at limited-resource settings, we investigated the nucleic acid capture ability of two chitosan-coated magnetic particles that are preparable by minimally trained personnel using only a water bath and a magnetic stirrer within 6-8 h. We quantitatively probed the efficiency of the passive (without any electrical shaking or vortex-aided) DNA magnetocapture (i.e., binding to chitosan magnetic particles, physical separation from its sample of origin, and release from the particles) using UV260. To explore their suitability towards clinically relevant sensitive downstream NAATs, 100-1000 copies (i.e., in the order of zeptomole) of Escherichia coli (E. coli) or human genomic DNA from aqueous solution, crude cell lysate, and fetal bovine serum were extracted by them and then successfully detected using quantitative real-time loop-mediated isothermal amplification (LAMP) or real-time polymerase chain reaction (PCR). Alongside, their suitability with gel-based LAMP, colorimetric LAMP, and in situ (on beads) LAMP was also probed. The required optimization of the amplification methods has been discussed. Overall, the turnaround time for the magnetocapture combined with NAAT was 1.5-2 h and is thus expected to aid in rapid clinical decision making. With the ease of preparation, reproducibility, and compatibility with downstream NAATs, we anticipate that these magnetic particles would facilitate the expansion and decentralization of nucleic acid-based diagnosis for limited-resource settings.