Direct capture and amplification of nucleic acids using a universal, elution-free magnetic bead-based method for rapid pathogen detection in multiple types of biological samples

Abstract

Nucleic acid amplification tests (NAATs) have become an attractive approach for pathogen detection, and obtaining high-quality nucleic acid extracts from biological samples plays a critical role in ensuring accurate NAATs. In this work, we established an elution-free magnetic bead (MB)-based method by introducing polyethylene-polypropylene glycol (PEPPG) F68 in lysis buffer and using NaOH solution instead of alcohols as the washing buffer for rapid nucleic acid extraction from multiple types of biological samples, including nasopharyngeal swabs, serum, milk, and pork, which bypassed the nucleic acid elution step and allowed the nucleic acid/MB composite to be directly used as the template for amplification reactions. The entire extraction process was able to be completed in approximately 7 min. Even though the nucleic acid/MB composite could not be used for quantitative real-time PCR (qPCR) assays, this elution-free MB-based method significantly improved the sensitivity of the loop-mediated isothermal amplification (LAMP) assay. The sensitivity of the quantitative real-time LAMP (qLAMP) assays combined with this elution-free MB-based method showed an improvement of one to three orders of magnitude compared with qLAMP or qPCR assays combined with the traditional MB-based method. In addition to manual operation, like the traditional MB-based method, this universal, rapid, and facile nucleic acid extraction method also has potential for integration into automated robotic processing, making it particularly suitable for the establishment of an analysis platform for ultrafast and sensitive pathogen detection in various biological samples both in centralized laboratories and at remote sites.

Keywords: Elution-free; Magnetic beads; Nucleic acid amplification tests; Nucleic acid extraction; Pathogen detection; Polyethylene-polypropylene glycol F68.

Fig. 1

Optimization of the protocol for elution-free MB-based method. Effect of (a) the MB size, (b) the MB dosage, (c) the PEPPG F68 concentration in lysis buffer, (d) the incubation time for MBs and samples in lysis buffer, (e) the NaOH concentration in washing buffer, and (f) the sample volume applied for nucleic acid extraction on the efficiency of LAMP assays

Fig. 2

Application of the elution-free MB-based method on pathogen detection from multiple types of simulated samples. Fluorescence curves of the qLAMP assays for the nucleic acid products prepared by the elution-free MB-based method and the commercial kit from simulated (a) SARS-CoV-2-positive nasopharyngeal swabs, (b) S. aureus-infected serum, (c) E. coli O157:H7-contaminated milk, and (d) S. typhimurium-contaminated pork. NTC represents no template control

Fig. 3

Influence of PEPPG F68 in lysis buffer on the absorption capability of the Si-OH MBs to (a) protein and (b) nucleic acid content. PEPPG F68+ and PEPPG F68− represent lysis buffers with and without PEPPG F68, respectively. MBs+ and MBs− represent mixtures with and without MBs, respectively

Fig. 4

Specificity validation of the qLAMP assays combined with the elution-free MB-based method for pathogen detection in multiple types of biological samples. (a) SARS-CoV-2 pseudovirus detection in nasopharyngeal swabs spiked with SARS-CoV-2 pseudovirus, M. pneumoniae, influenza A (H1N1) virus, and influenza B (Victoria) virus. (b) S. aureus detection in serum spiked with S. aureus, E. coli O157:H7, V. parahaemolyticus, and P. aeruginosa. (c) E. coli O157:H7 detection in milk contaminated with E. coli O157:H7, nonpathogenic E. coli, S. aureus, and S. typhimurium. (d) S. typhimurium detection in pork contaminated with S. typhimurium, E. coli O157:H7, S. aureus, and V. parahaemolyticus. NTC represents no template control

Fig. 5

Sensitivity validation of (a–d) the fluorescence qLAMP assays combined with the elution-free MB-based method, (e–h) the fluorescence qLAMP assays combined with the commercial kit, and (i–l) the fluorescence qPCR assays combined with the commercial kit for pathogen detection in multiple types of biological samples. (a, e, and i) SARS-CoV-2 pseudovirus detection in nasopharyngeal swabs. (b, f, and j) S. aureus detection in serum. (c, g, and k) E. coli O157:H7 detection in milk. (d, h, and l) S. typhimurium detection in pork. NTC represents no template control

Fig. 6

Sensitivity validation of the colorimetric LAMP assays combined with the elution-free MB-based method and the commercial kit for pathogen detection in multiple types of biological samples. (a) SARS-CoV-2 pseudovirus detection in nasopharyngeal swabs. (b) S. aureus detection in serum. (c) E. coli O157:H7 detection in milk. (d) S. typhimurium detection in pork. EF and CK represent the elution-free MB-based method and commercial kit, respectively. NTC represents no template control