Silica-coated magnetic particles for efficient RNA extraction for SARS-CoV-2 detection

Abstract

Molecular diagnostic methods to detect and quantify viral RNA in clinical samples rely on the purification of the genetic material prior to reverse transcription polymerase chain reaction (qRT-PCR). Due to the large number of samples processed in clinical laboratories, automation has become a necessity in order to increase method processivity and maximize throughput per unit of time. An attractive option for isolating viral RNA is based on the magnetic solid phase separation procedure (MSPS) using magnetic microparticles. This method offers the advantage over other alternative methods of making it possible to automate the process. In this study, we report the results of the MSPS method based on magnetic microparticles obtained by a simple synthesis process, to purify RNA from oro- and nasopharyngeal swab samples of patients suspected of COVID-19 provided by three diagnostic laboratories located in the Buenos Aires Province, Argentina. Magnetite nanoparticles of Fe₃O₄ (MNPs) were synthesized by the coprecipitation method and then coated with silica (SiO₂) produced by hydrolysis of tetraethyl orthosilicate (TEOS). After preliminary tests on samples from the A549 human lung cell line and swabs, an extraction protocol was developed. The quantity and purity of the RNA obtained were determined by gel electrophoresis, spectrophotometry, and qRT-PCR. Tests on samples from naso- and oropharyngeal swabs were performed in order to validate the method for RNA purification in high-throughput SARS-CoV-2 diagnosis by qRT-PCR. The method was compared to the spin columns method and the automated method using commercial magnetic particles. The results show that the method developed is efficient for RNA extraction from nasal and oropharyngeal swab samples, and also comparable to other extraction methods in terms of sensitivity for SARS-CoV-2 detection. Of note, this procedure and reagents developed locally were intended to overcome the shortage of imported diagnostic supplies as the sudden spread of COVID-19 required unexpected quantities of nucleic acid isolation and diagnostic kits worldwide.

Keywords: Functionalized magnetic beads; MBs based protocol; Magnetic separation; Ribonucleic acid isolation; SARS-CoV-2 detection.

Graphical abstract

Fig. 1

(a) MNPs@SiO₂ 3720 SEM micrograph shows the formation of irregular clusters with micrometric size. (b) A single MB composed of several MNPs. (c–d) HAADF-STEM images of a single MB. (e) Si and Fe EDS elemental mapping reflecting the SiO₂ coverage on the MNPs.

Fig. 2

(a) Microscope photography taken to calculate size distributions of MNPs@SiO2. Ellipses were drawn manually with ImageJ software. (b) Histogram of particle size distribution obtained from the ellipses data. The number of particles analyzed was 102.

Fig. 3

(a) Magnetization curves for bare MNPs (black and red circles) and silica-coated MNPs@SiO₂ (green and blue dots). MNPs exhibit similar saturation magnetization values while in coated particles the magnetization decreases with increasing silica percentage. (b) Evolution of saturation magnetization as a function of silica percentage. (c) Magnetic force per unit weight (dimensionless) versus silica percentage. (d) Z potential a function of silica percentage, the dash-dotted line is only to guide the eye.

Fig. 4

(a) Schematic representation of RNA extraction protocol by the MB–UNLP method. The numbers below correspond to the extraction steps, which are described in detail in Supplementary file 1. (b) Quantification of RNA samples by Nanodrop™ and UV absorption spectra (typical spectra are presented) to determine the absorbance ratios at 260 and 280 nm (A₂₆₀/A₂₈₀ ratio). (c) Lines 1, 2, and 4 correspond to RNA extractions using three batches of MBs; lane 3 is the 1 kb ladder (Guangzhou Dongsheng Biotech, R.P. China), and lane 5 corresponds to RNA extraction using the GeneAid kit column method. The original gel is provided in “Supplementary Information”. (d) Comparative qRT-PCR results of RNA extraction and amplification curves of the human β–actin gene in RNA samples purified either with different MBs batches or Viral Nucleic Acid Extraction Kit column method (GeneAid Biotech, Taiwan). The horizontal line represents the fixed threshold chosen for the analysis.

Fig. 5

Scatter plot of the SARS-CoV-2 detection by qRT-PCR and comparison among commercial RNA extraction methods and MB–UNLP methodology. Cts obtained by the alternative commercial method are represented on the horizontal axis, and MB–UNLP Cts are represented on the vertical axis. For each panel, the upper left quadrant indicates D by both methods, the upper right quadrant indicates ND by both methods; the bottom left quadrant indicates D by a commercial method and ND by MB–UNLP, and the bottom right quadrant indicates D by MB–UNLP and ND by the commercial method. The full red line represents positions of identical Ct for both methods; the red dashed line represents the Ct limit established by each laboratory. (a) and (b) Rossi Hospital Ct results for the internal control RNase P and ORF1ab gene (N = 30); (c) and (d) Public Health Laboratory Ct results for the internal control β-actin and ORF1ab gene (N = 46, * indicates negative control); (e) and (f) Evita Hospital Ct results for the internal control RNase P (N = 24) and the ORF1ab gene (N = 46).