Improved Detection of Herpesviruses from Diluted Vitreous Specimens Using Hydrogel Particles

Abstract

Infectious uveitis is a sight-threatening infection commonly caused by herpesviruses. Vitreous humor is often collected for molecular confirmation of the causative agent during vitrectomy and mixed in large volumes of buffered saline, diluting the pathogen load. Here, we explore affinity-capture hydrogel particles (Nanotrap^®) to concentrate low abundant herpesviruses from diluted vitreous. Simulated samples were prepared using porcine vitreous spiked with HSV-1, HSV-2, VZV and CMV at 10⁵ copies/mL. Pure undiluted samples were used to test capturing capability of three custom Nanotrap particles (red, white and blue) in a vitreous matrix. We found that all particles demonstrated affinity to the herpesviruses, with the Red Particles having both good capture capability and ease of handling for all herpesviruses. To mimic diluted vitrectomy specimens, simulated-infected vitreous were then serially diluted in 7 mL TE buffer. Diluted samples were subjected to an enrichment protocol using the Nanotrap Red particles. Sensitivity of pathogen detection by qPCR in diluted vitreous increased anywhere between 2.3 to 26.5 times compared to non-enriched specimens. This resulted in a 10-fold increase in the limit of detection for HSV-1, HSV-2 and VZV. These data demonstrated that Nanotrap particles can capture and concentrate HSV-1, HSV-2, VZV and CMV in a vitreous matrix.

Keywords: PCR; herpesviruses; nanoparticles; uveitis.

Figure 1

Creating simulated vitreous samples. The serial dilution process to prepare samples to be either enriched with Nanotrap Red particles or not enriched (Created with BioRender.com).

Figure 2

Binding of herpesviruses to the Nanotrap Red particles (Created with BioRender.com).

Figure 3

A visual protocol demonstrating the use of Nanotrap Red particles to enrich the samples and the processes of DNA extraction and PCR that followed (Created with BioRender.com).

Figure 4

A comparison of the three particle types tested (Nanotrap Red, Nanotrap White, and Nanotrap Blue) against the four herpesviruses tested in this study.

Figure 4

A comparison of the three particle types tested (Nanotrap Red, Nanotrap White, and Nanotrap Blue) against the four herpesviruses tested in this study.

Figure 5

A comparison of samples enriched with Nanotrap Red particles versus those untreated in terms of copy number per mL.