A miniaturized and integrated gel post platform for multiparameter PCR detection of herpes simplex viruses from raw genital swabs

Abstract

Herpes simplex virus (HSV) is one of the most prevalent viruses, with acute and recurrent infections in humans. The current gold standard for the diagnosis of HSV is viral culture which takes 2-14 days and has low sensitivity. In contrast, DNA amplification by polymerase chain reaction (PCR) can be performed within 1-2 h. We here describe a multiparameter PCR assay to simultaneously detect HSV-1 and HSV-2 DNA templates, together with integrated positive and negative controls, with product detection by melting curve analysis (MCA), in an array of semi-solid polyacrylamide gel posts. Each gel post is 0.67 mL in volume, and polymerized with all the components required for PCR. Both PCR and MCA can currently be performed in one hour and 20 min. Unprocessed genital swabs collected in universal transport medium were directly added to the reagents before or after polymerization, diffusing from atop the gel posts. The gel post platform detects HSV templates in as little as 2.5 nL of raw sample. In this study, 45 genital swab specimens were tested blindly as a preliminary validation of this platform. The concordance of PCR on gel posts with conventional PCR was 91%. The primer sequestration method introduced here (wherein different primers are placed in different sets of posts) enables the simultaneous detection of multiple pathogens for the same sample, together with positive and negative controls, on a single chip. This platform accepts unprocessed samples and is readily adaptable to detection of multiple different pathogens or biomarkers for point-of-care diagnostics.

An enclosed in-gel PCR amplification cassette with multi-target, multi-sample detection for platform molecular diagnostics

“…16 Previously, we reported in-gel PCR in 670-860 nL of cylindrical or conical hydrogels (gel posts). 17,18 HSV was detected with raw genital swabs in an open array of freshly made gel posts. 18 We performed a study of 45 clinical samples that were blinded to the operator and obtained a concordance of 91% with the gold standard diagnostic and confirmed the reliability of gel-based DNA amplification in individual reaction units configured as an array.…”

“…17,18 HSV was detected with raw genital swabs in an open array of freshly made gel posts. 18 We performed a study of 45 clinical samples that were blinded to the operator and obtained a concordance of 91% with the gold standard diagnostic and confirmed the reliability of gel-based DNA amplification in individual reaction units configured as an array. However, gel posts cannot be manufactured and must be enclosed if they are to be used in the clinic.…”

“…Applying pressure to seal the perimeter of the chamber is widely used where a lid makes a tight seal or a pressured finger is used to pinch the channels. Another way is to add mineral oil to immerse the reagents as described for the gel post system [17][18][19] and also by Reboud et al 20 The use of wax to enclose capillaries provides a vapour barrier that avoids the need for complex pressure-based sealing techniques and bypasses the need for fluid agents in a packaged reaction cassette. After PCR is complete, the wax solidifies again to enclose the used capillaries for safe disposal.…”

Advances in Directly Amplifying Nucleic Acids from Complex Samples

“…Rodriguez et al used a similar approach in detecting clinical levels of H1N1 by passing an in-house elution buffer with the sample of interest through a filter paper-based setup, then amplifying on the filter membrane [205]. Future developments could focus on direct reactions with swabs that integrate extraction, Typically, elution either at room temperature [51, 176,[182][183][184][185][186] or with heating [43,44, [187][188][189][190][191] is sufficient to generate PCR-amplifiable template from swabs in solution. These methods of dilution or heating can save over thirty minutes of processing time, as with Nihonyanagi et al's heating protocol to release MRSA in CellEaseII (Biocosm Inc., Hyogo, Japan) diluents [169].…”

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…Another example of PCR amplification within acrylamide gel pads (fabricated using soft lithography) with "pseudo immobilized" PCR reagents was demonstrated by Manage et al [83]. The authors performed amplification and melting curve analysis in free standing gel posts (pillar like gel elements with a volume of 0.67 µL) containing all components of the PCR reaction including specific primers for Herpes Simplex Virus (HSV) genotype discrimination and an intercalating dye for fluorescent detection of amplification products.…”