Impact of an unusual disulfide transformation on detection of isothermal amplification products

Abstract

Detection of infectious pathogens such as HIV-1, HPV, and SARS-CoV-2 from biospecimens is critical to healthcare. Particularly sensitive and specific diagnostic techniques to accomplish this include molecular amplification and detection tests of nucleic acids from pathogens. Such tests are comprised of reagent compositions to facilitate hybridization of primers and probes that are complementary to specifically amplified sequences of the analyte target. One of these reagents from an isothermal molecular assay occasionally changed its physical appearance over time, generating interest into the cause of the transformation and suitability of the reagent in diagnostic testing. A preliminary hypothesis was that the 2,2′-dithiodipyridine component was the pre-chromophoric compound of its distinctly yellow reduced form, 2-thiopyridine. However, under oxidizing conditions, 2-thiopyridine is a minor constituent of hybridization reagents and not a major contributor to the yellow colour. Instead, a new yellow compound was isolated from coloured hybridization reagent, identified as 1-(2′-pyridyl)-2-thiopyridone and determined to be the result of an intramolecular cyclic rearrangement and sulfur extrusion from 2,2′-dithiodipyridine under acidic and oxidizing conditions. Neither the appearance of 1-(2′-pyridyl)-2-thiopyridone, nor the concomitant depletion of 2,2′-dithiodipyridine reduced the sensitivity or specificity of in vitro diagnostic screening assay results for detecting amplified nucleic acids from viral pathogens, ensuring the safety of tested blood transfusion products.