Nanoparticle-assisted pyrrolidonyl arylamidase assay for a culture-free Group A Streptococcus pyogenes detection with image analysis

Abstract

Existing techniques for the detection of Group A Streptococcus pyogenes (GAS) have drawbacks in rapidness, accuracy or in high-cost. Considering the clinical importance of GAS, we have developed a culture-free detection method based on pyrrolidonyl arylamidase (PYR) activity with the aid of magnetic gold nanoparticles (AuNPs). GAS is the reason for pharyngitis and sampling starts from the throat with cotton swabs. After swab sampling, the target was collected with antibody modified magnetic AuNPs and transferred into 500 μL of PYR-broth without any antigen extraction or pure colony isolation. Then, the assay was finished by adding 25 μL of 4-(dimethylamino)-cinnamaldehyde (DMACA) reagent after 4-h incubation. A red color formation was evaluated as the presence of GAS comparing to blank, however, image analysis was employed for the interpretation of color changes clearly. For this purpose, a formula related to image data was proposed and analytical validation parameters were defined. Thus, the correlation was found to be linear with the R² of 0.9685 between the log of bacteria concentration and the image data with the limit of detection of 3.3 × 10² CFU/mL of GAS. In addition, the assay worked efficiently in the abundance interference of Enterococcus faecalis. The results represent a new feature to nanoparticles eliminating the selective growth media for a bacteria and this study provided a detection with intact cells of bacteria without any antigen or DNA/RNA extraction. The proposed work has been the most similar to the gold standard but a faster method in this field.

Keywords: Culture-free detection; Group A Streptococcus; Image analysis; Magnetic nanoparticle; PYR; Pyrrolidonyl arylamidase.