Rapid Detection of Staphylococcus aureus Genomic DNA Using Peptide Nucleic Acid and Gold Nanoparticles

Abstract

Staphylococcus aureus is a Gram-positive bacterium and is known to affect almost all mammalian species. This organism causes a multitude of cutaneous and systemic human diseases and is the main etiological agent in causing bovine mastitis causing heavy economic loss to dairy industry. Microbiological detection of S. aureus is time consuming and molecular diagnostic technique requires skilled personnel and costly equipment. Peptide nucleic acids (PNA) are DNA analogues with charged neutral polyamide backbone. Free PNA induces aggregation of citrate capped colloidal gold nanoparticles. Using this property of PNA, rapid, simple, and an easy method of detecting S. aureus genomics was developed in the present study. PNA concentration of 1 μM induced clear colour change of gold nanoparticles from brick red to blue colour. Visual detection limit of complementary target DNA inhibiting PNA induced agglomeration of gold nanoparticles was 0.25 μM whereas, spectral detection limit was even at lower concentration of 0.1 μM. PNA induced aggregation was gradually reduced with increasing concentration of non-complementary DNA and concentration of non-complementary DNA of 2.5 times more than PNA concentration was able to prevent the PNA induced aggregation of gold nanoparticles. Sensitivity detection limit of this colorimetric assay in detecting genomic DNA of S. aureus was 106 cells visually and 104 cells spectrophotometrically. The test was performed with 20 field isolates of staphylococcal species. Out of 20 isolates, 16 were identified as S. aureus specifically. This assay was robust and had the advantage of being simple, fast and sensitive.