A Simple Gold Nanoprobe Assay for the Identification of Staphylococcus Aureus, Listeria Monocytogenes and Salmonella Enteritidis in Food Specimens

Abstract

In the present study, we developed a gold nanoprobe assay, which relies on the colorimetric differentiation of specific DNA sequences, based on different aggregation profiles. We evaluated the assay on DNA extracted from pathogen cultures and from contaminated food specimens. The targets used were the femA gene for the identification of Staphylococcus aureus, the hly gene of the Listeria monocytogenes listeriolysin and the invA sequence for Salmonella spp. Comparison was performed with the reference assay, as described in the respective ISO guidelines for each pathogen, and a direct PCR amplification and detection. The minimum detection limit of the assay was defined at 123 fg/μL DNA, for all species, lower than PCR detection. Specificity was 100%. Sensitivity was 100%, as compared το the reference method, whereas the sensitivity of PCR was 93.3%. The evaluated assay could be used as a sensitive screening method, which does not require major infrastructure, for the detection and identification of pathogens in food specimens. In addition, it can accommodate detection of multiple species, thus allowing multiplexing and expedite turnaround time.

Sensitive and selective colorimetric detection of Staphylococcus aureus-SPA gene by engineered gold nanosensor

“…Protein A, found in all strains of S. aureus, is the most studied cell surface protein in Gram-positive bacteria encoded by the SPA gene, which can be used as a genetic marker to detect S. aureus by PCR ( Kim et al, 2012 ) . Our results showed successful identification of the SPA gene with a molecular size previously reported in the literature ( Houhoula et al 2017 ) .…”

“…In the current study, a single probe was designed for SPA gene detection with sequence (5′SH-GCCTAACTTGAACGAAGAAC3′). In contrast, Houhoula et al (2017) used a probe with sequence 5′SH-CATTAGTGGAAAGATGGAATG3′. However, our designed probe seemed to have a higher affinity to the target gene, as revealed by the stability of Au-Ns after the addition of concentrated NaCl.…”

“…Indeed, AuNPs are easy to synthesize, simple to modify, and highly biocompatible ( Yeh et al, 2012 ). Moreover, AuNPs have a high affinity to the thiol group, so they could be easily functionalized with thiolated oligonucleotides without amplifying this targeted DNA ( Baptista et al, 2011 , Houhoula et al, 2017 ) . Depending on this phenomenon, AuNPs-based sensors have been extensively used for detecting specific DNA sequences.…”

“…For detecting S. aureus colorimetrically, AuNPs have been reported to conjugate Aptamer ( Zhu et al, 2021 , Raji et al, 2021 ) ; however, these methods suffer from low-temperature storage conditions and relatively high cost. On the other hand, ( Houhoula et al, 2017 , Shahbazi et al, 2018 ) described utilizing AuNPs-single DNA probe with DL of 123 fg/μL and 8.73 ng/ μL, respectively. The current study aimed to synthesize a novel Au-Ns for detecting S. aureus by a colorimetric method using three components: target DNA, Au-Ns, and sodium chloride solution.…”

“… Detection method Microorganisms of interest Detection limit Reference Colorimetric Salmonella enterica 37 fM ( Kalidasan et al, 2013 ) Staphylococcus aureus 500 ng amplicon. ( Chan et al, 2014 ) Pseudomonas aeruginosa 9.899 ng/μL ( Amini et al, 2018 ) Staphylococcus aureus, Listeria monocytogenes, Salmonella Enteritidis 123 fg/μL ( Houhoula et al, 2017 ) Staphylococcus aureus 8.73 ng/μL ( Shahbazi et al, 2018 ) Shigella spp. 8.14 ng/μL ( Elahi et al, 2019 ) Fluorescence Vibrio cholera O1 2.34 pg/μL ( Narmani et al, 2018 ) Electrochemical Staphylococcus aureus – ( Spain et al, 2011 ) …”