Real-Time PCR Method for the Rapid Detection and Quantification of Pathogenic Staphylococcus Species Based on Novel Molecular Target Genes

Abstract

Coagulase-positive Staphylococcus aureus is a foodborne pathogen considered one of the causes of food-related disease outbreaks. Like S. aureus, Staphylococcus capitis, Staphylococcus caprae, and S. epidermidis are opportunistic pathogens causing clinical infections and food contamination. The objective of our study was to develop a rapid, accurate, and monitoring technique to detect four Staphylococcus species in food. Four novel molecular targets (GntR family transcriptional regulator for S. aureus, phosphomannomutase for S. epidermidis, FAD-dependent urate hydroxylase for S. capitis, and Gram-positive signal peptide protein for S. caprae) were mined based on pan-genome analysis. Primers targeting molecular target genes showed 100% specificity for 100 non-target reference strains. The detection limit in pure cultures and artificially contaminated food samples was 102 colony-forming unit/mL for S. aureus, S. capitis, S. caprae, and S. epidermidis. Moreover, real-time polymerase chain reaction successfully detected strains isolated from various food matrices. Thus, our method allows an accurate and rapid monitoring of Staphylococcus species and may help control staphylococcal contamination of food.

Rapid identification of Staphylococcus aureus based on a fluorescence imaging/detection platform that combines loop mediated isothermal amplification assay and the smartphone-based system

“…S1 ). This sensitivity analysis showed that qPCR was more sensitive than LAMP at detecting S. aureus DNA and CFU as a template, which is consistent with published results 30 . However, in terms of reaction time, LAMP reactions are very fast (< 60 min) as no thermocycling steps and specialized equipment are necessary; and results can be obtained in as little as 15 min 31 .…”

“…Nevertheless, LAMP is less sensitive than nested PCR and qPCR 29 . To compare the sensitivity of LAMP versus qPCR for detecting S. aureus , we reproduced the assay reported by Kim et al 30 with genomic DNA from S. aureus . We were able to detect 1 pg of DNA and calculated a limit of detection (LOD) of 1.3 pg with qPCR.…”

Antibiofilm Effect of Silver Nanoparticles in Changing the Biofilm-Related Gene Expression of Staphylococcus epidermidis

“…The primer sequences are shown below in Table 1 . PCR amplification was performed according to the manufacturer’s protocol: pre-incubation occurred at 98 °C for 30 s, followed by 40 amplification cycles with denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s and final melting from 65 °C to 95 °C with an increase of 0.5 °C every 5 s [ 37 ].…”

Establishment of a real‐time fluorescence and visual colorimetric detection method for Staphylococcus aureus based on loop‐mediated isothermal amplification

“…The conventional culture‐based detection method is regarded as the gold standard for the detection of S. aureus , which mainly includes pre‐enrichment, selective growth, plate isolation, biochemical testing, and serotype identification. However, these technologies are labor‐intensive, time‐consuming, and not appropriate for on‐site analysis (Kim et al, 2021). Nucleic acid detection technology overcomes these constraints and has been applied to the detection of many pathogenic microorganisms.…”