Comparison of quantitative real-time PCR targeting <i>nuc</i> gene and culture-based plate count methods for quantification of <i>Staphylococcus aureus</i> in raw cow milk

Abstract

Staphylococcal food poisoning (SFP) is caused by ingestion of enterotoxins produced by enterotoxigenic Staphylococcus aureus when the cell population exceeds 5 Log CFU per gram/ml of food. The Objectives of this study were to evaluate the performance of SYBR Green 1-based quantitative real-time PCR (qPCR) targeting the nuc gene for the quantification of S. aureus in milk and to compare the assay with the plate count method. The qPCR and the plate count were applied for the quantification of S. aureus in 92 naturally contaminated and artificially spiked bulk milk samples. Optimized standard curves were generated as the qPCR employed the absolute quantification method. The qPCR assay discriminates S. aureus from other Staphylococcus species with a large difference in quantification cycle (Cq) (Mean S. aureus Cq = 13.83± 0.93; other staphylococci Cq= 30.34 ± 2.65). The standard curve showed 91 % amplification efficiency and 0.98 coefficients of correlation (R2 ). The detection and quantification limit of the assay was 18 copies of the nuc gene. The precision of the assay as expressed by standard deviation was 0.12 – 0.3 for intra-assay and 0.29 – 0.5 for inter-assay variability. In artificially contaminated milk, the R2 between CFU ml-1 and S. aureus cell equivalent (SCE) ml-1 was 0.95, which implies, the estimation of CFU ml-1 in raw milk by qPCR is possible. A statistically significant (p< 0.05) difference in S. aureus count was documented between qPCR and plate count. The average SCE (5.59 ±1.55 Log SCE ml-1) estimated by qPCR was one Log higher than CFU (4.46 ± 1.06 Log CFU ml-1) estimated by plate count. Furthermore, 28% of the samples with < 5 Log ml-1 S. aureus by plate count had > 5 Log ml-1 by qPCR. Hence, the qPCR is recommended for routine and research work for its advantage of rapid, sensitivity, and reliability. Further study on validation of the qPCR protocol in different food matrixes for quantification of foodborne pathogens and cost-benefit analysis of the assay is required.