Rapid detection of Staphylococcus aureus by real-time PCR using high resolution melting curve analysis
A rapid real-time PCR method for detecting Staphylococcus aureus(SA) by high resolution melting curve analysis(HRM) was developed. A pair of specific primers was designed targeting the sa442 gene of SA. After optimizing the conditions, the specificity of the method was validated with 8 target SA strains and 8 non-target strains. The solutions with different copies of purified recombinant plasmids PMD18-sa442 were analyzed by the HRM real-time PCR method to evaluate its sensitivity. The reproducibility of the method was also evaluated. The method was also applied to detect artifi ciallyinoculated chicken samples added with 5, 10, 20 cfu/25 g Staphylococcus aureus. The results showed that:(1)The developed HRM real-time PCR method showed a good specificity by detecting only SA with the Tm value of(77.34±0.287) ℃ and was not affected by other normal food-borne pathogenic bacteria such as Salmonela, Listeria monocytogenes, E.coli O157, et al.(2)The sensitivity of this method was 2.00×102 copies/mL.(3)The developed protocol of HRM real-time PCR method showed a high reproducibility with the variations(CVs) of(0.76±0.52)% and(1.34±0.45)% inner and between tests, respectively.(4)The method showed a good performance in the detection of artifi cially-inoculated chicken samples. The established HRM real-time PCR assay exibited suffi cient specifi city, high sensitivity and good reproducibility. It is rapid, simple and cheap and would be useful in food microbiology laboratory for detecting SA.