ObjectiveTo establish a simple, rapid, sensitive and accurate method for detection of Staphylococcus aureus (SA).MethodsAccording to SA thermostable nuclease nuc gene, a pair of universal primers and two specific probes were designed. 5′ end of the universal primers were biotinylated. The probes were fixed on the nitrocellulose,then the probes were hybridized with PCR products. ResultsSA DNA concentration of 2 ng/μL could be detected by reverse linear hybrid method. The specificity and accuracy were 100%.ConclusionThe reverse linear hybrid method has a high sensitivity and specificity. It can be used for rapid detection of SA.