AimsTo develop and evaluate cross-priming amplification (CPA) combined with immuno-blotting for the detection of coagulase-positive Staphylococci including Staphylococcus aureus. Methods and ResultsTwenty-four sets of cross and detection primers were designed according to four sequences of coagulase gene in Staph.aureus. The most specific primer pair was screened out for the next amplification and interaction. The specificity was evaluated in a total of 53 species of Staph.aureus and non-Staph.aureus. Two red lines indicating positive were always observed on the BioHelix Express strip for 12 subspecies of Staph.aureus. In contrast, only one signal line showing negative results was detected in all of non-Staph.aureus samples. The limit of detection (LOD) of CPA was 3627fg for the genomic DNA, which is about 100 and 10 times sensitive than those of PCR and loop-mediated isothermal amplification respectively. For the pure culture of Staph.aureus and milk powders, the LODs of CPA were about 134CFU per reaction and 5237CFU per 100g of milk powder respectively. The CPA method was also successfully applied to evaluate the contamination of Staph.aureus in 318 samples of daily food. ConclusionsCPA is a very sensitive and rapid method to detect Staph.aureus by simple laboratory instrument. Significance and Impact of the StudyIt is the first report on the application of the CPA with immuno-blotting for detection of coagulase-positive Staphylococci including Staph.aureus.