Abstract: Based on multicolor melting curve analysis, an efficient and low-cost simultaneous detection protocol of five mouse pathogens for the rapid detection of infection in experimental animals (Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Pasteurella pneumotropica,andProteus) was established. Meanwhile, the capsicum genome was added as an internal reference to exclude false negatives and make the results more accurate and reliable. By designing the probes, the melting peaks of different pathogens appeared at different Tm values in different fluorescence channels: in the FAM fluorescence channel, the melting peak Tm values ofS. aureusandK. pneumoniaewere in the range of （53.51±0.94） ℃ and （61.23±0.34） ℃, respectively; in the ROX channel, the melting peaks ofP. aeruginosaandP. pneumotropicawere in the range of （61.70±0.66） ℃, （55.98±0.10） ℃; in CY5 channel, the melting peak Tm values ofProteusandCapsicum annuumL. were at （63.91±0.19） ℃, （57.43±0.31） ℃, respectively. The method was verified to have different detection limits for different pathogens with 102copies forS. aureus, 101copies forK. pneumoniae, 102copies forP. aeruginosa, 102copies forP. pneumotropica, and 102copies forProteus. In summary, the multicolor melting curve method established in this work was capable of rapid and effective detection of a wide range of common mouse pathogens in a single tube.

Key words: multicolor melting curve analysis, multiple PCR, mouse pathogens, molecular detection, rapid detection