实验动物,荧光定量PCR z-bo 2020-12-10 322

　　Objective To establish a fluorescent quantitative PCR method that can detect common mycoplasma in laboratory animals.

　　 Method Design a pair of primers and probes for the more conservative regions in the 16 s rRNA genes of a variety of large and mouse susceptible Mycoplasma, establish a fluorescent quantitative PCR method and test its specificity, sensitivity and repeatability. Results The method showed a good linear relationship between the template concentration of 5×100-5×106copies/μL (R²=0.9972) and the amplification efficiency was 98.11%; it has strong specificity and can distinguish and detect common Staphylococcus aureus Pathogenic bacteria; high sensitivity, the lower limit of detection is 5 copies/μL. The detection sensitivity is 100 times higher than that of conventional PCR; the repeatability is good, the coefficient of variation of the repeatability test within and between groups for different concentrations of templates is less than 2%. Fluorescence quantitative PCR and ordinary PCR methods were used to detect 120 clinical samples from different sources and types. The results showed that the positive rates of mycoplasma detection in mice were 3.33% (2/60) and 1.67% (1/60) respectively. The positive rate of detection was 5% (3/60).

　　 Conclusion The fluorescence quantitative PCR method established in this study is fast, specific, sensitive, and reproducible, and can be used for rapid diagnosis of common mycoplasma in laboratory animals.