Specific quantitative detection of and in co-infection and mixed biofilms.

Respiratory infections seriously affect the swine industry worldwide. Co-infections of two vital pathogenic bacteria () and (), colonizing the respiratory tract often occurs in veterinary clinical practice. Moreover, our previous research found that and can form biofilm . The formation of a mixed biofilm not only causes persistent infections, but also increases the multiple drug resistance of bacteria, which brings difficulties to disease prevention and control. However, the methods for detecting and in co-infection and biofilm are immature. Therefore, in this study, primers and probes were designed based on the conservative sequence of gene and IVA gene. Then, a TaqMan duplex real-time PCR method for simultaneous detection of and was successfully established optimizing the reaction system and conditions. The specificity analysis results showed that this TaqMan real-time PCR method had strong specificity and high reliability. The sensitivity test results showed that the minimum detection concentration of and recombinant plasmid was 10 copies/μL, which is 100 times more sensitive than conventional PCR methods. The amplification efficiencies of and were 95.9% and 104.4% with R value greater than 0.995, respectively. The slopes of the calibration curves of absolute cell abundance of and were 1.02 and 1.09, respectively. The assays were applied to cultivated mixed biofilms and approximately 10 CFUs per biofilm were quantified when 10 CFUs planktonic bacteria of either or were added to biofilms. In summary, this study developed a TaqMan real-time PCR assay for specific, accurate quantification of or in mixed biofilms, which may help for the detection, prevention and control of diseases caused by a bacterial mixed infection involving and .