This study aimed to evaluate the threshold of detection of fluorescent multiplex PCR coupled with capillary electrophoresis for detection of infectious agents in semen samples from experimentally infected with decreasing concentrations of the bacteria Brucella abortus, Leptospira interrogans serovar pomona, Campylobacter fetus and Haemophilus somnus. Samples of bovine semen were experimentally infected with decreasing concentrations of bacteria obtained from serial dilutions in the base 10 so as to obtain samples containing a long time until 10-7 bacteria/mL from the initial concentration of Lepstospira pomona, Brucella abortus, Haemophilus somnus and Campylobacter fetus. The dilutions were made individually for each bacterium, as well as in different concentrations needed to standardize the multiplex PCR test. DNA extractions of all solutions containing sperm and bacteria analyzed in this study were performed according to protocol described by Heinemann et al. (2000). The multiplex PCR products were analyzed by electrophoresis on 8% polyacrylamide gel and capillary electrophoretic separation system for automated equipment in the analysis of DNA fragments MegaBACE. We observed amplification of fragments of 193pb, 330pb, 415pb and 400bp from the DNA of B. abortus, L. pomona, H. somnus, C. fetus respectively. The analysis by capillary electrophoresis of multiplex PCR products of DNA for simultaneous detection of the four pathogens was observed by detecting the dilution of 10-3 bacteria / mL times the initial concentration of the stock solution of each bacterium. The multiplex PCR coupled with capillary electrophoresis was first used for the direct diagnosis of four pathogenic bacteria in semen, proving to be a rapid method to detect bacteria that cause reproductive disorders.
Cattle; semen; bacteria; diagnosis; fluorescent PCR; capillary electrophoresis
