engleski

Comparison of antigen capture ELISA and PCR for detection of Mycoplasma bovis in milk samples

Mycoplasma bovis is a significant cause of bovine mastitis at large dairy farms in Croatia. In our laboratory, we routinely use antigen capture ELISA combined with culture on modified Hayflick agar for detection of M. bovis in milk, nasal swabs or lung tissue. It takes at least three days to detect M. bovis using this technique, and the presence of antimicrobials in samples may have a negative effect on the growth and sensitivity of the method. PCR based techniques have great sensitivity, are faster, and are not influenced by antimicrobial therapy. In this work we compared antigen capture ELISA and PCR combined with two different methods of DNA extraction to detect M. bovis in bulk tank or individual milk samples. The aim was to develop a cheap and quick method for DNA extraction from milk without using commercial kits or expensive chemicals for purification of DNA. Approximately 1000 milk samples were examined for the presence of M. bovis using antigen capture ELISA. A smaller subset consisting of ELISA-positive and ELISA-negative milk samples was further investigated using PCR. Extraction of DNA from milk samples was performed using Chelex-100 resin (Bio-Rad, USA) or proteinase K digestion. PCR was performed using primers specific for uvrC or 16S rRNA gene of M. bovis. Although the PCRs performed after Chelex or proteinase K based DNA extraction showed similar efficiency, antigen capture ELISA outperformed both methods when testing either bulk tank or individual milk samples. In addition, the sensitivity of PCR performed after the investigated DNA extraction methods was below 80%, rendering them unsuitable for detection of M. bovis, possibly because of the presence of PCR inhibitors due to the absence of purification step, or too low DNA content.

PCR; ELISA; M. bovis detection

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