Evaluation of two DNA extraction techniques in the detection of Mycoplasma hyopneumoniae by PCR in lungs samples of pigs with and without pneumonia

Abstract

Swine Enzootic Pneumonia (PES) has as the primary etiologic agent the Mycoplasma hyopneumoniae. The diagnosis of PES involves clinical manifestations on farms, macroscopic lesions visualized in the slaughterhouse and laboratory techniques, such as isolation by culture, histopathology, immunohistochemistry, ELISA and other serological tests and PCR. This work aimed to establish the comparison between PCR results and typical macroscopic findings in lungs with PES and to evaluate the efficacy of PCR results with extraction by phenol-chloroform and commercial kit in fragments of swine lungs. The efficacy of PCR was evaluated by comparing the classic method of DNA extraction by phenol-chloroform and by commercial Kit. Fragments of lungs of 18 pigs with characteristic lesions of PES and 17 without lesions were subjected to DNA extraction and posterior PCR. The pulmonary lesions with PES were PCR-positive in 94.44%, compared to 76.47% of the non-PES lungs. Kappa concordance between macroscopic and PCR was 18%, which was not significant (P > 0.05).   Of the 35 lung fragments processed by phenol-chloroform, 85.71% were positive by PCR, compared to 28.57% by the commercial kit. Kappa agreement between DNA extraction methods was 57%, which was statistically significant (p <0.05), but Odds ratio was 15 (CI 4.52 to 49.68), which means that the probability of PCR positivity by the phenol-chloroform method was 15 times higher than that by the commercial kit. Macroscopy and PCR results were discordant, with phenol-chloroform extraction being the best between both procedures.