Evaluation of Four DNA Extraction Protocols forBrucella abortusDetection by PCR in Tissues from Experimentally Infected Cows with the 2308 Strain

Abstract

This study compared 4 protocols for DNA extraction from homogenates of 6 different organs of cows infected with the Brucella abortus 2308 strain. The extraction protocols compared were as follows: GT (guanidine isothiocyanate lysis), Boom (GT lysis with the carrying suspension diatomaceous earth), PK (proteinase K lysis), and Santos (lysis by boiling and freezing with liquid nitrogen). Positive and negative gold standard reference groups were generated by classical bacteriological methods. All samples were processed with the 4 DNA extraction protocols and amplified with the B4 and B5 primers. The number of positive samples in the placental cotyledons was higher than that in the other organs. The cumulated results showed that the Santos protocol was more sensitive than the Boom (p=0.003) and GT (p=0.0506) methods and was similar to the PK method (p=0.2969). All of the DNA extraction protocols resulted in false-negative results for PCR. In conclusion, despite the disadvantages of classical bacteriological methods, the best approach for direct diagnosis of B. abortus in organs of infected cows includes the isolation associated with PCR of DNA extracted from the cotyledon by the Santos or PK methods.

Molecular, serological, pathological, immunohistochemical and microbiological investigation ofBrucellaspp. in marine mammals of Brazil reveals new cetacean hosts

“…Samples from 129 animals were analysed, consisting of manually homogenized frozen tissues from 123 marine mammals and formalin‐fixed, paraffin‐embedded (FFPE) tissue sections from six cases (without available frozen tissue). DNA was extracted from the samples described previously using the DNeasy Blood & Tissue Kit (QIAGEN®, Valencia, CA, USA) and/or the CTAB phenol‐chloroform method (Table S1; Kamel, Helmy, & Hafez, ; Vejarano et al, ). DNA concentration and purity was determined via spectrophotometry with Epoch (Biotek®, Winooski, VE, USA).…”

Use of Chelex-100 for the molecular diagnosis of five animal pathogens

“…alcohol (PhChIA), 25: 24: 1 (Thermo Fisher Scientific, USA) was used for E. tenella, L. infantum, and B. melitensis. For the latter, 50 mg of tissue was placed in a 1.5 µl microtube with 750 µl of lysis buffer (20 mM Tris-HCl (Promega, USA), 5 mM EDTA pH 8 (Promega, USA), 400 mM NaCl, 1% SDS (Promega, USA), proteinase k 400 µg/ ml (Promega, USA) and incubation was carried out for 8 h at 55 °C (Vejarano et al 2013). E. tenella sporulated oocysts were broken with glass beads as mentioned above and following supernatant recovery from the disruption, it was mixed with 0.33 vol of 10% sodium dodecyl sulphate solution (Promega, USA) and incubated at 37 °C for 2 h in the presence of 100 µg /ml of proteinase K (Promega, USA) as described Blake et al (2003).…”