Molecular Investigation for Bacteria
Due to the finding of granulomas revealed by the histopathological
investigation, the presence of mycobacterial DNA was investigated in
frozen (pool made of liver, spleen, heart, and kidney), and in
formaldehyde‐fixed paraffin‐embedded (FFPE) samples. Frozen samples were
processed for DNA extraction using the Purelink Genomic DNA kit
(Invitrogen, USA) following the manufacturer’s instructions. FFPE
samples were processed for DNA extraction using the Purelink Genomic DNA
kit (Invitrogen, USA) following the manufacturer’s instructions with
minor modifications. Particularly, unstained sections, serial to
sections showing granulomas, were used for DNA extraction: 5–10 mg of
sliced FFPE tissue was placed in 1 mL of xylene (J.T. Baker) and a
pre-extraction step to remove paraffin from the sample was applied as
previously described (Sirri et al., 2018). Samples were deparaffinized
in xylene for 5 min; following centrifugation, samples were washed twice
in 100% Ethanol. The pellet was dried at 37 °C for 10 minutes, and DNA
extraction was subsequently undertaken using the aforementioned kit.
Bacterial presence was investigated through a PCR method targeting the
HSP65 gene using primers common to all mycobacteria (Telenti et al.,
1993). For the amplification, a reaction mixture was assembled
containing 1X PCR Buffer, 1 mM MgCl2, 200µM dNTP, 0.4 µM
of each primer, 2.5U of Taq polymerase and 5µl of DNA in a final volume
of 25 µl. The amplification cycle was conducted at 94 °C for one minute,
45 cycles consisting of denaturation at 94 ° C for one minute, annealing
at 60 °C for one minute, extension at 72 ° C for one minute and a final
extension step at 72 °C for 10 minutes. The PCR results were visualized
by electrophoresis on 1.5% agarose gel by running the samples with a
reference marker (100 bp ladder Invitrogen). PCR products of positive
samples were purified using the ExoSAP-IT PCR Product Cleanup Reagent
(Invitrogen, USA), and sequenced through the Bio‐Fab Sequencing Service
(Rome, Italy). The sequences were then manually corrected and subjected
to BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for
identification.