08 fg μL−1 DNA. These DNA samples were then used as templates for the nested PCR. A 5-μL aliquot of the PCR products was separated electrophoretically in a 2% agarose gel (Sigma, Milan, Italy) stained with ethidium bromide (0.5 μg mL−1) in 0.5 × TBE buffer (0.045 M Tris-borate; 0.001 M EDTA, pH and compared with a Molecular Weight Marker Palbociclib cell line (Sigma). Amplicons obtained from 90-day samples (S90) and 60-day samples (S60) were purified using
the QIAquick PCR Purification Kit (Qiagen), dried and sent to the MWG sequencing centre (Eurofins MWG GmbH, Martinsried, Germany) for sequencing. The samples of freshly collected intestinal content of trout at 90 days were positive for the presence of segmented filamentous bacteria (SFB, C. arthromitus) under microscopic examination (Fig. 1). Filaments containing endospores were clearly visible in phase-contrast microscopy under × 1000 magnification. This result allowed us to consider these selleck chemicals llc samples as positive reference samples. The primer pair CAF–CAR showed specificity for C. arthromitus as no PCR products were obtained when the DNA from microorganisms reported in Table 1 were used, representing indigenous microbial communities of freshwater fish as a template in the PCR assay. The expected PCR products of 515 bp were obtained for the samples at 90 days, as reported in Fig. 2. They indicated the presence of C. arthromitus in the fish intestinal content either in the initial ileum
tract or in the final ileum tract. This result confirmed the presence of the microorganism obtained by microscopic examination. No PCR products were obtained for control samples
and for the samples at 30 and 60 days. The sensitivity tests results obtained by nested PCR were in agreement with the first PCR protocol, applied using CAF and CAR primers for all the S90 samples showing the presence of C. arthromitus and confirming the positive results obtained before. The expected amplicon of 270 bp shown in Fig. 3 for the 90-day samples was also obtained for 16 out of 18 60-day samples using the nested PCR. The samples were positive for the presence of C. arthromitus, showing the importance of a method able to decrease the detection limit in the presence of heterogeneous DNA as the template. The control samples (SC) and S30 samples were also negative after Cetuximab nested PCR, as summarized in Table 2. The sensitivity tests obtained by nested PCR are reported in Fig. 4. PCR products were obtained when 80 ng μL−1 to 0.08 pg μL−1 DNA were used as the template, whereas no amplicons were produced when 8–0.08 fg μL−1 DNA were used as the template. This can be considered a good result because the medium DNA content of a single prokaryote cell is 5.5–10 fg. The results suggest that the method can detect a single cell of the microorganism tested: C. arthromitus. In fact, nested PCR allowed for the detection of C. arthromitus in asymptomatic trout at 60 days of growth.