Fig. 3: Development of a PCR-based assay for Fusobacterium lineages L1 and L5 detection. | npj Biofilms and Microbiomes

Fig. 3: Development of a PCR-based assay for Fusobacterium lineages L1 and L5 detection.

From: Fusobacterium lineage profiling facilitates the clarification of the associations between non-nucleatum Fusobacterium and colorectal cancer

Fig. 3

A Amplification performance of the designed primers in Fusobacterium strains of different lineages. The primer sets selected for further analyses are marked in red. B The selected primer sets did not amplify DNA from human whole blood or Fusobacterium-free faecal or tissue samples. The presence of bacteria but the absence of Fusobacterium was confirmed by amplification with the primer sets V6-mix and fuso-rsub-F2/R2, respectively. F. nucleatum ATCC 25586 and F. varium THCT13E1 were used as positive controls. HC healthy control, CRC colorectal cancer. C qPCR with the selected primer sets in the corresponding L1 (F. nucleatum ATCC 25586, Fn) and L5 (F. varium THCT13E1, Fv) reference strains loaded with defined CFUs (colony-forming units). D qPCR with the selected primer sets in a given culture of Fn or Fv and its tenfold serial dilutions, with or without DNA from a Fusobacterium-free faeces/tissue sample and strains of other lineages. Fm, F. mortiferum THCT6B2. NC, negative control. For C, D the experiments were performed in triplicate, and the data are expressed as the means and standard deviations. Spearman correlation between qPCR- and FrpoB-seq-obtained L1 (E) or L5 (F) levels in clinical samples. Samples with L1 or L5 detected by FrpoB-seq were subjected to qPCR with L1_746-F/R or L5_1621-F/R, respectively. For (E), the faecal samples were from 50 CRC patients and 24 healthy controls, while the tissue samples included 45 tumour samples and 43 normal samples. For (F), the faecal samples were from 32 CRC patients and 28 healthy controls, while the tissue samples included 43 tumour samples and 39 normal samples.

Back to article page