Quantitative PCR of Fusobacteriota abundance
Siblings of the surface fish and Pachón cavefish used for microbiome
sequencing (SMR1, PMR1) were shipped from Harvard Medical School to
University of Nevada, Reno in March 2021 when they were 20 months old.
Fish were housed in 9L tanks with ten fish per tank under 6500K daylight
LEDs and fed ⅛ tsp of New Life Spectrum Thera+A Regular Pellet Enhanced
Non-Medicated Fish Food per day for four months prior to fecal
collections. To collect feces, individual fish were moved to 2L tanks
and fed five pellets of New Life Spectra Thera A+ per day. On the third
day of isolation, tanks were cleared of any debris, the fish were fed,
and the inlet water was turned off. Feces were collected from the tank
after 24 hours. The water inlet was turned on to recirculate fresh water
and the process was repeated over three days. Fecal samples were
centrifuged, excess water was removed, and they were stored at -80°C
until DNA extraction. DNA was extracted from the fecal samples using
Zymo Quick-DNA Fecal/Soil Microbe Microprep (D6012). DNA concentration
was quantified using an Invitrogen Qubit 4 Fluorometer with the 1X dsDNA
High Sensitivity assay kit (Q33230). Quantitative PCR was performed on a
BioRad CFX96 Touch Real-Time PCR machine using the following primers to
amplify 16s rRNA from Fusobacteriota (Fwd:
5’-GGATTTATTGGGCGTAAAGC-3’; Rev: 5’-GGCATTCCTACAAATATCTACGAA-3’). We
also included reactions to amplify 16s rRNA from all Eubacteriato normalize the results to the total amount of bacterial DNA (Fwd
5’-GGTGAATACGTTCCCGG-3’; Rev 5’- TACGGCTACCTTGTTACGACTT-3’). Each
reaction contained 1× iTaq™ Universal SYBR® Green Supermix, 100 nM each
primer, and 1.5ng of DNA. The cycle conditions were 95°C for 5 min,
followed by 40 cycles of 95°C for 5 s and 57°C for 30 s. Melt curve
analysis was performed at 65-95°C with 5°C increments 5 s/step.
Reactions without fecal DNA showed amplification of Eubacterial
sequences at high cycle number (>29) consistent with the
manufacturing of Taq DNA polymerase . Fusobacteriota was not
detected in samples without fecal DNA.