Results

The following table summarises the total bacterial quantification from samples.

MBI.Sample.ID Your.Sample.ID copies.per.ul.of.DNA copies.per.ul.of.sample
S00OQ-0001 KL-1 12753.382 27634.51
S00OQ-0002 KL-2 14221.577 30815.86
S00OQ-0003 KL-3 12703.799 27527.08
S00OQ-0004 KL-4 5569.768 12068.79
S00OQ-0005 KL-5 24732.565 53591.47
S00OQ-0006 SI-1 145573.829 315434.93
S00OQ-0007 SI-2 113227.123 245344.86
S00OQ-0008 SI-3 82953.159 179746.07
S00OQ-0009 SI-4 59527.284 128985.99
S00OQ-0010 SI-5 88108.351 190916.54
S00OQ-0011 IL-1 101259.832 219413.67
S00OQ-0012 IL-2 145319.799 314884.49
S00OQ-0013 IL-3 119632.409 259224.07
S00OQ-0014 IL-4 65460.798 141842.96
S00OQ-0015 IL-5 58276.891 126276.59

Statistical analyses (NONE)

Visualization of the Results

Appendix



Quantitative PCR

Bacterial-specific (300 nM 27F, 5’-AGAGTTTGATCCTGGCTCAG-3’) forward primers coupled to (300 nM 519R, 5’-ATTACCGCGGCTGCTGG-3’) reverse primers were used to amplify Bacterial 16S rRNA. 20 μl SsoAdvanced Universal SYBR® Green Supermix (Bio‐Rad) were run on Applied Biosystems StepOne Plus instrument in duplicate.

Step Temperature Time Cycles
Initial Denaturation 95 oC 3:00
Denaturation 95 oC 0:15
Annealing 55 oC 0:30 40
Extension 72 oC 0:30

Standards

Full-length bacterial 16S rRNA gene was cloned into a pCR4-TOPO vector, with Kanomycin-Ampicillin resistance. The total plasmid fragment size is expected to be 5556 bp. An bacterial standard was prepared via. 10-fold serial dilutions, and the copies of 16S was determined by the following: Copy# = (DNA wt. x 6.02E23)/(Fragment Size x 660 x 1E9)

Linear regression was used to determine copy numbers of samples, based on CT of standards.

Melt Curve

Reaction specificity was assessed using a melt curve from 55 oC to 95 oC, held at 0.5 oC increment for 1s.