For SEPT9 we observed a 100% detection down to 25 pg of spiked methylated template (corresponding to 1 methylated copy in 500 to 2,000 unmethylated copies) (see Additional file 1, supplemental Figure 2A, supplemental Figure 2B and supplemental table 1).
In addition, barcoded, spiked-in synthetic templates were used to measure the degree of sequencing coverage and residual PCR bias.
We spiked defined amounts of methylated templates (6.25, 12.5, 25, 50 and 100 pg) into unmethylated background (50 ng) and performed primer extension reactions on 10 to 24 HM-PCR reactions for each 'dilution'dilution
Tumour samples from male patients in which no mutation was identified on initial screening were re-screened after spiking with DNA from a male wild-type control sample to a concentration of ∼20% of amplifiable templates in the spiked sample.
The Ab@e-RGO immunoassay template also shows good selectivity and practical applicability, with the spiked sweat and saliva samples of cortisol and lactate.
The results showed that reaction products were detectable from as little as 100 fg and 4.8 CFU of pure K. pneumoniae templates, and from approximately 480 CFU in 1 mL of spiked clinical samples.
A pure culture of strain and a Shigella spiked faecal sample served as positive controls whilst a PCR reaction mixture without bacterial DNA template and an unspiked faecal sample from a healthy individual were incorporated as negative controls.
All spiked-in controls had internal MseI sites at their ends, and for each pair, one template was methylated in vitro for protection against MSRE activity.
A pure culture of strain TH13/00 and a Shigella-spiked faecal sample served as a positive control, whilst a PCR reaction mixture without bacterial DNA template and an unspiked faecal sample from a healthy individual acted as a negative control.
A possible explanation of the lower amplification efficiency with LNA-spiked primers is that for short targets such as miRNAs the primers that are incorporated into the template during amplification will lead to a high proportion of LNA in the template that will decrease the efficiency of subsequent PCR cycles.
