Paired Cq values of tcdB and 16S rDNA genes

Published: 23-02-2019| Version 1 | DOI: 10.17632/brrbs5sgr6.1
Ivan Brukner


Paired Cq (cycle threshold) samples from tcdB positive samples (toxinogenic C difficile) reflecting total bacterial load and tcdB Cq value


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Sample processing was equivalent to the one described in BD GeneOhm TM C diff Assay Manual ( All oligonucleotides were synthesized by Integrated DNA Technologies (IDT) (Iowa, US): (5’TGCAGCCAAAGTTGTTGAAT3’) and (5’GCTCTTTGA TTGCTGCAC CT3’) tcdB tprimers, probe (/56-FAM/TCTGAAGGA/ZEN/TTACCTRTAATT GCAA/3IABkFQ/; QuantiNova Probe PCR kit (Cat No./ID: 208254, Qiagen, Canada); The full sequence of Inhibition Control Target was (5’TGCAGCCAAAGTTGTTGA ATGCAATGGTCCCAATGGCTAACGCGCAGAGCCTTCAGGTCAGAAATTTTTGCCATC CGAGACATCAGGTGCAGCAATCAAAGAGC3’), detected by hydrolysis probe /56-FAM/TTCTGACCT/ZEN/GAAGGCTCTGCGCG/3IABkFQ/. The relative normalization of sample was obtained using universal 16S rDNA amplicons, as a measure of total bacterial load, as described [23]. The qPCR was performed on Roche Light Cycler 480 instrument, following PCR program: hold at 94oC for 2 minutes and cycling of 45x (94oC, 10sec, following by priming, elongation and acquisition of fluorescence at 50oC for 20 seconds). The RFU (Relative Fluorescence Units) versus time/cycles curve was visually analyzed. Internal PCR positive control was genomic DNA ATCC 43255, adjusted to produce positive and reliable/stabile Cq value. LC480 software (V SP3) was used to calculate Cq values using second derivate analyses and high sensitivity mode. Decisions of the result was made by operator (positive/negative/ inhibition), based on interplay between results of toxB and/or inhibition assays and curve shape, and/or generated Cq value. Total bacterial load quantifications were performed on additionally 10 times diluted sample (comparing to equivalent toxB sample) to assure that inhibition of 16S rDNA reaction is not a dominant factor in defining Cq value. The difference in Cq values among 16S and toxB reactions (each done in separate wells) was calculated and plotted, after correction for sample dilution.