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TcdB_Quantification.md

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qPCR-based detection and quantification of C. difficile TcdB

Materials and Equipment

  • iTaq™ Universal Probes Supermix (BioRad 1725132)
  • CFX384 thermocycler (Biorad)
  • 384 Plates for qPCR (Biorad #HSP3865)
  • Optically clear Plate Seals (Biorad Microseal ‘B’ #MSB1001)
  • 10x Assay mix (prepare 2µM of each oligo below in nuclease-free water)
  • 10-fold dilution series of gDNA (from a TcdB+ C. difficile). Use the QuBit DNA concentration to calculate genome copy number based on genome size to genome copy number. For example: 2.14ng of C. difficile JBZPo1 (4,097,778bp) ~ 5.5E5 genome copies = 5.5e5 TcdB copies.
  • Extracted gDNA and knowledge of the original weight/volume extracted and the final elution volume. Check the gDNA using a nanodrop and if there is any indiciation of suspected ethanol carryover, dilute all samples 10x before running.

Oligos

Oligo Sequence Stock [µM] Working [µM] Final [nM]
TcdB_F TACAAACAGGTGTATTTAGTACAGAAGATGGA 100 2 200
TcdB_R CACCTATTTGATTTAGMCCTTTAAAAGC 100 2 200
TcdB_P [6FAM]TTTKCCAGTAAAATCAATTGCTTC[BHQ1] 100 2 200

Setting up qPCR RXNs

The master mix we are using has an antibody-mediated hot-start, and as such can be set up at RT in the PCR flow-hood in a 384 well plate.

Reaction mixture

Component [stock] µL/rxn [final]
iTaq Supermix 2X 5 1X
10X assay mix 2µM 1 200nM
nuclease free water - 2 -
DNA template variable 2 varable

After setting up the plate, breifly centrifuge to mix and remove air bubbles before transfering to the CFX384.

Cycling Parameters

Cycle Temperature (˚C) Time
Initial Denaturation 95 5min
40 cycles:
Denature 95˚C 5 sec
Anneal/Extend 56.6˚C 15 sec

When setting up the plate using the BioRad software, select the 384 plate template with all channels (not the FAM/SYBR only). Using the plate editor, change your standard curves from unknown, to standards and enter the copy number you have calculated.

Interpretation

To convert from the assay concentration to the genome copies per gram or mL it is essential to know the original mass/volume that was extracted. Assuming the DNA was extracted from 100 mg of feces and the final elution volume during the DNA extraction was 50 ul, and a 10x dilution was performed before the assay see below:

5e5 TcdB copies/ul (from qPCR assay) * (50 ul elution volume / 2 ul template in rxn) * (10 Xdilution) / (0.1g) = 1.25e9 GE/g.