TITLE: Extraction and qPCR of Environmental Surveillance samples for the detection of Salmonella Typhi
AUTHORS: Dilip Abraham, Nicola Elviss, Nicholas Feasey, Nick Grassly, Jacob John, Gagandeep Kang, John Scott Meschke, Venkata Raghava Mohan, Satheesh Nair, Jonathan Rigby, Rajan Srinivasan, Catherine Troman, Christopher Uzzell, Nicolette Zhou

[DESCRIPTION]
The following protocol is for use on environmental samples that have been collected by Moore swab or grab sampling methods and processed up to the stage of DNA extraction. The protocol includes DNA extraction and qPCR analysis of the samples to detect three gene targets for identifying Salmonella Typhi, a human faecal indicator (HF183), and a commercial spike-in control.

The qPCR uses fluorescence of Taqman probes for measuring the product. Care must be taken to ensure that the fluorescent dyes chosen for the probes are suitable for the machine being used or can be calibrated for use on the machine. If the dyes selected are different from those described in the protocol, make sure that their excitation/emission spectra do not overlap to avoid crossover between targets.

Generation of standard curves is mentioned in this protocol but is described in more detail in a separate protocol in the Typhoid ES workspace.



[STEPS]
SECTION: DNA Extraction
1. For extraction from Moore swabs, empty the contents of a PowerBead tube provided in the extraction kit into the PowerBead tube used for storing the filter.

For the pellet from membrane filtration, resuspend the pellet in 800µl CD1 from the first step of the DNA extraction protocol, then transfer to the PowerBead tube for homogenisation.

For an extraction negative control, carry out the extraction protocol starting with just the CD1 (step2.1) and no sample.
SECTION: DNA Extraction
2. Carry out the DNA extraction according to the manufacturer's protocol provided with the DNA extraction kit. 
Note that x g and rcf are the same and may be named either way depending on your centrifuge.

In brief:
SECTION: DNA Extraction
2.1. Add 800µl of Solution CD1 to the PowerBead tube and 1ul of validated SPC dilution from the Extraction and Inhibition control kit.

1) Before first use of the SPC, prepare a 1/10th and a 1/100th dilution of the control DNA in pure water and store them on ice

2) Evaluate the non-diluted and the 2 diluted control DNA solutions in separate extractions by adding 1μL of control DNA into your reference sample before parallel extractions. Perform the extraction and qPCR and select the dilution factor generating Ct values between 30 and 33
SECTION: DNA Extraction
2.2. Homogenize using a vortex adapter at maximum speed for up to 10 minutes
SECTION: DNA Extraction
2.3. Centrifuge the PowerBead tube at 15000 x g, 1 min

SECTION: DNA Extraction
2.4. Transfer the supernatant to a clean Microcentrifuge tube, add 200µl of Solution CD2 then vortex for 5s
SECTION: DNA Extraction
2.5. Centrifuge at 15000 x g, 1 min and transfer up to 700µl of supernatant to a clean 2ml Microcentrifuge tube, avoiding the pellet

SECTION: DNA Extraction
2.6. Add 600µl of Solution CD3 and vortex for 5s
SECTION: DNA Extraction
2.7. Load 650µl onto an MB Spin Column and centrifuge at 15000 x g, 1 min

SECTION: DNA Extraction
2.8. Discard the flow-through and repeat until all the lysate has been passed through the spin column
SECTION: DNA Extraction
2.9. Carefully place the spin column into a clean 2ml collection tube. Avoid splashing any flow-through onto the column
SECTION: DNA Extraction
2.10. Add 500µl of Solution EA to the spin column and centrifuge at 15000 x g, 1 min

SECTION: DNA Extraction
2.11. Discard the flow-through and add 500µl of Solution C5 to the spin column and centrifuge at 15000 x g, 1 min 

SECTION: DNA Extraction
2.12. Discard the flow through and place the column in a clean 2ml collection tube
SECTION: DNA Extraction
2.13. Centrifuge at 16000 x g, 2 min then place the column in a clean 1.5ml collection tube

SECTION: DNA Extraction
2.14. Add 50µl of Solution C6 to the center of the filter membrane and let it sit for 1 minute
SECTION: DNA Extraction
2.15. Centrifuge at15000 x g, 1 minand discard the spin column

SECTION: DNA Extraction
2.16. The DNA is now ready for qPCR, or storage at -20°C
SECTION: Preparation of Primers and Probes
3. S. Typhi genes being targeted are staG, tviB, and ttr. Primers and probes have also been included for HF183 human faecal indicator to use as a positive control for faecal contamination in the samples and sample sites.

Before ordering probes, please make sure the selected fluorophores are compatible with the machine you will be using (see guidance note).

 

SECTION: Preparation of Primers and Probes
4. Resuspend the probes and primers in the appropriate volume of nuclease free water and make stock dilutions of each primer at 20µM and each probe at 5µM.
SECTION: Preparation of Standards
5. Using gBlocks gene fragments (IDT) with the target amplicon sequences, a standard curve can be obtained by running a qPCR on a dilution series of the DNA fragments.

This allows determination of potential Ct cut off values to help with determining a positive result in the qPCR with real samples.

A separate protocol for carrying out this experiment is provided in the Typhoid ES workspace.

 

SECTION: Quantitative PCR
6. Thaw qPCR reagents and samples on ice and briefly spin down.
SECTION: Quantitative PCR
7. Set up a master mix for the number of samples to be tested plus a negative control and one extra to allow for pipetting error.

If you wish to carry out single-plex reactions for any of the gene targets, input the same amount of the mastermix, target primer and probe, and substitute the remaining volume with nuclease free water.


 

SECTION: Quantitative PCR
8. Make up another master mix for the HF183 qPCR and internal control


 

SECTION: Quantitative PCR
9. Aliquot 20µl of mastermix to each required well in a 96-well plate.
SECTION: Quantitative PCR
10. Add 5µl of sample, or 5µl of nuclease free water for negative controls.
SECTION: Quantitative PCR
11. Seal the plate carefully with a plate seal the spin the plate down briefly to gather all reagents at the bottom of the well and remove bubbles.
SECTION: Quantitative PCR
12. Load the plate into the real-time PCR machine after setting it up appropriately and carry out cycling using the following conditions:

 

SECTION: Quantitative PCR
13. Once the run is complete assess the normalised Ct values for each gene target for each sample, determining positivity using the cut-off Ct values chosen from running the standards.
SECTION: Quantitative PCR
13.1. If all gene targets have Ct values higher than the cut-off, check the Ct values for the HF183 target and the spike-in control.
If the spike-in control is negative or lower than expected this may indicate an issue with PCR inhibition or during DNA extraction
If the HF183 is negative and spike-in control was positive, then this may mean that there was no or insufficient faecal contamination in the sample site
SECTION: Quantitative PCR
13.2. If all three targets are positive, then the sample is considered positive for S.Typhi.

If the sample is positive for ttr and only one of either staG or tviB, repeat the target that was negative in a singleplex reaction as this may result in a positive if the target is just very close to the limit of detection.

The following table gives expamples for interpretation of the qPCR results (after singleplex repeats):