Extraction and qPCR of Environmental Surveillance samples for the detection of Salmonella Typhi

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.

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:

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

Add 500µl of Solution EA to the spin column and centrifuge at 15000x g

Discard the flow-through and add 500µl of Solution C5 to the spin column and centrifuge at 15000x g

Discard the flow through and place the column in a clean 2ml collection tube

Centrifuge at 16000x g then place the column in a clean 1.5ml collection tube

Add 50µl of Solution C6 to the center of the filter membrane and let it sit for 1 minute

Centrifuge at15000x gand discard the spin column

The DNA is now ready for qPCR, or storage at -20°C

Homogenize using a vortex adapter at maximum speed for up to 10 minutes

Centrifuge the PowerBead tube at 15000x g

Transfer the supernatant to a clean Microcentrifuge tube, add 200µl of Solution CD2 then vortex for 5s

Centrifuge at 15000x g and transfer up to 700µl of supernatant to a clean 2ml Microcentrifuge tube, avoiding the pellet

Add 600µl of Solution CD3 and vortex for 5s

Load 650µl onto an MB Spin Column and centrifuge at 15000x g

Discard the flow-through and repeat until all the lysate has been passed through the spin column

Carefully place the spin column into a clean 2ml collection tube. Avoid splashing any flow-through onto the column

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).

A	B
Primer/Probe Name	Sequence (5' -3')
staG_F	CGC GAA GTC AGA GTC GAC ATA G
staG_R	AAG ACC TCA ACG CCG ATC AC
staG_P	[Cy5] - CA TTT GTT CTG GAG CAG GCT GAC GG - [BHQ2]
ttr_F	CTC ACC AGG AGA TTA CAA CAT GG
ttr_R	AGC TCA GAC CAA AAG TGA CCA TC
ttr_P	[FAM] - CA CCG ACG GCG AGA CCG ACT TT - [BHQ1]
tviB_F	TGT GGT AAA GGA ACT CGG TAA A
tviB_R	GAC TTC CGA TAC CGG GAT AAT G
tviB_P	[JOE] - TG GAT GCC GAA GAG GTA AGA CGA GA - [BHQ1]
HF183_F	ATC ATG AGT TCA CAT GTC CG
HF183_R	CTT CCT CTC AGA ACC CCT ATC C
HF183_P	[FAM] - CT AAT GGA ACG CAT CCC - [BHQ1]

Table1: Primer (F or R) and probe (P) sequences. Fluorescent dyes and quenchers are shown in square brackets.

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.

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.

A	B
gene target	gBlocks sequence (5' - 3')
staG	CGGCGCGAAGTCAGAGTCGACATAGGCATAGATTTTCAGGCCATACATTAATTTGCCAAGGTTGCTATAAACATTTGTTCTGGAGCAGGCTGACGGAAATTCCGTGAACTCGCTGGTGATCGGCGTTGAGGTCTTATC
ttr	GAAACGCTGAACGGACTCACCAGGAGATTACAACATGGCTAATTTAACCCGTCGTCAGTGGCTAAAAGTCGGTCTCGCCGTCGGTGGGATGGTCACTTTTGGTCTGAGCTACCGTGATGTGGCGA
tviB	CTTGATTTGACTTCCGATACCGGGATAATGCCATACTCTCGTCTTACCTCTTCGGCATCCACCCATGGATCAAAAATATCCACTTTACAACTATATTTACCGAGTTCCTTTACCACATCAATAAT
HF183	GGGATCATGAGTTCACATGTCCGCATGATTAAAGGTATTTTCCGGTAGACGATGGGGATGCGTTCCATTAGATAGTAGGCGGGGTAACGGCCCACCTAGTCAACGATGGATAGGGGTTCTGAGAGGAAGGTC

Table 2: Sequences used for gBlocks gene fragments. Sequences for the S.Typhi targets were taken from the CT18 reference genome (AL513382.1) and the HF183 sequence was taken from a Bacteroides dorei 16S rRNA partial gene sequence (MT464394.1). 3 additional bases were added either side of the staG and HF183 amplicon sequences. 15 and 8 bases were added to either end of the ttr and tviB sequences respectively to reach the minimum length for the gBlocks fragment.

Thaw qPCR reagents and samples on ice and briefly spin down.

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.

A	B	C
Reagent	Volume per reaction (uL)	Final Concentration (uM)
ttr_F	0.25	0.2
ttr_R	0.25	0.2
ttr_P	0.5	0.1
tviB_F	0.5	0.4
tviB_R	0.5	0.4
tviB_P	1	0.2
staG_F	0.5	0.4
staG_R	0.5	0.4
staG_P	1	0.2
2x MasterMix with ROX	12.5	1x
Nuclease free water	2.5	-

Table 3: Mastermix composition for the S.Typhi qPCR

Make up another master mix for the HF183 qPCR and internal control

A	B	C
Reagent	Volume per reaction (uL)	Final Concentration (uM)
HF183_F	0.5	0.4
HF183_R	0.5	0.4
HF183_P	1	0.2
2x MasterMix with ROX	12.5	1x
10x Control mix (Eurogentec)	2.5	1x
Nuclease free water	3	-

Table 4: Mastermix composition for the HF183 and extraction control qPCR

Aliquot 20µl of mastermix to each required well in a 96-well plate.

Add 5µl of sample, or 5µl of nuclease free water for negative controls.

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.

Load the plate into the real-time PCR machine after setting it up appropriately and carry out cycling using the following conditions:

A	B	C
Cycle	Temperature (ºC)	Duration
1	50	2 minutes
1	95	2 minutes
40	95	15 seconds
	60	30 seconds
	72	30 seconds

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.

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

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):

A	B	C
qPCR result	Low Ct values	High Ct values
ttr+ staG+ tviB+	S.Typhi	S.Typhi (or a more complex mixture but unlikely)
ttr+ staG- tviB+	Mixture (NTS)	Mixture (NTS)
ttr+ staG+ tviB-	Mixture (NTS)	Mixture or S.Typhi where Vi gene is lost