Protocol for detection of Salmonella Typhi and Salmonella Paratyphi A in Soil

The following steps describe processing of soil up to enrichment stage.

Clean your work surface with 70% ethanol.

Let the sample settle for 0h 30m 0s.

Put on gloves and spray hands with 70% ethanol and rub hands together to sanitize all surfaces of the gloves.

Spray the outside of the Whirl-Pak bag containing the sample with 70% ethanol and rub it well.

Rotate the Whirl-Pak bag with the sample five times to mix the sample.

Open the Whirl-Pak bag by untwisting the ties and pulling gently outwards until the mouth of the bag opens.

Using a weighing scale and a sterile spatula, measure 10 grams of sample.

Transfer the sample to a sterile 250 mL flask.

Using a graduated cylinder, add 100mL of PBS to the measured sample.

Shake vigorously on a rotator or shaker for 0h 30m 0s at room temperature.

The following steps are for optional enrichment of the sample. If enrichment is not performed, supernatant from settled sample (after step 1.10) can be used directly for membrane filtration and DNA extraction (Section 3).

Transfer 180mL of Universal Pre-enrichment (UP) Broth (USEPA Standard Analytical Protocol for Salmonella Typhi in Drinking Water) to a sterile 500 mL flask.

With a sterile pipette, carefully remove 20mL of supernatant from the top of the 250 mL flask, without sucking up particulate matter and debris.

Transfer the supernatant to the flask containing the UP Broth.

Incubate the flask at 37°C in a shaking incubator overnight.

The following steps are for membrane filtration and DNA extraction. They can be performed following step 1.10 or step 2.4.

Clean your workspace and set up your filter units.

Transfer the folded filter to a bead tube (from Qiagen DNeasy PowerWater kit).

Add 1mL of Buffer PW1 (Qiagen DNeasy PowerWater kit) to the bead tube and vortex for 0h 5m 0s.

Proceed with the DNA Extraction according to manufacturer’s protocol (Qiagen DNeasy PowerWater kit).

Using sterile forceps, place a clean membrane filter on the base of the filter unit.

Place the cup on top of the filter.

Add 20mL of enriched sample to the cup and turn on the vacuum.

Allow the sample to filter until liquid is no longer visible on the filter.

Turn off the vacuum and remove the cup from the base.

Using a sterile forcep, remove the filter from the base.

Using two sterile forceps, fold the filter in half inward, so the cells are now contained inside the folded filter.

Fold the filter in half again.

Test DNA extracts for S . Typhi and S . Paratyphi A using Taqman-based quantitative real-time PCR (qPCR) platform.

Detection of S . Typhi

S . Typhi is detected using duplex PCR protocol developed by researchers at the University of Washington (Scott Meschke and team) usIng primers and probes targeting the tviB and staG genes (Nair et al., 2019).

tviB _F 5’TGTGGTAAAGGAACTCGGTAAA-3’;

tvi B_R 5’-GACTTCCGATACCGGGATAATG-3’;

tvi B_P HEX - TGGATGCCGAAGAGGTAAGACGAGA-BHQ1;

sta G ___ F 5’- CGCGAAGTCAGAGTCGACATAG-3’;

sta G ___ R 5’-AAGACCTCAACGCCGATCAC-3’;

sta G ___ P FAM-5’-CATTTGTTCTGGAGCAGGCTGACGG-3’-BHQ1

The reaction mixture contain 0.65 µl of tviB_F (20µM), 0.75 µl each of tviB_R (20µM), staG_F(20µM),and staG_R (20µm), 0.5 µl each of the probe tviB_P (10µM) and staG_P (10 µM), 12.5 µl of SsoAdvanced Universal Probes Supermix (Bio-rad), and 5 µl of DNA in a final volume of 25 µl. The PCR reaction conditions include initial denaturation at 95^oC for 5 min, followed by 45 cycles of 95^oC 30 sec, 64^oC 30 sec, 72^oC 10 sec, and final extension at 72^oC for 5 min.

Detection of S . Paratyphi A

S . Paratyphi A Is detected using primers and probe targeting SPA2308 (Nga et al., 2010)

SPA2308_F 5’- ACGATGATGACTGATTTATCGAAC-3’;

SPA2308_R5’-TGAAAAGATATCTCTCAGAGCTGG-3’;

SPA2308_PCY5 - CCCATACAATTTCATTCTTATTGAGAATGCGC-BHQ2

The reaction mixture containing 1 µl of each primer (10µM), 0.4 µl of probe (10µM), 200µM of dNTPs, 5mM of MgCl2, 5U of HotStar Taq DNA polymerase (Qiagen), and 5 µl of DNA in a final reaction volume of 25 µl. The PCR reaction conditions include initial denaturation at 95^oC for 5 min, followed by 45 cycles of 95^oC 30 sec, 60^oC 30 sec, 72^oC 30 sec, and final extension at 72^oC for 10 min.