Protocol for detection of Salmonella Typhi and Salmonella Paratyphi A in Street Food

The following steps describe processing of street food up to enrichment stage.

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

Clean your work surface(s) with 70% ethanol.

Weigh 25 grams of street food.

Use a sterile spatula to transfer the sample to a BagPage+ full-page filter bag (Interscience Inc.).

Rinse spatula and weigh boat with 25mL of 1x PBST to add to the sample bag.

Add 200mL of 1x PBST to the sample in the bag.

Place bag into the BagMixer 400 CC and mix for 0h 3m 0s at settings of speed 4 with gap at -3 mm.

The following steps describe enrichment culture process for street food samples

Add 90mL of Universal Pre-enrichment (UP) Broth (USEPA Standard Analytical Protocol for Salmonella Typhi in Drinking Water) to a 250 mL flask.

Using a sterile disposable pipette, immediately transfer 10mL of homogenized solution from the filtered side of the bag ( do not allow particles to settle ) to 250 ml flask containing 90 mL of UP broth.

Incubate the flask in shaking incubator 37°C overnight.

The following steps are for membrane filtration and DNA extraction.

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 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.