TITLE: Protocol for detection of Salmonella Typhi and Salmonella Paratyphi A in Produce
AUTHORS: Renuka Kapoor, Ashutosh Wadhwa, Christine Moe

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The protocol describes method for qualitative detection (presence/ absence) of </span><span style = "font-style:italic;">Salmonella</span><span> Typhi and </span><span style = "font-style:italic;">Salmonella </span><span>Paratyphi A on produce by enrichment culture followed by real-time PCR.</span></div><br/></div>

[STEPS]
?. [1. Processing of produce sample]
The following steps describe processing of produce samples up to enrichment stage.
?. [2. Enrichment culture]
The following steps are for optional enrichment of the sample. If enrichment is not performed, the produce wash can be used directly for membrane filtration and DNA extraction (Section 3).
?. [1. Processing of produce sample]
Clean your work surface with 70% ethanol. Steps 3-6 should be done inside a biosafety hood.
?. [1. Processing of produce sample]
Spray the outside of the Whirl-Pak bag containing the sample with 70% ethanol and rub it well.
Inspect the bag to see if liquid can be added without overflowing. If the bag is more than 2/3rds full with produce, open the bag by untwisting the ties and pulling them gently outwards until the mouth of the bag opens.  Remove extra produce items one at a time by pressing upward underneath them from the outside of the bag and move them to the top. Never stick your hands into the bag. Discard removed produce, and only process what remains.
?. [1. Processing of produce sample]
Open the Whirl-Pak bag by untwisting the ties and pulling gently outwards until the mouth of the bag opens.
?. [1. Processing of produce sample]
Add  PBST to the bag.
500 mL
?. [1. Processing of produce sample]
Seal the bag, trapping minimal amounts of air inside.
?. [1. Processing of produce sample]
Incubate the bag for  at 37°C.
?. [1. Processing of produce sample]
Vigorously shake the bag with the produce for .
?. [1. Processing of produce sample]
Gently massage the surface of each piece of produce through the bag for .
For delicate items like lettuce or onions, try to rub at least the outer leaves. Try not to break open any items.
?. [1. Processing of produce sample]
Shake the bag again for .
?. [1. Processing of produce sample]
Open the Whirl-Pak bag.
?. [1. Processing of produce sample]
Remove the produce from the Whirl-Pak bag by gently pressing upwards underneath each item from the outside of the bag and move it to the top.
Never stick your hands inside the bag. Take care not to lose any water or smash any produce.
?. [1. Processing of produce sample]
Set aside produce removed from bag.
?. [1. Processing of produce sample]
Close the Whirl-Pak bag containing produce wash.
?. [1. Processing of produce sample]
Weigh the produce (using aluminum foil) and record the weight.
?. [1. Processing of produce sample]
Put on gloves and spray hands with 70% ethanol and rub hands together to sanitize all surfaces of the gloves.
?. [2. Enrichment culture]
Fill a 1 liter flask with  of Universal Pre-enrichment (UP) Broth (USEPA Standard Analytical Protocol for Salmonella Typhi in Drinking Water).
450 mL
?. [2. Enrichment culture]
Add  of produce wash to the flask containing 450 mL of UP Broth.
50 mL
?. [2. Enrichment culture]
Incubate the flask at 37°C in a shaking incubator overnight.
?. [3. DNA Extraction]
The following steps are for membrane filtration and DNA extraction. They can be performed following step 1.15 or step 2.3.
?. [3. DNA Extraction]
Clean your workspace and set up your filter units.
If enrichment was performed, remove the flask from the incubator.
?. [3. DNA Extraction]
Using sterile forceps, place a clean membrane filter on the base of the filter unit.
?. [3. DNA Extraction]
Place the cup on top of the filter.
Make sure the cup is placed flush against the base. If there are any gaps the sample will spill out.
?. [3. DNA Extraction]
Add  of enriched sample to the cup and turn on the vacuum.
20 mL
For samples processed without enrichment, use 100 mL of produce wash from step 1.15 for membrane filtration.
?. [3. DNA Extraction]
Allow the sample to filter until liquid is no longer visible on the filter.
The time needed for this step varies depending on the sample type and dirtiness or turbidity. Another way to tell if it is finished is when the ridges of the filter unit base are visible on the filter.
?. [3. DNA Extraction]
Turn off the vacuum and remove the cup from the base.
?. [3. DNA Extraction]
Using a sterile forcep, remove the filter from the base.
?. [3. DNA Extraction]
Using two sterile forceps, fold the filter in half inward, so the cells are now contained inside the folded filter.
?. [3. DNA Extraction]
Fold the filter in half again.
?. [3. DNA Extraction]
Transfer the folded filter to a bead tube (from Qiagen DNeasy PowerWater kit).
?. [3. DNA Extraction]
Add  of Buffer PW1 (Qiagen DNeasy PowerWater kit) to the bead tube and vortex for .
1 mL
?. [3. DNA Extraction]
Proceed with the DNA Extraction according to manufacturer’s protocol (Qiagen DNeasy PowerWater kit).
?. [4. Real-time PCR]
Test DNA extracts for S. Typhi and S. Paratyphi A using Taqman-based quantitative real-time PCR (qPCR) platform.
?. [4. Real-time PCR]
Detection of S. TyphiS. 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’; tviB_R 5’-GACTTCCGATACCGGGATAATG-3’; tviB_P HEX-TGGATGCCGAAGAGGTAAGACGAGA-BHQ1; staG_F 5’- CGCGAAGTCAGAGTCGACATAG-3’;staG_R 5’-AAGACCTCAACGCCGATCAC-3’; staG_P FAM-5’-CATTTGTTCTGGAGCAGGCTGACGG-3’-BHQ1The 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 95oC for 5 min, followed by 45 cycles of 95oC 30 sec, 64oC 30 sec, 72oC 10 sec, and final extension at 72oC for 5 min.
?. [4. Real-time PCR]
Detection of S. Paratyphi AS. 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 95oC for 5 min, followed by 45 cycles of 95oC 30 sec, 60oC 30 sec, 72oC 30 sec, and final extension at 72oC for 10 min.