TITLE: River water laboratory processing Protocol
AUTHORS: Sneha Shrestha, Christopher LeBoa

[DESCRIPTION]
These are the qPCR conditions used for the S. Typhi and Paratyphi qPCR used for environmental surveillance by our lab team

[STEPS]
SECTION: Turbidity Testing
1. 2ml of sample (from 5ml separated for Phage Screening) is taken in a sterile glass
tube
SECTION: Turbidity Testing
2. Turbidity is measured by densitometry.
SECTION: Differential Centrifugation
3. Spin down 45ml of collected water sample at 2000rpm for 1 min to separate out larger
debris.
SECTION: Differential Centrifugation
4. Transfer the supernatant to a new sterile 50ml tube.
SECTION: Differential Centrifugation
5. Centrifuge at 4000rpm for 25mins.
SECTION: Differential Centrifugation
6. Discard the supernatant and resuspend the pellet in 0.5ml of sterile D/w.
SECTION: Enrichment and DNA extraction
7. Transfer 0.5ml of resuspended pellet into 10ml of Selenite F broth.
SECTION: Enrichment and DNA extraction
8. Transfer 1ml of this suspension to a sterile 1.5ml tube for DNA extraction for T0.
SECTION: Enrichment and DNA extraction
9. Incubate the inoculated Selenite F broth overnight (16hrs) at 37°C.
SECTION: Enrichment and DNA extraction
10. At the end of incubation, transfer 1ml of the o/n growth to 1.5ml tube for DNA
extraction at T16.
SECTION: DNA extraction (DNEasy Blood and Tissue Kit Cat# 69504)
11. Transfer 1ml of bacterial suspension in SF broth to 1.5ml tube.
SECTION: DNA extraction (DNEasy Blood and Tissue Kit Cat# 69504)
12. Centrifuge at 2000rpm for 2mins.
SECTION: DNA extraction (DNEasy Blood and Tissue Kit Cat# 69504)
13. Discard the Supernatant.
SECTION: DNA extraction (DNEasy Blood and Tissue Kit Cat# 69504)
14. Resuspend pellet in 200 μl PBS.
SECTION: DNA extraction (DNEasy Blood and Tissue Kit Cat# 69504)
15. Add 20 μl proteinase K.
SECTION: DNA extraction (DNEasy Blood and Tissue Kit Cat# 69504)
16. Add 200 μl buffer AL. Mix thoroughly by vortexing.
SECTION: DNA extraction (DNEasy Blood and Tissue Kit Cat# 69504)
17. Incubate for 10mins at 55°C.
SECTION: DNA extraction (DNEasy Blood and Tissue Kit Cat# 69504)
18. Add 200 μl ethanol (96-100%) and mix thoroughly by vortexing.
SECTION: DNA extraction (DNEasy Blood and Tissue Kit Cat# 69504)
19. Transfer the mixture into a DNeasy Mini spin column placed in a 2ml collection tube. Centrifuge at 8000rpm for 1min. Discard the flow-through and replace a new collection tube.
SECTION: DNA extraction (DNEasy Blood and Tissue Kit Cat# 69504)
20. Add 500 μl Buffer AW1 to the column and centrifuge at 8000rpm for 1 min. Discard flow-through and replace a new collection tube.
SECTION: DNA extraction (DNEasy Blood and Tissue Kit Cat# 69504)
21. Add 500 μl Buffer AW2 to the column and centrifuge at 14000 rpm for 3 mins. Discard flow-through.
SECTION: DNA extraction (DNEasy Blood and Tissue Kit Cat# 69504)
22. Transfer the collection tube to a new 1.5ml microcentrifuge tube.
SECTION: DNA extraction (DNEasy Blood and Tissue Kit Cat# 69504)
23. Elute the DNA by adding 60 μl Buffer AE to the center of the spin column membrane. Incubate for 1 min at room temperature. Centrifuge at 8000 rpm for 1 min.
SECTION: Real time PCR (Adapted from Tran Vu Thieu Nga)
24. Primer preparation
Primers/probes are shipped in lyophilized form and need to be constituted with TE buffer or nuclease
free dH2O. Make the primary primer/probe stock solution of 100 μM.
Amount of nuclease free dH2O= 10 µl × X ng
Here, X is the amount of primer/probe provided by the manufacturer.
Generally, use the following formula to find unknown volume or concentration.
C1*V1 = C2*V2
Where,
C1 = Initial concentration.
V1 = Initial volume.
C2 = Final concentration.
V2 = Final volume.
Here, for this protocol, we used a portion of the 100 μM primers/probes to make 10 μM/μl solutions
to use as working solutions. Aliquot the working solution into 5 separate tubes and store at -20 °C to
avoid contamination and multiple freeze-thawing cycles.
SECTION: Real time PCR (Adapted from Tran Vu Thieu Nga)
25. Master mix  setup
Master mix hi/Paratyphi-A qPCR: 

SECTION: Real time PCR (Adapted from Tran Vu Thieu Nga)
26.