TITLE:  qPCR based multipathogen detection for Salmonella Paratyphi &#34;A&#34; and Vibrio cholerae from wastewater samples
AUTHORS: Dilip Abraham, Blossom Benny Sam, Nirmal Kumar, Raju Ravi, Venkata Raghava Mohan

[DESCRIPTION]
The following protocol is optimized for the detection of Salmonella Paratyphi A and Vibrio cholerae using multiplex qPCR assays from TNA extracted from environmental samples (wastewater samples). Total Nucleic Acid (TNA) extracted from the pellet particles is used as template in a real-time PCR assay which uses primer-probes specific for Salmonella Paratyphi A or Vibrio cholerae gene targets. Three different multiplex PCR assays were designed following standardization and validation of the primer probe combinations. 

[BEFORE_START]
Ensure that the fluorescent dye used for TaqMan probes is compatible with the machine being used and properly calibrated for it.

[GUIDELINES]
This protocol describes qPCR based detection of Salmonella Paratyphi "A" and Vibrio cholerae gene targets in three PCR panels from TNA extraction of processed environmental samples that were spiked with internal controls (SPC EGT control DNA - referenced below) . Additionally, HF183 which serves as a marker of fecal contamination in environmental samples, is included in the qPCR assays. Due to the similarity in cycling conditions, these assays can be conducted simultaneously for a sample as three distinct panels.

The positive controls (PCs) used here are linear oligos (gBlocks) that have been used in generating standard curves. Ideally the concentration of PCs used should yield Ct values that fall in the linear phase of the amplification curve.


 



[STEPS]
SECTION: Primer-Probe panel
1.2. PCR 2: Vibrio cholerae_1 PCR panel


 

SECTION: Primer-Probe panel
1.3. PCR 3: Vibrio cholerae_2 PCR panel


 

SECTION: Primer-probe reconstitution
2.1. Multiply nmol value by 10 to get the required volume of Nuclease Free Water (NFW) needed to reconstitute the lyophilized primer/probes.
Ex: For a primer with 30 nmoles, to make 100 micromolar (µM) stock solution:
30 nmol x 10 = 300 µL of NFW to make 100 micromolar (µM) stock solution.
SECTION: Primer-probe reconstitution
2. To reconstitute the lyophilized primers/probes, use the nmole information on the specification sheet received with primers/probes.
SECTION: Primer-probe reconstitution
2.2. Add the required volume of nuclease free water, pulse vortex and spin down. This is the primer/probe stock with 100 micromolar (µM) concentration. 
SECTION: Primer-probe dilution
3. Prepare a working stock from 100 micromolar (µM) stock solution.
SECTION: Primer-probe reconstitution
2.3. Store at -20 °C for long term storage.
SECTION: Primer-probe dilution
3.2. Store at 4 °C for frequent usage or -20 °C for long-term storage.


SECTION: Primer-probe dilution
3.1. In a fresh tube add 10 µL  of 100 micromolar (µM) primer/probe stock and 90 µL of nuclease free water to give 100 µL  of 10 micromolar (µM) working primer/probe.
SECTION: qPCR controls
4. Positive control: gBlocks gene fragments corresponding to each gene target is included in qPCR assay to use as positive control. 


 

SECTION: Quantitative PCR
7.  [PCR 1] Salmonella Paratyphi A (SPA2308) and ctxA
SECTION: Primer-Probe panel
1. The following primers and probes are employed  for the detect Salmonella Paratyphi A and Vibrio cholerae specific gene targets.
SECTION: Primer-Probe panel
1.1. PCR 1: Salmonella Paratyphi A (SPA2308) and V. cholerae ctxA PCR panel


 

SECTION: qPCR controls
5. Negative control: 3 µL of extraction blank of each batch of extraction.
NTC: Master mix alone used for no template control.

SECTION: Quantitative PCR
6. Thaw qPCR reagents and samples on ice and briefly spin it down. 
Prepare master mix for each of the 3 primer-probe panels.
SECTION: Quantitative PCR
9. [PCR 3] Vibrio cholerae_2 PCR   

SECTION: Quantitative PCR
8.1.   Prepare the master mix as follows for the number of samples, positive and negative controls, NTC and one extra reaction to account for any pipetting error.


 

 

SECTION: Quantitative PCR
8.  [PCR 2] Vibrio cholerae_1 PCR
SECTION: Quantitative PCR
7.1. Prepare the master mix as follows for the number of samples, positive and negative controls, NTC and one extra reaction to account for any pipetting error.

 


SECTION: Quantitative PCR
9.1. Prepare the master mix as follows for the number of samples, positive and negative controls, NTC and one extra reaction to account for any pipetting error.


 

SECTION: Quantitative PCR
10. For each PCR, aliquot 22 μl of master mix to each required well in a 96-well plate.
Add 3μl of sample, or 3 μl of nuclease free water for negative controls.
SECTION: Quantitative PCR
11. Seal the plate with a roller sealer and then centrifuge the plate for 1 min at 2,000g.
SECTION: Quantitative PCR
15. Once the run is complete, adjust the thresholds and baseline if any abnormal baseline at the start or at the end is observed, which may lead to a false-positive curve. Verify if the PC is within the range using the cut-off Ct values chosen from running the standards.
SECTION: Quantitative PCR
13. Set up the experiment properties with 96-well block, TaqMan reagents, 0.2 ml PCR plate and standard run. Define sample ID and define the targets as described for respective PCR panels. Assign targets and sample ID to each well.
SECTION: Quantitative PCR
12. Load the plate into Quantstudio7 flex instrument after proper initiation of the instrument. Open QS7 software, then select "New Experiment set up".
SECTION: Quantitative PCR
16. Export the result to excel/csv file and upload both run and csv files.
SECTION: Quantitative PCR
14. Set up the PCR cycling method as described below.
Cycling conditions remain the same for all 3 qPCRs.


 

Start the qPCR by clicking “Run.”
SECTION: Quantitative PCR
17. The threshold for each target can be set such that the PC for that target falls within the pre-defined range obtained with the standard curves. 


The sample is considered positive if the amplification curve is appropriate and the Ct value falls below the defined cut-off thresholds for each target.


A separate protocol, provided in the Typhoid ES workspace, serves as an example and can be followed to generate Ct cut-off values: