qPCR based multipathogen detection for Salmonella Paratyphi "A" and Vibrio cholerae from wastewater samples

The following primers and probes are employed for the detect Salmonella Paratyphi A and Vibrio cholerae specific gene targets.

PCR 1: Salmonella Paratyphi A (SPA2308) and V. cholerae ctxA PCR panel

A	B	C
TARGET	PRIMERS	SEQUENCES 5' TO 3'
ctxA	Forward	TCCGGAGCATAGAGCTTGGA
	Reverse	TCGATGATCTTGGAGCATTCC
	Probe	[CY5] - CCGTGGATTCATCATGCACCGC - [BHQ2]
SPA2308	Forward	ACGATGATGACTGATTTATCGAAC
	Reverse	TGAAAAGATATCTCTCAGAGCTGG
	Probe	[FAM] - CCCATACAATTTCATTCTTATTGAGAATGCGC - [BHQ1]

Table1: SPA and ctxA Primer and probe sequences. Fluorescent dyes and quenchers are shown in square brackets.

PCR 2: Vibrio cholerae _1 PCR panel

A	B	C
TARGET	PRIMERS	SEQUENCES 5' TO 3'
HF183	Forward	ATCATGAGTTCACA GTCCG
	Reverse	CTTCCTCTCAGAACCCCTATCC
	Probe	[FAM] - CTAATGGAACGCATCCC - [BHQ1]
wbfO139	Forward	AGAAGCCAGTCGCAGTAAAG
	Reverse	TCGCCATCTTCCAGCATAAA
	Probe	[TAMRA] - TGGTGGTACAGCTTAGCCGCATTA - [BHQ2]
tcpA classic	Forward	GCGTAATGCAGCAGCTAATAAA
	Reverse	TATGGGAACATATCACCGACAC
	Probe	[JOE] - ATGGTCTGACACAGGCTCAATGCA - [BHQ1]

Table2: Vibrio cholerae_1 PCR Primer and probe sequences. Fluorescent dyes and quenchers are shown in square brackets.

PCR 3: Vibrio cholerae _2 PCR panel

A	B	C
TARGET	PRIMERS	SEQUENCES 5' TO 3'
OmpW	Forward	TCAATGATAGCTGGTTCCTCAAC
	Reverse	CGATGATAAATACCCAAGCATTGA
	Probe	[JOE]TGGTATGCCAATATTGAAACAACG[BHQ1]
wbeO1	Forward	GTTGAGAAGGGCGGTCTAATAA
	Reverse	TGTCTGGTACTTGAGTTGGTAAG
	Probe	[FAM] - TGCCTCAGCAATGGA - [BHQ1]
tcpA Eltor	Forward	ATCCTTTCACTGGTACAGCTATG
	Reverse	GTCAAGCCACCGACTGTAAT
	Probe	[TAMRA]- ACGAAACTCTGCAGCGAATAAAGC-[BHQ2]

Table3: Vibrio cholerae_2 PCR Primer and probe sequences. Fluorescent dyes and quenchers are shown in square brackets.

To reconstitute the lyophilized primers/probes, use the nmole information on the specification sheet received with primers/probes.

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 100micromolar (µM) stock solution:

30 nmol x 10 = 300µL of NFW to make 100micromolar (µM) stock solution.

Add the required volume of nuclease free water, pulse vortex and spin down. This is the primer/probe stock with 100micromolar (µM) concentration.

Store at -20°C for long term storage.

Prepare a working stock from 100micromolar (µM) stock solution.

In a fresh tube add 10µL of 100micromolar (µM) primer/probe stock and 90µL of nuclease free water to give 100µL of 10micromolar (µM) working primer/probe.

Store at 4°C for frequent usage or -20°C for long-term storage.

Positive control: gBlocks gene fragments corresponding to each gene target is included in qPCR assay to use as positive control.

A	B	C	D
gBlock gene	Sequence(5'-3')	Accession No:	bp size
SPA2308	ACGATGATGACTGATTTATCGAACAACGACTCTCCCATACAATTTCATTCTTATTGAGAATGCGCTTATGTAATTTATACCCCAGCTCTGAGAGATATCTTTTCA	FM200053.1	105
ctxA	TCCGGAGCATAGAGCTTGGAGGGAAGAGCCGTGGATTCATCATGCACCGCCGGGTTGTGGGAATGCTCCAAGATCATCGA	AF463401.1	80
HF183	GGGATCATGAGTTCACATGTCCGCATGATTAAAGGTATTTTCCGGTAGACGAT GGGGATGCGTTCCATTAGATAGTAGGCGGGGTAACGGCCCACCTAGTCAACG ATGGATAGGGGTTCTGAGAGGAAGGTC	MT464394.1	132
wbfO139	AGAAGCCAGTCGCAGTAAAGCACTAGGGCGCATGGTGGTACAGCTTAGCCGCATTATGCGAGATGAGCCGGGTGCGGATTTTATGCTGGAAGATGGCGA	AB012956.1	99
tcpA classic	GCGTAATGCAGCAGCTAATAAAGCATTTGCAATTTCAGTGGATGGTCTGACACAGGCTCAATGCAAGACACTTATTACCAGTGTCGGTGATATGTTCCCATA	M33514.1	102
OmpW	TCAATGATAGCTGGTTCCTCAACGCTTCTGTGTGGTATGCCAATATTGAAACAACGGCAACCTACAAAGCAGGTGCAGATGCCAAATCCACGGATGTTGAAATCAATCCTTGGGTATTTATGATCG	X51948 modified	126
wbeO1	GTTGAGAAGGGCGGTCTAATAACACCTAAAGAGTTTGCAGAGAAGCTTGCCTCAGCAATGGATAAGGCTCTTGTACGCTTACCAACTCAAGTACCAGACA	KC152957.1	100
tcpA El-Tor	ATCCTTTCACTGGTACAGCTATGGGGATTTTCTCATTTCCACGAAACTCTGCAGCGAATAAAGCATTCGCAATTACAGTCGGTGGCTTGAC	KP187623.1	91

Table4: Sequences used for gBlocks gene fragments

Negative control: 3µL of extraction blank of each batch of extraction.

NTC: Master mix alone used for no template control.

Thaw qPCR reagents and samples on ice and briefly spin it down.

Prepare master mix for each of the 3 primer-probe panels.

[PCR 1] Salmonella Paratyphi A (SPA2308) and ctxA Salmonella Paratyphi A (SPA2308) and ctxA

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.

A	B
REAGENT	VOLUME FOR 1 REACTION (μL)
UDG Mix	12.5
MgCl2	1
Rox dye	0.05
ctxA F Primer (10 µM)	0.5
ctxA R Primer (10 µM)	0.5
ctxA Probe (10 µM)	0.25
SPA2308 F (10 µM)	0.5
SPA2308 R (10 µM)	0.5
SPA2308 Probe (10 µM)	0.25
NFW	5.95

Table5: Mastermix composition for SPA2308 and ctxA gene targets

[PCR 2] Vibrio cholerae_1 PCR Vibrio cholerae _1 PCR

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.

A	B
REAGENT	VOLUME FOR 1 REACTION (μL)
UDG Mix	12.5
MgCl2	1
ROX dye	0.05
HF183 F Primer (10 µM)	0.5
HF183 R Primer (10 µM)	0.5
HF183 Probe (10 µM)	0.25
wbfO139 F Primer (10 µM)	0.5
wbfO139 R Primer (10 µM)	0.5
wbfO139 Probe (10 µM)	0.25
tcpA classic F Primer (10 µM)	0.5
tcpA classic R Primer (10 µM)	0.5
tcpA classic Probe (10 µM)	0.25
SPC (10 X EGT Control Mix)	2.5
NFW	2.2

Table6: Mastermix composition for Vibrio cholerae_1 PCR gene targets

[PCR 3] Vibrio cholerae_2 PCR Vibrio cholerae _2 PCR

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.

A	B
REAGENT	VOLUME FOR 1 REACTION (μL)
UDG Mix	12.5
MgCl2	1
ROX dye	0.05
OmpW F Primer (10 µM)	0.5
OmpW R Primer (10 µM)	0.5
OmpW Probe (10 µM)	0.25
wbeO1 F Primer (10 µM)	0.5
wbeO1 R Primer (10 µM)	0.5
wbeO1 Probe (10 µM)	0.25
tcpA El-Tor F Primer (10 µM)	0.5
tcpA El-Tor R Primer(10µM)	0.5
tcpA El-Tor Probe (10 µM)	0.25
NFW	4.7

Table7: Mastermix composition for Vibrio cholerae_2 PCR gene targets

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.

Seal the plate with a roller sealer and then centrifuge the plate for 1 min at 2,000g.

Load the plate into Quantstudio7 flex instrument after proper initiation of the instrument. Open QS7 software, then select "New Experiment set up".

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.

Set up the PCR cycling method as described below.Cycling conditions remain the same for all 3 qPCRs.

A	B	C
Step ( Hold Stage)	Temperature	Time
Reverse Transcription	50°C	2 MIN
PCR initial heat activation	95°C	2 MIN
2-step cycling (40 cycles)
Denaturation	95°C	15 SEC
Combined annealing/extension	60°C (data collection step)	30 SEC

Start the qPCR by clicking “Run.”

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.

Export the result to excel/csv file and upload both run and csv files.

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:

Generating Ct cut-off values using gBlocks gene fragments