RT-qPCR Detection of SARS-CoV-2 from Wastewater Using the AB 7500

Prepare Master Mix for all sample and control reactions based on the volumes per 25 µl reaction in this table. Composition of mixes are listed here: Reagent Mixes for RT-qPCR Detection of SARS-CoV-2 from Wastewater (protocols.io) and should be prepared in advance and stored appropriately. Alternatively, Master Mixes can be prepared from individual components as described here: Master Mix Tables for SARS-CoV-2 Assays .pdf.

Thaw Master Mix reagents in bench top cool block (chilled at 2-8°C) or 4On ice in master mix preparation hood.

Vortex reagent tubes for 0h 0m 3sat setting medium high to high (if vortex has settings).

Briefly centrifuge all reagents 0h 0m 5s in a personal microcentrifuge to bring liquid to the bottom of tube.

Return all reagents to bench top cool block (chilled at 2-8°C) or 4On ice.

Proceed to hood/area or room where the template is added and thaw IC RNA and sample RNA in this hood/area.

Briefly centrifuge IC RNA 0h 0m 5s in a personal microcentrifuge to bring liquid to the bottom of tube.

Add appropriate volume of IC RNA (0.2µLper reaction) to Master Mix from Step 1.4 in cold block/on ice.

Vortex briefly and centrifuge 0h 0m 5s in a personal microcentrifuge.

Add 22µL of Master Mix to each designated reaction tube or sample wells.

Briefly centrifuge sample RNA 0h 0m 5s in a personal microcentrifuge to bring liquid to the bottom of tube.

Add 3µL of sample RNA template to each of three reaction tubes or wells.

Ensure each plate or run has appropriate controls (positive and negative controls) included.

Seal sample plate or strip tubes. Then, briefly spin 0h 0m 5s.

Start run on Applied Biosystems 7500 Fast instrument.

Use the following settings for the Experiment Properties:

"7500Fast (96 wells)"

"Quantitation Standard Curve"

"TaqMan Reagents"

"Standard (~2 hours to complete run)"

Identify the appropriate target reporters and leave all quenchers as "NFQ-MGB".

Select appropriate passive reference dye (FAM for the triplex assay).

Assign targets and samples.

Use the following settings for Run Method:

25µL reaction volume

Holding stage 1: 50°C for 0h 50m 0s

Holding stage 2: 95°C for 0h 15m 0s

Cycling stage: 45 cycles of 95°C for 0h 0m 10s, 56°C for 0h 0m 25s, 62°C for 0h 1m 0s

Enable data collection on Step 3 of Cycling stage

Adjust analysis settings to appropriate thresholds. For the triplex assay, N1 and N2 should be set at 0.01 and the IC set at 0.1 . Baseline start cycle should be set at 3 and baseline end cycle should be set at 10.

Verify positive and negative calls for each reaction using either linear or log amplification plots.

RT-qPCR run is invalid if any of the following are observed:

1. Negative RT-qPCR control is positive (Ct value indicated) for any of the expected SARS-CoV-2 targets;

2. Positive RT-qPCR control is negative (undetermined) for expected SARS-CoV-2 targets; or

3. IC is negative (undetermined) in the negative RT-qPCR control; or

Sample is negative if:

1. Negative and positive RT-qPCR control reactions give appropriate results;

2. Sample reaction is negative (undetermined) for expected SARS-CoV-2 target/s; and

3. Internal amplification control (IC) is positive.

Sample is positive if:

1. Negative and positive RT-qPCR control reactions give appropriate results; and

2. Sample reaction is positive (Ct value indicated) for expected SARS-CoV-2 target/s.