TITLE: RT-qPCR Detection of Process Controls (Murine noroviurs and crAssphage) from Wastewater using AB 7500
AUTHORS: Jacquelina.Woods  , rachel.rodriguez  

[DESCRIPTION]
This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This protocol describes the murine norovirus (MNV; extraction control) and crAssphage (human indicator) RT-qPCR assay developed for use on the AB 7500 platform using software version 2.0 or 2.3. The assay incorporates an internal amplification control (IC) to prevent the reporting of false negatives due to inhibition or failure of the RT-qPCR. This multiplexed detection assay was developed for the determination crAssphage extracted from wastewater, as an endogenous control, and MNV as an extraction control. The assay is designed to be used in conjunction with the SARS-CoV-2 RT-qPCR detection assay. Valid sample results for SARS-CoV-2 detection are contingent upon the detection of the MNV extraction control from the sample being tested.  

[BEFORE_START]
Always wear gloves during this procedure and never wear the same gloves when going between master mix and samples. 

Always use aerosol resistant pipette tips for PCR.

 


[STEPS]
SECTION: Master Mix Preparation
1. 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 Process Controls from Wastewater Samples (protocols.io) and should be prepared in advance and stored appropriately. Alternatively, Master Mixes can be prepared from individual components as described here:  .

       
 


SECTION: Master Mix Preparation
1.1. Thaw Master Mix reagents in bench top cool block (chilled at 2-8 °C) or on ice in master mix preparation hood.

 

  
SECTION: Master Mix Preparation
1.2. Vortex reagent tubes for 3 sat setting medium high to high (if vortex has settings).  
SECTION: Master Mix Preparation
1.3. Briefly centrifuge all reagents 5 s in a personal microcentrifuge to bring liquid to the bottom of tube.

SECTION: Master Mix Preparation
1.4. Return all reagents to bench top cool block (chilled at 2-8 °C) or on ice.
 


SECTION: Master Mix Preparation
2. Proceed to hood/area or room where the template is added and thaw IC RNA and sample RNA in this hood/area.  

 

SECTION: Master Mix Preparation
2.1. Briefly centrifuge IC RNA 5 s in a personal microcentrifuge to bring liquid to the bottom of tube.
SECTION: Master Mix Preparation
2.2. Add appropriate volume of IC RNA (0.2 µLper reaction) to Master Mix from Step 1.4 in cold block/on ice.
SECTION: Master Mix Preparation
2.3. Vortex briefly and centrifuge 5 s in a personal microcentrifuge.
SECTION: Reaction Set-Up
3. Add 22 µL of Master Mix to each designated reaction tube or sample wells.

SECTION: Reaction Set-Up
4. Briefly centrifuge sample RNA 5 s in a personal microcentrifuge to bring liquid to the bottom of tube.
SECTION: Reaction Set-Up
5. Add 3 µL of sample RNA template to each of three reaction tubes or wells.

SECTION: Reaction Set-Up
6. Ensure each plate or run has appropriate controls (positive and negative controls) included.  

 

SECTION: Reaction Set-Up
7. Seal sample plate or strip tubes. Then, briefly spin 5 s.
SECTION: Reaction Set-Up
8. Start run on Applied Biosystems 7500 Fast instrument.

 

SECTION: Reaction Set-Up
8.1. Use the following settings for the Experiment Properties:
"7500Fast (96 wells)"
"Quantitation Standard Curve"
"TaqMan Reagents"
"Standard (~2 hours to complete run)"

   

SECTION: Reaction Set-Up
8.2. Identify the appropriate target reporters (MNV-Cy5, crAss-JOE, IC-TexasRed) and leave all quenchers as "NFQ-MGB".

  

SECTION: Reaction Set-Up
8.3. Select appropriate passive reference dye (FAM for the triplex assay). 
  

SECTION: Reaction Set-Up
8.4. Assign targets and samples. 
  

SECTION: Reaction Set-Up
8.5. Use the following settings for Run Method:
25 µL reaction volume
Holding stage 1: 50 °C for 50 min
Holding stage 2: 95 °C for 15 min
Cycling stage: 45 cycles of 95 °C for 15 s, 55 °C for 20 s, 62 °C for 1 min
Enable data collection on Step 3 of Cycling stage
  

SECTION: Data Analysis
9. Adjust analysis settings to appropriate thresholds. All thresholds should be set at 0.01. Baseline start cycle should be set at 3 and baseline end cycle should be set at 10.

   


SECTION: Data Analysis
10. Verify positive and negative calls for each reaction using either linear or log amplification plots.
SECTION: Data Analysis
10.1.   

SECTION: Data Analysis
10.2.   

SECTION: Data Analysis
10.3.   

SECTION: Data Analysis
10.4.   

SECTION: Data Analysis
11. Sample is invalid if any of the following are observed: 

1. Negative RT-qPCR control is positive (Ct value indicated) for any of the expected targets (MNV or crAssphage);
2. Positive RT-qPCR control is negative (undetermined) for expected target/s (MNV or crAssphage); 
3. Sample is negative (undetermined) for MNV; or
4. IC is negative (undetermined) in the sample, or the average of the IC Ct values from the sample replicates are greater than 4 Cts than the IC Ct of the negative RT-qPCR control.

 

SECTION: Data Analysis
12. Sample is valid if all of the following are observed: 

1. Negative RT-qPCR control is negative (undetermined) for expected target/s (i.e., MNV and crAssphage); 
2. Positive RT-qPCR control is positive (Ct value indicated) for expected target/s (i.e., MNV and crAssphage); and  
3. Internal amplification control (IC) is positive in all sample reactions with the average IC Ct values for the sample is less than 4 Cts greater than the IC Ct of the negative RT-qPCR control.