TITLE: Mirimus COVID-19 Pool Surveillance RT-PCR Testing
AUTHORS: Prem Premsrirut, Ana Vasileva, Huiting Cheng

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Overview</span></div><div class = "text-block">At the start of the Pandemic in the US, New York was hit with more cases of COVID-19 than any single country in the world. Although case numbers are finally at an all-time low, the question of how to safely reopen the economy, school and businesses still remains unclear. Today, SARS-CoV-2 detection via RT-PCR methods remains the gold standard to detect active infection and recommend isolation of individuals in order to prevent further spread. Unfortunately, the cost and bottlenecks created from repeated massive testing efforts have become unsustainable. As a way to address this issue, we have devised a strategy of pooling samples and testing entire populations together as one unit. This cuts the cost of testing dramatically and still enables the detection of a susceptible group that would need to undergo individual testing.</div><div class = "text-block">To meet the COVID-19 testing demands, we created an organizational-based pooling strategy, whereby organizations can enroll in surveillance testing to monitor groups of people through pooled testing. By using organizational-based pooling, we avoid random pooling of samples. Instead, we pool groups of known contacts from a defined workplace, school or organization where people regularly interact. Therefore, a positive case would likely have an impact on the entire pooled population and the entire pooled population can be treated as an infected cohort until further individual testing, potentially, through a healthcare provider is recommended or offered.</div><div class = "text-block"><span style = "font-weight:bold;">Method of Pooled Testing</span></div><div class = "text-block">For pooled surveillance testing to be effective and widely adopted, sample collection methods must also be simplified and non-invasive in order to achieve high compliance and enrollment into a surveillance program. The method of testing must also be highly sensitive to accurately detect one positive case in a large pool of negative samples. For these reasons, we developed a simple saliva collection method with a validated pooled saliva specimens using RT-PCR and an acceptable limit of detection, which will be the most sensitive diagnostic test to date. We established automated methods to pool up to 24 saliva specimens and if a positive case is detected, the pools can be further broken down to pools of 2 specimens. The remaining 2 individuals within a pool will be referred to or, potentially, offered further clinical diagnostic testing by a healthcare provider.</div></div>

[STEPS]
?. [Sample Pooling]
Place 24 tubes on rack.  Place pool tube in position F8. Place organization tube in position F1. Barcode scan the rack. Initiate automated decapping on the Hamilton.
?. [Sample Pooling]
Initiate automated decapping. Move decapped rack to the Integra assist.
?. [Sample Pooling]
Place 8 tubes of 400mM DTT in position B on the assist. Place the specimen rack in position A. Place tip box and pool reservoir in their proper position.
?. [Sample Pooling]
Initiate the pooling automated program on the Integra Assist
?. [Sample Pooling]
Repeat pooling for each cohort
?. [RNA extraction]
Create the Wash Buffer plate 2.Add   of Wash Buffer to each well.
500 µl
?. [RNA extraction]
Create the  ethanol plate 3.Add  of to each well.
1000 µl
?. [RNA extraction]
Create the Elution plate 4.Add  of elution buffer to each well.
50 µl
?. [RNA extraction]
Scan the pool tube for QC. Initiate automated decapping.
?. [RNA extraction]
Aspirate and mix each sample 5 times. Aspirate and dispense  from each pool tube into Sample Plate 1.
200 µl
?. [RNA extraction]
Add  of  to the negative control well.
200 µl
?. [RNA extraction]
Load the King Fisher with each plate and initiate the RNA extraction. Use the standard program: MVP_2Wash_200_Flex
?. [Sample Pooling]
?. 
?. [Sample Pooling]
1) Aspirate   of    DTT2) Dispense   of  DTT into each sample3) Mix samples 5 times, using speed setting 4) Aspirate  from each specimen5) Dispense  into the reservoir6) Repeat for each row until all specimens are pooled7) Transfer the pooled specimen to the pool tube8) Place the pool tube in a new specimen rack
50 µl
50 µl
200 µl
200 µl
?. [Sample Pooling]
Rack all pool tubes and scan again. This scan will autopopulate the qPCR template for the run.
?. [Sample Pooling]
?. [RNA extraction]
Set up RNA extraction using the reagents provided in the
?. [RNA extraction]
Create the sample plate 1 by combing the following from the For each pool specimen (N), use the following recipe1)  of Binding Solution [ X (N+1) X 1.1]2)  of MVPII Binding Beads [ X (N+1) X 1.1]. Be sure to vortex well before using.3)  of Proteinase K [ X (N+1) X 1.1]4)  of MS2 Phage [ X (N+1) X 1.1] from the Vortext and then add  of the mixture to each sample well using the multichannel pipettes.
265 µl
265 µl
10 µl
10 µl
5 µl
5 µl
5 µl
5 µl
285 µl
?. Transfer the purified RNA from the KingFisher Elution plate to the qPCR using the Add in the positive control
?. [RT-PCR setup]
Run the program on the Mantis, to aliquot  of Master Mix to each well. Plate in triplicates for each pool sample into  . Include an additional reaction for the positive control.
5 µl
?. [RT-PCR setup]
Generate  TaqPath COVID-19 Master Mix according to the recipe per pool sample plus 1 positive control:1)  of 2)   of primers from the 3)of The following formula can also be use:50% Taqpath 1-step master mix10% Taqpath combo kit primers40% H20
7.5 µl
1.5 µl
6 µl
?. [RT-PCR setup]
PERFORM THE NEXT STEPS IN THE CLEAN ROOM. Set-up the RT-PCR plate using the . Select program and determine the volume required.
?. [RNA extraction]
Label  4 Deep Well Plates and generate1) Samples2) Wash Buffer3)  Ethanol4) Elution
?. Seal the plate with
?. Vortex for 30 sec. Centrifuge the plate for 10 sec.
?. Place the plate in the Use the program: 1 cycle 1 cycle 1 cycle45 cycles of:
25 °C
53 °C
95 °C
95 °C
60 °C