COVID-19 Sample Pooling: From RNA Extraction to Quantitative Real-time RT-PCR

Materials and Reagents

1. Personal protective equipment (PPE) appropriate for working with SARS-CoV-2

2. 0.2 ml low-profile PCR tube strips with caps (Bio-Rad, catalog numbers: TLS0851 and TCS0903)

3. 1.5 ml microcentrifuge tubes (Axygen, catalog number: MCT-150-C-S)

4. 0.5-10 µl, 0.5-50 µl, 1-200 µl, 100-1,000 µl filtered pipette tips (Axygen, catalog numbers: TF-400-R-S, TF-50-R-S , TF-200-R-S, TF-1005-WB-R-S)

5. Geneaid Viral Nucleic Acid Extraction Kit (Geneaid Biotech, catalog number: VR300)

6. Qiagen DNeasy Blood and Tissue Kit (Qiagen, catalog number: 69506)

7. SuperScript^TM III One-Step RT-PCR System with Platinum^TM Taq DNA Polymerase (Invitrogen, catalog number: 12574-026) (aliquot and store at -20°C)

8. DNase- and RNase-free water (Integrated DNA Technologies, catalog number: 11-05-01-04) (aliquot into clean 1.5-ml microcentrifuge tubes)

9. Primers for SARS-CoV-2 (Integrated DNA Technologies) (Table 1)

   
   

   Table 1. Primers and probes targerting RNA-dependent RNA polymerase (RdRP), SARS-CoV-2 detection^a

   Catalog number	Oligonucleotide	Sequenceb
   CV001	RdRp_SARSr-F	5’-GTGARATGGTCATGTGTGGCGG-3’
   CV002	RdRp_SARSr-R	5’-CARATGTTAAASACACTATTAGCATA-3’
   CV101	RdRP_SARSr-P1	5’-FAM-CCAGGTGGWACRTCATCMGGTGATGC-BBQ-3’
   CV102	RdRp_SARSr-P2	5’-FAM-CAGGTGGAACCTCATCAGGAGATGC-BBQ-3’

   ^a Oligonucleotide sequences were adapted from the WHO-Charité protocol (Corman et al., 2020).

   ^b W is A/T; R is G/A; M is A/C; S is G/C. FAM: 6-carboxyfluorescein; BBQ: blackberry quencher.

   
   

10. Primer mix (see Recipes）

Equipment

1. 1 µl-10 µl, 10 µl-100 µl, 20 µl-200 µl, 100 µl-1,000 µl micropipettors (Eppendorf, catalog numbers: 3123000020, 3123000047, 3123000055, 3123000063)

2. CFX96 RealTime System (Bio-Rad, model: C1000 touch)

3. Thermo Aluminium Bath (Fine PCR, model: ALB128) to be used with the Qiagen DNeasy Blood and Tissue Kit

4. Centrifuge 5425 (Eppenddorf, catalog number: 5405000417)

Procedure

All procedures in (A) sample pooling, (B) preparation of positive sample-pool, (C) ribonucleic acid (RNA) extraction, and (D) quantitative reverse transcription polymerase chain reaction (RT-qPCR) were carried out separately inside different biosafety cabinets. All clinical waste and consumables were autoclaved prior to being sent for incineration.

1. Sample pooling (see Figure 1)

   1. We chose 10 sample-pools since the positive detection rate in our laboratory is less than 2%. You may optimize the sample-pool size according to the positive detection rate in your laboratory (Abdalhamid et al., 2020).

   2. Pipette 60 µl each of the 10 clinical NP/OP swabs into a single 1.5-ml microcentrifuge tube. The total volume is 600 µl. The original volume of the specimens ranged from 1 ml to 3 ml.

   3. Aliquot 300 µl pooled swabs to each of two clean 1.5-ml microcentrifuge tubes and label with the same sample ID.
      
      
      Figure 1. Pooling of 10 individual samples




2. Preparation of positive sample-pool

   1. Identify a positive NP/OP swab that has a C_T value ≥15.00 but ≤36.00 (refer to Table S2 in Lim et al., 2020).

   2. Aliquot the identified positive NP/OP swab and store at -20°C.

   3. Prepare the positive sample-pool according to Steps A2-A3 by mixing 1 positive with 9 unknown samples.




3. RNA extraction

   1. Choose either of the kits below for RNA extraction

      1. Geneaid Viral Nucleic Acid Extraction Kit

         1. Add 400 µl VB Lysis Buffer to each of the 1.5-ml microcentrifuge tubes. Shake the tube virgorously for 10 s.

         2. Incubate at room temperature for 10 min.

         3. Add 450 µl AD Buffer (make sure that the ethanol has been added) to each of the 1.5-ml microcentrifuge tubes. Shake the tube vigorously to mix.

         4. Transfer 700 µl lysate mixture to the VB column in a 2-ml collection tube. Centrifuge at 14,000 × g for 30 s. Discard the flowthrough and place the VB Column back into the 2-ml collection tube. Repeat Step (iv) until all the mixture (two 1.5-ml microcentrifuge tubes prepared in Step A3) has completely flowed through the VB column.

         5. Add 400 µl W1 Buffer to the VB column and centrifuge at 14,000 × g for 30 s. Discard the flowthrough and place the VB column back into the 2-ml collection tube.

         6. Add 600 µl Wash Buffer (make sure that the ethanol has been added) to the VB column. Centrifuge at 14,000 × g for 30 s. Discard the flowthrough and place the VB column back into the 2-ml collection tube.

         7. Centrifuge at 14,000 × g for 3 min to dry the column matrix.

         8. Place the dried VB column in a clean 1.5-ml microcentrifuge tube. Add 35 µl DNAse RNase-free water to the center of the VB column matrix and incubate for 3 min at room temperature (15-25°C).

         9. Centrifuge at ≥6,000 × g for 1 min to elute the purified nucleic acid.

      2. Qiagen DNeasy Blood and Tissue Kit

         1. Pipette 20 µl Proteinase K into each 1.5-ml microcentrifuge tube.

         2. Add 200 µl Buffer AL and mix thoroughly by shaking vigorously.

         3. Incubate at 56°C for 10 min.

         4. Add 200 µl ethanol (96-100%) and mix thoroughly by shaking vigorously.

         5. Pipette 700 µl mixture into a DNeasy Mini spin column placed in a 2-ml collection tube. Centrifuge at ≥6,000 × g for 1 min. Repeat Step (v) until all the mixture (two 1.5-ml microcentrifuge tubes prepared in Step A3) has completely flowed through the spin column. Discard the flowthrough and collection tube.

         6. Place the spin column into a clean 2-ml collection tube.

         7. Add 500 µl Buffer AW1 and centrifuge for 1 min at ≥6,000 × g.

         8. Discard the flowthrough and collection tube.

         9. Place the spin column in a clean 2-ml collection tube, add 500 µl Buffer AW2, and centrifuge for 3 min at 14,000 × g.

         10. Discard the flowthrough and collection tube.

         11. Transfer the spin column into a clean 1.5-ml microcentrifuge tube.

         12. Elute the RNA by adding 35 µl DNAse- and RNAse-free water to the center of the spin column membrane. Incubate for 3 min at room temperature (15-25°C).

         13. Centrifuge for 1 min at ≥6,000 × g to elute the purified nucleic acid.




4. RT-qPCR

   1. Prepare the RdRP-Primer/Probe mixture (10 µM) (see Recipe).

   2. Prepare 3 tests: (1) positive sample-pool; (2) non-template control (NTC); and (3) unknown sample-pool.

   3. Prepare the RT-qPCR mixture using the SuperScript^TM III One-Step RT-PCR System with Platinum^TM Taq DNA Polymerase as described in Table 2.
              
      Table 2. RT-qPCR mixture preparation

      Reagent	Final concentration	Volume (µl) for one reaction
      2× reaction buffer	1×	10.0
      SuperScriptTM III RT/PlatinumTM Taq Mix	-	0.5
      RdRP-Primer/Probe mixture (10 µM)	2 µM	1.0
      Purified nucleic acid (10 sample pool)a	-	8.5
      Total volume		20.0

      ^a Replace purified nucleic acid with DNAse- and RNAse-free water for NTC.

   
   

   4. Run the RT-qPCR cycles as described below:

      55°C for 15 min

      95°C for 2 min

      95°C for 15 s and 56°C for 30 s for 50 cycles

Data analysis

1. Intepret and record the results using the criteria below (see Notes for explanation):

   1. If the C_T value is ≤14.00, consider it as contamination. Repeat the RT-qPCR of the pooled purified nucleic acid. Upon detection of the same C_T value in the repeated RT-qPCR, deconvolute the positive pool(s) and repeat the nucleic acid extraction and RT-qPCR on individual samples from the positive pool(s). Test results are confirmed by a conventional RT-qPCR clinical test on a single sample.

   2. If the C_T value is >14.01 but ≤45, record the C_T value and consider positive detection of SARS-CoV-2 in the pooled samples. Deconvolute the positive pool(s) and repeat the nucleic acid extraction and RT-qPCR on individual samples from the positive pool(s). Test results are confirmed by a conventional RT-qPCR clinical test on a single sample.

   3. If the C_T value is ≥45.01 or no C_T value is detected, record the pooled samples as negative. Each single sample is reported as negative in this pool.

2. An example of the analysis is shown in Tables 3, 4, and 5 (extracted Dataset S1 from Lim et al., 2020). Here, we briefly describe the data shown in Tables 3-5.

       On 6^th April 2020, the total number of specimens to be extracted was 101 (including an internal positive control). We performed sample pooling according to Steps A1-A3. Groups A-I were pools of 10, while Groups J and K were pools of 5 (total volume 300 µl) and 6 (total volume 360 µl), respectively. The RNA was extracted from the pooled samples as per Steps C1a.i-ix. The RT-qPCR was performed as per Steps D1-D4. Results of pooled testing are indicated in Table 3. SARS-CoV-2 was detected in Groups I and J. Individual specimens from both groups were individually extracted again according to the manufacturer’s protocol and RT-qPCR was performed as per Steps D1-D4. The results of individual testing are stated in Tables 4 and 5 for Groups I and J, respectively.

   
   

   Table 3. April 7^th, 2020. IMU COVID-19 pooled testing results
   
   

   ^aGroup I was prepared as per step B3, spiked with one internal positive control (9 samples + 1 internal +ve control).
   ^bGroups J and K were pools of 5 (total volume 300 µl) and 6 (total volume 360 µl), respectively. Aliquots of 150 µl (Group J) and 180 µl (Group K) were pipetted into two 1.5-ml microcentrifuge tubes as per step A3.
   
   Table 4. Individual testing RT-qPCR results for pooled Group I

   Group I	IMU Ref#	CT value
   		Pooled testing (1st qRT-PCR)	Individual (2nd qRT-PCR)
   1	1458	COVID19 Detected (23.71)	Not Detected
   2	1459		Not Detected
   3	1460		Not Detected
   4	1461		Not Detected
   5	1462		Not Detected
   6	1463		Not Detected
   7	1464		Not Detected
   8	1465		Not Detected
   9	1466		Not Detected
   10	Internal +ve control		COVID19 Detected (23.23)

   
   Table 5. Individual testing RT-qPCR results for pooled Group J

   Group J	IMU Ref#	CT value
   		Pooled testing (1st RT-qPCR)	Individual (2nd RT-qPCR)
   1	1467	COVID19 Detected (40.16)	Not Detected
   2	1468		Not Detected
   3	1469		Not Detected
   4	1470		Not Detected
   5	1471		Not Detected

Notes

C_T value: We chose the C_T value cut-off based on our preliminary data obtained by optimizing the pooling. We noticed that a weak positive C_T value of 38 may increase to 40 after pooling; thus, we recommend a C_T value cut-off of 45 to safeguard the accuracy and precision of the detection method. Any pooled samples considered to be positive, with C_T values detected below 45, will need to be individually tested for accurate reporting. Please see the example provided in Table 3.

Recipes

1. Primer mix

   1. Prepare each one of the primers and probes at 100 µM

   2. Primer/probe mix (100 µl):

      RdRp_SARSr-F 10 µl (10 µM final concentration)

      RdRp_SARSr-R 10 µl (10 µM final concentration)

      RdRP_SARSr-P1 10 µl (10 µM final concentration)

      RdRp_SARSr-P2 10 µl (10 µM final concentration)

   3. DNase- and RNase-free water 60 µl

   4. Aliquot and store at -20°C

Acknowledgments

This study was supported by a grant from the International Medical University (IMU), Malaysia (IMU 477-2020). We thank the National Public Health Laboratory (NPHL), Ministry of Health Malaysia for their generosity and support in sharing the necessary reagents for laboratory testing. This protocol was derived from our previous work (Lim et al., 2020).

Competing interests

The author(s) declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. While performing this research, no company had invested in the research and no commercialization was intended.

Ethics

This study obtained ethical clearance from the International Medical University Joint Research and Ethical Committee (EC/IRB Ref. No. 4.41/JCM-196/2020). All clinical specimens used in this study were fully anonymized and deidentified by assigning new laboratory reference numbers before access was given to the researchers. The researchers were blinded to patient information.

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