A Protocol for Simple, Rapid, and Direct Detection of SARS-CoV-2 from clinical samples, using Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP)

Material and Reagents

1. Personal protective equipment (PPE) appropriate for working with SARS-CoV-2
2. 0.2 ml PCR tube strips (Labcon, catalogue number: LC 3927-550)
3. Pipette tips
4. WarmStart^® Colorimetric LAMP 2x Master Mix (New England BioLabs Inc., catalog number: M1800 ) (aliquot and store at -20 °C)
5. Proteinase K (Seegene, catalog number: 744300.4.UC384, store at -20 °C)
6. Guanidine hydrochloride (Sigma, catalog number: G4505 )
7. DNase RNase free water (Biological Industries, catalog number: 01-869-1B)
8. Primers for SARS-CoV-2 (Table 1) (Zhang et al., 2020)
9. Primers for pop7–positive internal processing and amplification control (Table 2) (Curtis et al., 2018)
10. Primer mix (see Recipes)
11. Proteinase K (see Recipes)
12. Guanidine hydrochloride (see Recipes)
    
    Table 1. SARS-CoV-2 Primers for RT-LAMP
    

    
    
    Table 2. RNaseP pop7 Primers for RT-LAMP–Positive Control

Equipment

1. 1 µl-10 µl, 10 µl-50 µl, 20 µl-200 µl pipettes
2. PCR Thermocycler as heat source (Biometra, catalog number: T3000 )
   

Procedure

1. Prepare primer mix. (see Recipes)
   Prepare two mixes: (1) For SARS-Cov-2 test. (2) For pop7 positive internal processing and amplification control.


2. Sample lysis and preparation (see Figure 1A)
   1. For saliva samples: Resuspend in 1 ml of DNase RNase free water (in the same container of the saliva sample). For throat/nasal swab samples: Use the standard universal transfer media (UTM) for this test.
      
   2. Add 5 µl of the sample (from Step B1) to a PCR tube containing 40 µl of DNase RNase free water.
   3. Include a non-template control. Add 5 µl DNase RNase free water instead of the sample.
   4. Add 2 µl proteinase K (final concentration 1.22 mg/ml).
   5. Incubate at room temperature for 15 min.
   6. Incubate samples at 95 °C for 5 min in a PCR machine or any other heat source.
      Note: We have successfully used thermal cup and hot water.
      
   7. Cool your sample down to room temperature.
      
      
3. RT-LAMP reaction (see Figure 1A)
   Prepare three mixes: (1) For SARS-CoV-2 test. (2) For pop7 gene as positive control. For each mix use the relevant primers. (3) For non-template control (see Step B3) using the SARS-CoV-2 primers.
   
   1. Prepare reaction:
      LAMP mix                                                           10 µl
      Primer mix                                                           2 µl
      Guanidine hydrochloride                                   1 µl
      Sample lysate (product of Procedure B)          7 µl
   2. Incubate at 65 °C for 30-40 min.
   3. Read results:
      If the mix color changes from pink to yellow, the sample has tested positive (see Figure 1B).
      If the mix maintains its pink color, the sample has tested negative (see Figure 1B).
      Please note that the results are binary as positive or negative. Only bright yellow results are considered positive. Pink is considered negative, and any other range or gradient of color is considered negative.
      Positive control results were published at Ben-Assa^*, Naddaf^* et al., 2020 (Figure 3a).
      
      
      Figure 1. Procedure and results of SARS-CoV-2 RT-LAMP detection test. A. An illustration of the steps of the protocol: this illustration summarizes the main steps of the protocol. Illustration was created with BioRender.com. B. One negative sample (Neg S.) and one positive sample (Pos S.) before and after incubation. At t = 0 min (left panel), all samples were pink. After incubation of 30min (right panel), positive samples turned yellow while negative samples retained their pink color. Pictures are representative RT-LAMP test results of clinical diagnostic nasal and throat swabs of data published in Ben-Assa^*, Naddaf^* et al., 2020. Results were confirmed by conventional RT-qPCR clinical test for SARS-CoV-2 following RNA extraction and purification step.

Notes

1. Alternative to a PCR machine, any heating source (e.g., water bath) can be used for Steps B5 and C2. We have successfully used a thermos cup and a thermometer to adjust water temperature.
2. Longer incubation than 40 min may result in non-specific color change and interpreted as false positive results.
3. It is very important to use proper personal protective equipment when handling the samples starting from step B and throughout the rest of the protocol.
4. The color change in the protocol is an indication for a binary positive/negative result and does not represent any gradient indication of the clinical status of the patient.
   

Recipes

1. Primer mix
   
   1. Prepare each one of the primers at 100 µM
   2. Primer mix:
      F3                    2 µl (2 µM final concentration)
      B3                    2 µl (2 µM final concentration)
      LF                    4 µl (4 µM final concentration)
      LB                    4 µl (4 µM final concentration)
      FIP                   16 µl (16 µM final concentration)
      BIP                   16 µl (16 µM final concentration)
   3. DNase RNase free water 56 µl
   4. Aliquot and store at -20 °C
      
2. Proteinase K at stock concentration 28.67 (mg/ml)
   Soluble in DNase RNase free water
   Store at -20 °C
3. Guanidine hydrochloride
   Soluble in DNase RNase free water
   Stock concentration, 800 mM
   Store at room temperature
   

Acknowledgments

We thank the Geva-Zatorsky lab for fruitful discussions and contributions. We would like to thank the Rambam hospital for their support and for hosting us, especially to the infectious disease and virology teams. We would also like to thank Dr. Rich Roberts and Dr. Nathan Tanner for their valuable support, as well as Prof. Daniel King, Prof. Yehuda Chowers, Dr. Ronit Almog, Dr. Yuval Geffen, Dr. Dani Zvi-Bar and Prof. Oded Lewinson for their crucial help. In addition, we would like to thank Dima Abdu for preparing the figure for this paper. The illustration in this in Figure 1B was created with BioRender.com.
   This work was supported by the Technion Integrated Cancer Center, the Technion – Israel Institute of Technology, “Keren Hanasi”, Alon Fellowship for Outstanding Young Researchers, Horev Fellow (Taub Foundation). NGZ is an Azrieli Global Scholar at the Canadian Institute for advanced research (CIFAR).

Competing interests

The author(s) declared 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 was granted exemption from IRB approval of the Rambam Health Care Campus (# 0244-20-RMB) for use of de-identified COVID-19 tests performed for the purpose of the standard testing, and for 4 volunteers.

References

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