SARS-CoV-2 Rapid Colorimetric LAMP Detection Assay Protocol (NEB #E2019)

Warnings and Precautions

  • Visualization of color analysis should be performed in a separate location from assay setup
  • DO NOT open assay tubes following incubation at 65°C. Visualize assay results, record results and dispose of assays immediately upon completion.
  • Waste should be disposed of in compliance with local, state and federal regulations
  • Always use pipette tips containing aerosol barriers that are sterile and free of DNases and RNases
  • Appropriate safety procedures should be followed at all times
  • Reagents must be stored at -20°C when not in use

Introduction

The SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit is a rapid colorimetric assay for in vitro detection of SARS-CoV-2 RNA that relies on loop-mediated isothermal amplification (LAMP). The WarmStart Colorimetric LAMP Master Mix with UDG combines WarmStart Bst 2.0 DNA Polymerase and WarmStart RTx Reverse Transcriptase to enable nucleic acid amplification at a single reaction temperature. The 2X colorimetric master mix also contains a weakly buffered solution and a pH-sensitive dye that changes color upon acidification. Amplification in the presence of target nucleic acids results in the production of protons that cause a decrease in pH, resulting in a clear visual color change from pink to yellow that is easily detectable by eye.

The WarmStart activated enzymes allow dual control of enzyme activity by reversible, aptamer-based inhibition (SOMAmers®). This temperature-dependent activation helps prevent undesired non-specific priming and extension prior to isothermal incubation at 65°C, providing added security for setting up reactions at room temperature. The colorimetric mix is enabled with carryover prevention (dUTP/UDG). It is formulated with a mixture of dTTP and dUTP. This ensures both efficient isothermal amplification as well as the incorporation of dU into the reaction products. LAMP products containing dU serve as a substrate for Antarctic Thermolabile Uracil DNA Glycosylase (UDG) present in the master mix, allowing carryover contamination prevention. Antarctic Thermolabile UDG will be completely inactivated upon isothermal incubation at 65°C. Because LAMP can generate large quantities of DNA in very short periods of time, best practices to reduce contamination involve not opening LAMP reactions post amplification.

The SARS-CoV-2 LAMP Primer Mix provided within the kit contains a mixture of primers that target both the nucleocapsid (N) gene and envelope (E) gene of SARS-CoV-2. The primer sets perform well individually but mixing them improves detection (https://pubmed.ncbi.nlm.nih.gov/32635743). Upon successful amplification, the reaction mix will change color from pink to yellow, or yellow/orange color. An internal control (IC) primer set that amplifies rActin is included to ensure the absence of inhibition from human nucleic acid templates. A positive control (PC) template containing the N-gene is also included. Guanidine hydrochloride has been shown to improve colorimetric LAMP at a concentration of 40 mM and is provided to supplement in the reaction for any samples that do not already contain guanidine. Guanidine concentrations up to 60 mM (final concentration at 1X) are tolerated. A sample can only be judged for the presence or absence of SAR-CoV-2 RNA if all reactions are pink prior to incubation and post incubation, the NTC reaction is pink, the PC reaction is yellow and the IC reaction is yellow.

Sample Compatibility

  • Sample input should be purified total nucleic acid eluted in nuclease-free water for best results
  • Material stored in TE or similar elution buffer should be kept to less than 5 μl (20% v/v) of the final reaction volume as excess buffer may inhibit the color change
  • Up to 1 μl (4% v/v) of transport media (UTM or VTM) may be used without impacting the colorimetric reaction
  • Acidic samples may immediately turn the colorimetric LAMP reaction orange or yellow upon addition. Try adjusting the sample pH to ~8.0 prior to addition to the reaction.
  • Samples should not contain SDS as it is a strong inhibitor of amplification
  • Small amounts of Tween® 20 or Triton® X-100 (≤ 0.2%) are tolerated
  • If samples contain a source of guanidine, ensure that the final concentration of guanidine remains less than 60 mM to avoid inhibiting the LAMP reaction

Protocol

1. Thaw WarmStart Colorimetric LAMP 2X Master Mix with UDG, LAMP Primer Mixes, Positive Control, and Guanidine Hydrochloride at room temperature. Once thawed, place on cold rack at 4°C or on ice.

2. Mix each component thoroughly by gently vortexing. Ensure that any precipitation in the WarmStart Colorimetric LAMP 2X Master Mix with UDG (NEB #M1804) is completely resuspended. Briefly centrifuge all Components to collect liquid at the bottom of the tubes before opening.

3.  A total of four reactions are required for each nucleic acid sample being evaluated:

#1. No Template Control (NTC)
#2. Positive Control
#3. Internal Control (rActin)
#4. SARS-CoV-2 Test Sample

The SARS-CoV-2 LAMP Primers will be used for the NTC, Positive Control, and SARS-CoV-2 sample reactions. The Internal Control confirms the activity of provided reagents, proper sample handling and the presence of human nucleic acid template using the Internal Control LAMP Primer Mix (rActin).

An overview of the reaction setup for one sample is described in the table below. Do not assemble the reactions until Step 4.

COMPONENT

#1.
NO TEMPLATE CONTROL

#2.
POSITIVE CONTROL

#3.
INTERNAL CONTROL

#4.
SARS-CoV-2 TEST SAMPLE

WarmStart Colorimetric LAMP 2X Master Mix with UDG

12.5 µl

12.5 µl

12.5 µl

12.5 µl

SARS-CoV-2 Positive Control (N gene)

2.0 µl

Internal Control LAMP Primer Mix (rActin)

2.5 µl

SARS-CoV-2 LAMP Primer Mix (N/E)

2.5 µl

2.5 µl

2.5 µl

Nuclease-free Water

7.5 µl

5.5 µl

5.5 µl

5.5 µl

Guanidine Hydrochloride*

2.5 µl

2.5 µl

2.5 µl

2.5 µl

Sample Nucleic Acid**

2.0 µl

2.0 µl

*Guanidine hydrochloride should be added to a final concentration of 40 mM only when samples do not already contain guanidine. If guanidine will be carried into the reaction with the input material, this volume should be replaced with nuclease-free water.

** For information regarding sample compatibility with colorimetric LAMP, please see Sample Compatibility section above

4. Prepare the components indicated below at room temperature for the number of samples (n) being evaluated. Volumes include 10% overage to accommodate transfer loss from pipetting.


SARS-CoV-2 LAMP Reaction Mix (for 3 of 4 reactions per sample):

COMPONENT VOLUME FOR ONE SAMPLE VOLUME FOR
n SAMPLES
VOLUME FOR 24 SAMPLES
WarmStart Colorimetric LAMP 2X Master Mix with UDG

41.25 µl

41.25 µl x n

990.0 µl

Nuclease-free Water

18.15 µl

18.15 µl x n

435.6 µl

SARS-CoV-2 LAMP Primer Mix (N/E)

8.25 µl

8.25 µl x n

198.0 µl

Guanidine Hydrochloride*

8.25 µl

8.25 µl x n

198.0 µl


*Guanidine hydrochloride should be added to a final concentration of 40 mM only when samples do not already contain guanidine. If guanidine will be carried into the reaction with the input material, this volume should be replaced with nuclease-free water.

Internal Control LAMP Reaction Mix (for 1 of 4 reactions per sample):
COMPONENT VOLUME FOR ONE SAMPLE

VOLUME FOR
n SAMPLES

VOLUME FOR 24 SAMPLES
WarmStart Colorimetric LAMP 2X Master Mix with UDG

13.75 µl

13.75 µl x n

330.0 µl

Nuclease-free Water

6.05 µl

6.05 µl x n

145.2 µl

Internal Control LAMP Primer Mix (rActin)

2.75 µl

2.75 µl x n

66.0 µl

Guanidine Hydrochloride*

2.75 µl

2.75 µl x n

66.0 µl


*Guanidine hydrochloride should be added to a final concentration of 40 mM only when samples do not already contain guanidine. If guanidine will be carried into the reaction with the input material, this volume should be replaced with nuclease-free water.

Aliquot 23 μl of the LAMP reaction mixes in a PCR strip tube or 96-well PCR plate at room temperature. Pipette 2.0 μl of Nuclease- free Water, SARS-CoV-2 Positive Control, or Sample Nucleic Acid into the reaction according to the table below.
COMPONENT

#1.
NO TEMPLATE CONTROL

#2.
POSITIVE CONTROL

#3.
INTERNAL CONTROL

#4.
SARS-CoV-2 TEST SAMPLE

SARS-CoV-2 LAMP Reaction Mix (prepared above)

23 µl

23 µl

23 µl

Internal Control LAMP Reaction Mix (prepared above)

23 µl

Nuclease-free Water

2.0 µl

SARS-CoV-2 Positive Control (N gene)

2.0 µl

Sample Nucleic Acid

2.0 µl

2.0 µl

 

5. Gently vortex reactions to mix well. Verify that all starting reactions are pink. A color change to yellow or orange upon the addition of sample nucleic acid for the Internal Control or SARS-CoV-2 test sample reaction indicates the input material is incompatible with the assay. It should not be interpreted to signify the presence of target nucleic acid. See sample combability section above for additional information. If the NTC or PC reactions turn yellow or orange, repeat assay set up from Step 4.

6. Place reactions directly into a pre-heated thermocycler or heat block set to 65°C. Incubate at 65°C for 30 minutes. For instruments with a heated lid, we recommend its use at 105°C. NOTE: For very low copy samples or maximum sensitivity, time may need to be extended to 40 minutes.

7. Allow reactions to cool from 65°C by placing at room temperature for 5 min for improved color contrast, or place tubes on ice for 5 seconds. DO NOT open the reaction vessels. Inspect reaction tubes for color and record results. NOTE: results can be read for up to 6 hours after reactions have been run.

8. A sample can only be judged for the presence or absence of SAR-CoV-2 RNA if the NTC reaction is pink, the PC reaction is yellow and the IC reaction is yellow. (See diagram below for interpretation of results).

9. Discard all completed reactions as medical waste.