Researchers evaluate a loop-mediated isothermal amplification (LAMP) assay using dry reagents for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). They found the test kit to be highly specific and sensitive. This method could enable point-of-care diagnosis in low-cost settings.
The rapid spread of the COVID-19 pandemic caused by the SARS-CoV-2 virus has necessitated the development of diagnostic methods that can quickly identify if a patient has been infected with the virus. Rapid detection of the virus is essential, as many of the symptoms of the infection, such as fever and cough, are similar to seasonal influenza or other respiratory illnesses. In addition, many patients have mild symptoms or are asymptomatic.
The most commonly used test is the real-time reverse transcription-polymerase chain reaction (RT-PCR). The method needs a special thermal cycler and precision optics to measure the emitted fluorescence from the samples. These requirements generally preclude its use in point-of-care (POC) settings.
Another method called the loop-mediated isothermal amplification (LAMP) method can amplify DNA under isothermal conditions with high specificity and sensitivity. This method is cheaper and faster than RT-PCR and has been used in POC testing of many diseases.
Transmission electron micrograph of SARS-CoV-2 virus particles, isolated from a patient. Image captured and color-enhanced at the NIAID Integrated Research Facility (IRF) in Fort Detrick, Maryland. Credit: NIAID
Testing LAMP assay with dry reagents
In a new study published on the preprint server medRxiv*, researchers evaluated the suitability of the Loopamp SARS-CoV-2 Detection kit from Eiken Chemica, Japan, for detecting SARS-CoV-2. This is a LAMP method that uses dry reagents for RNA amplification.
Dry reagents are more stable than liquid reagents, do not require a freezer for storage, and are easier to handle.
This will allow the method to be used at low-cost and developing countries, as the reagents can be stored in the refrigerator at about 4 °C and do not require stringent cold-chain transport and storage.
For evaluation of the LAMP method, the researchers first extracted RNA from nose and throat swabs from patients. They performed reverse transcription (RT)-LAMP using the Loopamp SARS-CoV-2 detection kit from Eiken Chemical.
All the reagents, except the primers, are dried and immobilized inside the tube lid. The authors added the purified RNA, and the SARS-CoV-2 specific primer sets to the tube bottom and shook the tube several times to resuspend the enzyme and buffer. They incubated the mixture, collected at the bottom of the tube by a spin down, for 35 minutes at 62.5 °C.
To test the sensitivity of the kit, the authors tested 22 viruses, including SARS coronavirus, Middle East respiratory (MERS) syndrome coronavirus, other human coronaviruses, and influenza viruses.
They did not see any amplification of viruses other than SARS-CoV-2, indicating that the test was specific to the virus. The researchers confirmed these results using a turbidity assay and agarose gel electrophoresis analysis.
To define the sensitivity of the test, the researchers used in vitro transcribed RNA serially diluted in a buffer and used carrier RNA at a concentration of 50 ng/mL to define the detection limit.
The detection limit found by the authors was 10 copies/reaction using turbidity assays and color changes seen with the eye and using UV illumination. This is similar to or slightly higher than those for LAMP assays for SARS-CoV-2.
The authors also evaluated their test using 24 clinical samples, which included three asymptomatic people who were in close contact with COVID-19 patients. The samples were collected between 7 March and 30 April 2020.
Using RT-PCR, 19 of the 24 samples resulted positive for SARS-CoV-2, whereas 15 samples showed positive results using the LAMP test evaluated by the authors.
The four false-negative samples, collected more than 7 days after the onset of the disease, had low copies of viral RNA of fewer than 10 copies/reaction, which is the detection limit of the new test. Two of the four false-negative samples were collected on days 18 and 20 to confirm the absence of the virus before discharge from the hospital.
Hence, the authors calculated the specificity, sensitivity, positive predictive value, and negative predictive value to be 100%, 78.9%, 100%, and 55.6%, respectively.
“Given that both turbidity assay and colorimetric changes detected all samples with more than 4.4 copies/reaction, we believe that this SARS-CoV-2 dry LAMP method is reliable for clinical use in diagnosing COVID-19,” write the authors.
A previous study has reported that SARS-CoV-2 RNA can be amplified in the LAMP assay without extraction. Incorporating such a method without RNA extraction could help a direct detection version of the test method for POC tests.
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.