Even as COVID-19 continues its inexorable march, taking hundreds of thousands of lives and causing nearly 11 million cases, the diagnosis of the infection is still not sensitive or specific enough. Now, a new study by scientists at the Francis Crick Institute and published on the preprint server medRxiv* in June 2020 describes a standard operating procedure (SOP) to enable rapid and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by an RT-LAMP assay. This would enable a reliable, repeatable, and cost-effective method of detection.
The Reason for the Current Study
The current study was motivated by the experience of the researchers at their biomedical research institute, during the early part of the peak phase of the pandemic. They set up a diagnostic testing workflow in partnership with affiliate hospitals and an accredited diagnostic laboratory to increase testing capability, especially for health workers.
They first used a set of buffers for viral inactivation followed by RNA extraction and a web-based reporting system. Despite reporting the results of viral detection testing on over 40,000 RT-PCR samples, since the beginning of April 2020, they realized it was unable to keep up with demand and used up too many resources.
“As lockdown restrictions are lifted, testing people for this coronavirus and, where necessary, instructing them, and their close contacts to self-isolate are absolutely vital. Without effective testing, the virus can spread quickly throughout communities, causing many more deaths,” says study author Caetano Reis e Sousa.
The Study: RT-LAMP for SARS-CoV-2 Detection
The current study is the fruit of their second objective, to publish validated alternative strategies that would allow more independence of RT-PCR, increase the testing capacity, and perhaps allow point-of-care diagnostics. The researchers used loop-mediated isothermal amplification PCR along with reverse transcription, called RT-PCR, to detect SARS-CoV-2. The whole procedure is completed within 30 minutes and does not require the need for a thermocycler qPCR.
This technique has already been developed for RNA viruses and other microbes, and even for SARS-CoV-2. The current study used the method to allow either visual or DNA-dye mediated quantification. They used already developed primers to target the N gene of the virus along with primers that detect 18S RNA for parallel quality control.
Advantages of RT-LAMP
The results show that RT-LAMP is fully capable of being used in place of RT-qPCR as a means of SARS-CoV-2 detection. This method not only avoids non-specific and cross-reactive amplification by other human CoVs or respiratory viruses while being almost as sensitive as RT-qPCR.
The total cost of the reagent is reduced tenfold. It is also much faster, yielding results in 25 minutes, meaning that the output can be quadrupled. This would help determine if any tested individual should self-isolate and cut the risk of viral spread steeply.
Avoiding RNA Extraction Step
Early data from the current study also indicates that the RNA extraction step will not be necessary, further reducing the cost and allowing for more rapid results, even point-of-care testing. The whole method has been validated by public health authorities as well, and accredited to current standards, allowing immediate deployment.
The test was validated on 24 patient samples. All 12 positive samples were found to be positive, and all 12 negative samples did not display amplification, thus agreeing in all cases with the current diagnostic platform.
Reliable and Specific Results
The detection threshold was set, based on previous data, to allow a maximum run time of 25 minutes for the N gene and 20 minutes for 18S rRNA. With these criteria, there were zero false positives in the assay for SARS-CoV-2. The use of a conventional melting curve allowed false positives to be eliminated and increased the reliability of the results.
The specificity of the assay was evaluated using clinical samples from patients without COVID-19, but with several other seasonal coronaviruses, influenza A and B viruses, and other RNA viruses. None of the samples were positive with the RT-LAMP assay for the N gene, showing its high specificity.
RT-LAMP Has Comparable Sensitivity with PCR
To assay its sensitivity, the sample used was a standard specimen assessed by limiting dilution to find the limit of reliable detection. The limit of detection (LOD) was found to lie between 500-1,000 copies of the N gene. The reproducibility and precision of the technique were found by a five-round extraction and assay of RNA from a positive sample and then running the test for the N gene and the 18S rRNA.
Next, the assay was benchmarked against the RT-qPCR standard methods on 37 samples. The results showed 100% agreement for the detection of positives, but when the cycle time for positives was near the LOD, borderline positives failed to be reliably detected by the N gene assay, but were detected by the 18S rRNA assay.
Repeated experiments showed that RT-LAMP did not always identify viral RNA in samples run in duplicate when the cycle times were within 2 cycles of the LOD of the RT-PCR assays. This indicates a slightly lower sensitivity of RT-LAMP compared to PCR on extracted RNA. However, the former was run with only a third of the input RNA used for PCR. Secondly, some recent research suggests that the addition of guanidine could improve the sensitivity of detection with RT-LAMP.
Implications
Finally, the use of RT-LAMP without prior RNA extraction showed 94% agreement with RT-PCR results, which shows this step could be potentially omitted. However, the researchers caution that this finding requires validation with actual clinical specimens.
If also coupled with a colorimetric read-out from a dye-based reporting machine, the resulting reduction in cost and time could yield a very competitive and inexpensive diagnostic workflow capable of point-of-care application and use in remote and impoverished areas.
*Important Notice
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.