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UIC Researchers Using Optimized RT-Lamp Technology for Low-Cost COVID-19 Testing

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NEW YORK – While reverse transcription loop mediated isothermal amplification (RT-LAMP) has been used to test for infectious diseases for years, researchers from the University of Illinois Chicago have developed a method that requires no RNA isolation or purification, which could make it easier for use at a point-of-care setting, including a patient's home. 

In an early preclinical study published in EBioMedicine last month, the research team laid out the design of its RT-LAMP assay for SARS-CoV-2 that requires only two mixtures and a heat block to return results.

Previous RT-LAMP diagnostic tests have generally not performed as well as PCR-based tests, which was the impetus for creating this test, said Xiaowei Wang, a professor of pharmacology and bioengineering at UIC and one of the authors on the paper. "People still feel that LAMP is inferior to PCR," he said. 

And since there's "already high-quality PCR tests," he said, the world "no longer has a need for more." Instead, Wang and his team were "thinking we need a new method" and "wanted to see what we could improve [on] with LAMP," he added.

One issue Wang and his team noted in the paper is that "few tools are available publicly for LAMP primer design, and available tools did not sufficiently consider the complexity of LAMP reactions."

To work around this, Wang simply made his own. He designed a bioinformatics algorithm to automate the primer design process and improve the accuracy. Wang said he started by changing the biostructure of the primers, shortening the stem region to boost amplification efficiency. His team designed 20 primer sets with variable lengths and tested them to determine the optimal primer length. 

Wang also created new design criteria for the RT-LAMP test: Instead of using two primers that detect the virus, like in many PCR-based tests, he used six primers that detect eight regions, making it more accurate.

The researchers then used the algorithm to design multiple sets of primers, measured the performance of each of those sets, and then combined the best primers into one test to get the best performance. The algorithm considered the possible unintended cross-reactivity between the primers — which can cause false positive results — and looked at multiple different combinations to determine the best primers, he said.

The other key elements to the primers that the algorithm looked at were the presence of a primary secondary structure, which could impact the specificity of the test, and the potential interactions between dimers, which may also affect performance. The primers also needed to be stable at homogenous temperatures. Unintended cross-reactivity between primers can be a significant challenge for LAMP tests, so the algorithm filtered out "potential primer dimer formation or other mispriming events from all possible primer pairs among the six primers in the assay," the researchers wrote. 

The team then tested the performance of 22 primer sets for the N gene of the SARS-CoV-2 virus, six primer sets for the M gene, and seven primer sets for the S gene, narrowing it down to four tests. Those were then tested both by themselves and in multiplexed tests, with the two multiplex tests showing better performance than the single target tests. Ultimately, one test won out in sensitivity and specificity, and that was the test the researchers decided on using.

For this test, a user's saliva sample is directly treated with two mixtures — one proteinase mix and one RT-LAMP mix — without the need to purify or isolate the RNA. In laboratory-based molecular tests, such as PCR-based tests, the RNA usually must be extracted to receive results.

Once the sample has been treated, it is applied to a heat block and heated to between 60 and 65 degrees Celsius to incubate the reaction. The reaction starts out dark pink, but changes to yellow if the virus is detected, making it easy to see the result, Wang said. While the team heated the reaction for at least 30 minutes to make sure of the result, Wang noted that a positive result usually changes color sooner than that. 

The researchers intend the test to be used beyond the central lab, aiming for use at community clinics as the next step, with the eventual goal of developing a home kit, Wang said.

The "only limitation [for home use] is the heat block," he said, so the team is currently optimizing the assay for use in a kitchen oven to allow it to be used at home. With home use, Wang noted that the user would need to have some control of the oven temperature. The team is also working to lyophilize the test so it is stable at room temperature instead of requiring refrigeration before use, he added.

He said that while it would be a challenge for many consumers to use ovens for the test, the benefit of higher accuracy compared to antigen tests would outweigh the difficulty. "Available antigen tests are much less sensitive than PCR assays, and the false negatives could be a major concern, beating the purpose of having home testing in many cases," he said. "It is always important to strike a balance between ease of use and testing accuracy."

The RT-LAMP test is "much more sensitive than an antigen test," able to detect the SARS-CoV-2 virus at levels as low as 1.5 copies per microliter, Wang said. In the EBioMedicine study, the researchers used a PCR comparator assay to measure the analytical validity: For nine of the samples they tested, positive cases were detected by both PCR and the RT-LAMP assay. And in one sample, the RT-LAMP test detected SARS-CoV-2 where PCR did not, Wang said.

If a heat block is ultimately needed to run the test, community clinics may be more likely to shell out money for such a device. They come relatively cheap — Wang said some run at $200 or less. Regardless of use case, the reaction mixture would stay the same, he said. If used at a community clinic, the test would also not require a clinician or expert to use, he said. 

Although the specific algorithm he created is designed for SARS-CoV-2, the general algorithm could be applied to design tests for other viruses, such as human papillomavirus, he said. 

Chris Mason, a professor of physiology and biophysics at Weill Cornell Medicine who has developed RT-LAMP tests for COVID-19, said via email that the test "shows promise," particularly by avoiding the extraction step to increase speed and ease of use, but that it will need to be as automated as possible before it's commercialized. 

He added that using the test with a kitchen oven seems feasible — he's been running tests with his tea kettle, and it has worked fine. 

"The more tests like this out there, the better," he said.

Manu Prakash, an assistant professor of bioengineering at Stanford University who has also been working on LAMP-based COVID-19 tests, said that the optimization of the primers and assay "could be immediately incorporated into existing LAMP diagnostic designs." He noted that "a centralized PCR-based approach is not able to handle and support decisions that we need to make in our daily life — in a surge like right now."

With the SARS-CoV-2 test, Wang said the team will be seeking Emergency Use Authorization from the US Food and Drug Administration to commercialize it, after more extensive testing. They have received a grant from the National Institutes of Health to test the assay on a larger population of 1,500 patients and hope to apply for FDA approval by this summer, he said. For the larger study, Wang's team will once again use a PCR comparator assay to measure the validity, but he said they're not sure which test will be used. 

Wang said the team has used the test on additional patient saliva samples, and the unpublished results are "quite consistent" with the data from the EBioMedicine study. 

Wang noted that he does have a startup company, called Nawgen, which was formed several years ago and that could commercialize the test, but that it would need to partner with at least one bigger company to market and distribute the assay, likely through a licensing agreement, to make sure it can get into the hands of users who need it. Wang said it's too early for him to plan out the pricing, but that he expects it to be competitive with other home kits on the market.

The "final goal" of creating the test, Wang said, is to "bring PCR-level sensitivity to at-home tests."

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