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Cornell Researchers Develop Heat-Free Alkaline DNA Extraction Method for Tissue Samples


NEW YORK – Researchers at Cornell University have developed a heat-free alkaline DNA extraction technique that could help expand access to tissue-based molecular testing in low-resource areas starting with tests to aid in the earlier diagnosis of a virus-linked cancer in sub-Saharan Africa.

In a study published earlier this month in Scientific Reports, the team reported that it had optimized and tested an alkaline extraction technique that was modified from hot sodium hydroxide and tris solution (HotSHOT) extraction but performed at room temperature. They determined that the sodium hydroxide-based extraction method, which they dubbed "ColdSHOT," could be used with a loop-mediated isothermal amplification (LAMP) assay for the detection of the Kaposi's sarcoma herpesvirus (KSHV), or human herpes virus-8. The room-temperature extraction took about one hour, which is double the time needed for HotSHOT.

David Erickson, director of the Center for Point-of-Care Technologies for Nutrition, Infection and Cancer in Global Health at Cornell, said that his research team has been working for about 10 years on expanding access to diagnostics for Kaposi's sarcoma, which is a malignancy caused by KSHV. The disease can cause red, purple, or brown lesions of the skin, mucous membranes, and internal organs, typically beginning with lesions on the legs and feet.

While most people infected with the KSHV will not develop Kaposi's sarcoma or symptoms of infection, people with weakened immune systems due to HIV infection are at higher risk. Kaposi's sarcoma is one of the most common HIV-associated malignancies.

The Cornell team noted in the paper that Kaposi's sarcoma is also associated with high mortality. Healthcare providers can mitigate the effects of the disease with early detection and treatment with antiretroviral therapy and chemotherapy. However, the malignancy is typically diagnosed through pathology, a lengthy process that is typically unavailable outside of centralized clinics.

As part of the work on Kaposi's sarcoma tests, the research team has been studying ways to reduce the reliance on costly laboratory infrastructure to run the tests, Erickson said. He and his colleagues have partnered with 11 local labs in Malawi, Tanzania, Kenya, Uganda, Rwanda, and Botswana and plan to work with them to gather feedback on the methods and demonstrate that they can be used at scale.

Jason Manning, a PhD student at Cornell and the lead author of the Scientific Reports article, said that while many of those partner labs have heat blocks, some others have access only to heated water baths, which makes it difficult for labs to perform HotSHOT extraction methods that require heat of 95 degrees Celsius to increase the rate of DNA extraction and the final yield.

In the study, the researchers performed the ColdSHOT extraction and used LAMP assays to quantify DNA yields for the ORF26 gene of KSHV and the human GAPDH sequence, the latter of which is a housekeeping gene that was used in the study to determine whether the extraction methods were producing sufficient yields. The team performed the tests on a LAMP instrument called TINY, which Erickson's team had demonstrated the feasibility of in research published in 2023.

In that previous publication, the team reported that the assay had 97 percent sensitivity and 92 percent specificity compared to pathology in a study using 506 biopsies.

In their recent study, the researchers performed punch biopsies on frozen skin tissue specimens that were obtained from Weill Cornell Medicine and the New York Presbyterian Hospital in New York City. They found that the ColdSHOT room-temperature extraction methods consistently produced about 40,000 GADPH DNA copies per reaction, or DNA from about 20,000 cells per reaction, from 3 mg samples that were taken from seven skin punch biopsies. Each of those samples was comprised of two .75 mm biopsies.

The team also spiked three skin samples each of four different sizes with about 20,000 copies of the ORF26 gene per reaction using plasmid stock solution to analyze the performance of the LAMP assay following ColdSHOT extraction. They determined that the LAMP assay's performance was unaffected by varying quantities of cellular debris in the sample.

Based on DNA yields from prior studies on KSHV, the authors estimated that extracting the DNA from as few as 10,000 cells from a biopsy of a Kaposi's sarcoma tumor would yield about 6,700 copies of the viral DNA, which is far greater than the 96-copy threshold of detection for their LAMP assay.

The authors noted that they found that the DNA yield from ColdSHOT was comparable to a silica membrane spin column extraction using a Qiagen DNeasy Blood and Tissue Kit.

Taylor Moehling, senior technical staff member for systems biology at Sandia National Laboratories, said that the Scientific Reports article presents promising initial results that demonstrate the utility of eliminating a heating step to simplify the extraction process. Moehling was the lead author of a study that was published in March in the journal Diagnostics on the development of a rapid, low-cost LAMP assay for the detection of Trypanosoma cruzi in low-resource settings. She was not connected with the Cornell team's study.

Robert Meagher, manager of the systems biology department at Sandia and coauthor of the Diagnostics article, said that the methods described by the Cornell team would greatly simplify the DNA extraction process in comparison with use of a spin column or silica column, which require too many steps, consumables, and pieces of equipment to be run by many healthcare workers in point-of-care settings. He also noted that the supply chains that are used to supply spin columns in the US cannot be taken for granted in much of the developing world, whereas the chemicals used in the Cornell team's extraction method are simpler and easier to access. He was also not connected with the Cornell study.

Meagher said that he would like to see in future studies how well the extraction methods work in a sample-to-answer real-time workflow by a healthcare worker. Moehling said that she would like to see the performance of the extraction methods when they are applied to samples as they are collected from patients rather than from remnant tissue samples.

According to Erickson, using the ColdSHOT extraction methods and performing the LAMP assay on crude samples together will greatly simplify sample processing for the KSHV test and reduce partners' reliance on laboratory infrastructure.

"There are places, including in the United States … where we don't have the access to the kind of diagnostic infrastructure that we would like whether that's in terms of expertise or time to results or any number of different things," he said.

The researchers wrote that the reaction requires only two reagents and a pipette, and Manning said that running a test would cost pennies.

Erickson added that that the technology could also be adapted to detect other viral-caused cancers such as Burkitt's lymphoma, which is associated with Epstein-Barr virus. He said that his team has been pursuing multiple paths to bring the molecular testing methods to market, including commercialization through the lab-to-market Global Health Point-of-Care Diagnostics Accelerator hosted by Columbia University.

Manning said the Infectious Diseases Institute in Kampala, Uganda, will be the first to implement ColdSHOT extraction, and the Cornell team will compare the results against other extraction methods and pathological diagnosis prior to implementing it in other sites. Erickson hopes to show that the test can deliver fast and accurate results that will help healthcare providers to start treatments earlier.

"Sample processing for these types of diagnosis is definitely the hardest part," Erickson said. "I think we're really excited that we've found a way to simplify things significantly."