NEW YORK – In an effort to help alleviate pandemic-related sample prep bottlenecks, researchers at the National Institutes of Health have developed a direct-from-sample testing protocol utilizing a common molecular lab reagent: Chelex 100.
Initially utilized to aid in DNA detection for early PCR-based forensic assays, the NIH team adapted Chelex for use with RNA. They found their method to yield equivalent results as standard extraction-based techniques for potentially aneighth of the cost, and it uses fewer plastic consumables.
Although Bio-Rad Laboratories has been marketing Chelex for decades, the firm has reported that sales of the reagent have increased significantly during the pandemic, as labs seek ways to keep COVID-19 testing running despite supply shortages.
Chelex is an ion-exchange resin made of a styrene and divinylbenzene copolymer stuck to iminodiacetate ions. When mixed into a slurry with a sample, the resin sequesters ions like magnesium and calcium, as well as heavy metals. The insoluble resin particles can then be spun down and separated from the sample.
At the NIH's National Eye Institute molecular diagnostics lab, Bin Guan, Robert Hufnagel, and colleagues set about to see if they could improve COVID-19 molecular diagnostic testing. The lab typically does genetic testing for rare eye diseases, but lockdown in April of last year led them to brainstorm ways around the supply chain crimps that quickly began cropping up.
"We were thinking, what if we could obviate one of the steps and find a way to skip the RNA extraction and do a direct detection," Hufnagel said in an interview. "It would streamline the workflow, and you could still use the CDC probes and do RT-PCR," he said.
The team drafted a proposal, then screened many direct detection-enabling molecules using inactivated virus to test out their ideas.
Guan had experience in the past teaching an undergraduate molecular biology lab and knew that Chelex 100 was a cheap and effective trick for students to prepare their own DNA from a saline mouth rinse.
Chelex chelates the magnesium and calcium ions that DNAses need to function, and it is also hypothesized to absorb cell lysis products that may interfere with PCR, Guan said.
The Chelex method was initially developed in the late 1980s. Researchers at Cetus Corporation — where PCR had been invented and patented just a few years earlier — published in 1991 that it could be used to remove impurities and detect low concentrations of DNA in forensic samples.
Sean Walsh, now a molecular diagnostics product developer at Veracyte, was part of that early study under the direction of Russ Higuchi, who is currently affiliated with Cepheid. In an email, Walsh said the team was developing what would become the first PCR-based forensic test and knew it would need to be very sensitive and also need to be used on small and challenging samples, like single hairs.
The Cetus forensics team was very concerned about PCR contamination. "We absolutely wanted our users to operate contamination free," Walsh said, adding that the Chelex method can be done in a single tube "with few steps and no tube transfers." By minimizing the potential for contamination, the workflow was able to facilitate successful adoption of forensic PCR technology.
Chelex for RNA-based MDx
Although the Chelex-based method has been known to work on DNA and was commercialized in the early 1990s, Guan said there had been few studies of the reagent on RNA.
"RNAse also uses magnesium and calcium for optimum activity ... but the pH of Chelex suspended in water is 10 to 11," Guan said, adding that such alkaline conditions could degrade RNA, including the RNA-based genome of SARS-CoV-2.
The team adjusted the pH of the Chelex solution closer to neutral. "From there, it turns out to be great, and it preserves the RNA," Guan said.
Using digital droplet PCR and synthetic samples, the NEI team found that they could detect as little as one genome copy of virus per microliter using their direct protocol.
But as a rare disease molecular diagnostics lab, they needed collaborators in the clinic to handle infectious samples. Investigators at the National Institute of Dental and Craniofacial Research and the NIH's Department of Laboratory Medicine joined in to perform testing on samples collected from local patients.
The clinical results, published in iScience last month, showed the Chelex method has similar or better sensitivity compared to an RNA extraction method for both primary nasopharyngeal swabs and saliva samples.
Many other groups have published recipes for rapid and inexpensive direct detection of RNA during the pandemic. But Guan said the NEI method is unique in that it involves heat inactivation at 95 degrees for five minutes, which kills the virus and could be helpful to labs that aren't authorized to handle live virus or in any future pandemics where a pathogen is not yet characterized.
Samples can also be collected into Chelex directly, so the prep steps would not require any pipetting, saving on time and pipette tips.
The team also estimated that the cost of viral transport media, or VTM, collection to be $1.70 per tube, and RNA extraction may cost around $6 per sample. They estimated the total cost of a non-VTM collection tube, the Chelex, and two other standard reagents for their method — low ethylenediaminetetraacetic acid tris buffer, also called low TE, and dimethyl sulfoxide, or DMSO — to be less than $1 per sample.
"Thus, the Chelex method will save cost and reduce supply chain burden by eliminating the need for RNA extraction and VTM," the team wrote in iScience.
Although extraction produces pure, isolated RNA, NEI's Hufnagel said that the team compared extraction-based methods to Chelex using simulated samples and many different buffer conditions, as well as head-to-head comparisons using clinical samples. Overall, the team showed that their direct detection method did not lead to decreases in sensitivity or specificity when the samples were subsequently analyzed using RT-PCR tests.
The NIH is now pursuing licensing and co-development of the method.
Hufnagel said the team also wants to enable other laboratories to use the method and noted that it makes primary samples shelf stable and suitable for mailing without concerns about RNA degradation. "Outside of typical labs doing large-scale diagnostics for COVID, there may be others that have a need for remote collection and shipping where this could also be advantageous," he said.
The NEI team had early discussions with Bio-Rad about the work, and Hufnagel said the discussions are ongoing.
Bio-Rad Laboratories lists the NEI paper on its website, and Ertan Ozyamak, the firm's global product manager for lab chromatography, also described the work in an article about Chelex earlier this year.
Ozyamak said in an email that Bio-Rad offers different Chelex formulations for different applications. Its molecular biology grade Chelex resin is appropriate for nucleic acid extractions and allows the optimization of the Chelex suspension for a given application, but the firm also sells a specially formulated, ready-made 6 percent suspension of Chelex called Instagene Matrix.
"During this pandemic, these products have become popular for viral RNA extraction globally," Ozyamak said, adding that sales of Chelex have also increased significantly.
"We are projecting an almost doubling of sales numbers this year and interest is growing," he said, noting that Chelex-based extraction methods are appealing alternatives "particularly because of the reduced sample prep time and the significantly reduced cost per sample in comparison to commercial extraction kits."
Ozyamak said Bio-Rad has a continued interest in new approaches that customers may develop, but he also said the firm could not speak to future plans for SARS-CoV-2 diagnostic use of Chelex at this time.