The recent surge in reported COVID-19 cases in California this past month met with a testing capacity that has now proven its ability to administer over 125,000 tests on a single day.
However, accelerating demand for testing has exerted even more of a strain on the supply chain of testing material to testing networks, which has caused extensive delays across the state.
It took the brainpower of a handful of researchers at UC Santa Barbara to develop a promising solution that could potentially answer some of these issues that exist not only in their state, but across the entire globe.
A representation of the SARS-CoV-2 virion under an electron microscope. Courtesy of Wikimedia Commons
COVID-19 testing is a multilayered process that depends on the fluency of individual operations and supplies that include, but are not limited to, collecting specimens with swabs, securing viral transport media and executing tests in laboratories with multiple reagents and instruments. Insufficiencies in any of the many necessities employed by the testing system can clog testing schedules and hinder the expansion of testing capacities.
RNA extraction kits are just one out of the many necessities that have fallen victim to shortages.
Commercial RNA extraction kits approved by the Centers for Disease Control and Prevention are used by laboratories to isolate the genetic material of SARS-CoV-2 — RNA — from infected human host cells. This is followed by the next step that is a form of polymerase chain reaction (PCR) known as real-time reverse-transcription PCR, or RT-qPCR, where targeted viral sequences from the isolated RNA are transcribed into DNA copies, and then quantitatively amplified such that it allows scientists to detect these targeted viral sequences that would confirm the presence of the virus.
Researchers under the guidance of Diego Acosta-Alvear, Carolina Arias, Max Wilson and Kenneth Kosik — professors from UCSB’s Department of Molecular, Cellular, and Developmental Biology — recognized that a significant factor limiting the COVID-19 testing capacity was the supply shortage of commercial RNA extraction kits.
“Because RNA extraction is the first step for amplification and detection of SARS-CoV-2 genetic material, these kits can become ‘testing bottlenecks’ when their availability is limited, as has occurred recently,” Acosta-Alvear said in an email interview.
Extracting and isolating RNA from SARS-CoV-2 positive host cells are critical steps in the testing process, because if not executed optimally, it could compromise the performance of the subsequent RT-qPCR test used to detect viral infection. Maintaining the sensitivity of this test is pivotal to avoiding false negative results, as asymptomatic carriers who test false negative could unknowingly spread the coronavirus.
The steep global demand on kit manufacturers to match increasing testing capacities have motivated researchers to look for alternative methods to RNA extraction and COVID-19 testing.
To offer a solution to the dilemma, the research team developed a method called Precipitation Enhanced Analyte RetrievaL, or PEARL for short. This method involves subjecting infected host cells to their “PEARL lysis solution,” which can extract nucleic acids and proteins that can then be analyzed for the presence of viral genetic material or proteins.
“We designed PEARL primarily to eliminate the need for commercial RNA extraction kits which are relatively expensive and are in short supply. PEARL uses common laboratory reagents and is very easy to perform, which alleviates the problem of reagent/supplies accessibility,” Acosta-Alvear said.
“We use a nonionic detergent to disrupt cell membranes and viral envelopes, which allows us to retrieve nucleic acids and proteins,” he added. “We also use reagents that inactivate RNA-degrading enzymes and aid in the precipitation of nucleic acids and proteins, including salts and linear polyacrylamide (used as a co-precipitant). Adding isopropanol ‘completes’ the procedure by allowing the precipitation of RNA, DNA, and proteins.”
To validate their approach, they tested for the presence of two specific viral RNA sequences of interest in SARS-CoV-2 positive host cells. Performing side-by-side RNA extractions using both PEARL and a standard commercial RNA kit, they ultimately found that PEARL was as capable as the kit at extracting RNA for subsequent analysis.
However, their research did not stop there. PEARL’s extended capacity to extract DNA and proteins in addition to RNA opened up doors to broader applications, as these capabilities are not fully available in other commercial extraction kits.
“When we designed PEARL we wanted to make it versatile. One way to convince ourselves and the public of its usability was to test it using very different viruses.”
With the right tools, the research team recognized that PEARL could be further tailored to detect viruses beyond SARS-CoV-2. By adding enzymes that degrade one of the main types of nucleic acid — either DNA or RNA — PEARL can be specified to detect viruses whose genomes are made of one or the other main type of nucleic acid.
In an additional experiment, either DNA-degrading enzymes or RNA-degrading enzymes were independently added to different PEARL extracts of host cells that were infected with Kaposi’s sarcoma-associated herpesvirus (KSHV), a DNA-based virus, or Zika virus (ZIKV), an RNA-based virus.
As expected, PEARL extracts that were treated with DNA-degrading enzymes allowed detection of RNA but not DNA (such as host cell mRNA transcripts, viral KSHV mRNA transcripts or ZIKV RNA genes), and PEARL extracts that were treated with RNA-degrading enzymes allowed detection of DNA but not RNA (such as host cell and KSHV DNA genes).
“In this way we confirmed PEARL’s ability to retrieve DNA or RNA sequences, which allows detection of different viruses.”
Furthermore, PEARL’s ability to enrich proteins allowed the researchers to successfully detect both host and viral proteins in PEARL extracts of cells infected with these viruses using antibodies.
“Protein detection can be sensitive and in some ways, “easier” than detection of nucleic acid sequences by amplification, but it relies on antibodies with proven performance,” Acosta-Alvear commented.
“If you have a good antibody and you know which viral protein to look for, you got yourself an alternative to detect viruses.”
Acosta-Alvear concluded, “These results highlight the broad applicability of PEARL and make us optimistic that PEARL could become a widespread tool for detection of many viruses.”
In the scope of COVID-19 testing, the ability to retrieve viral proteins, as Acosta-Alvear further noted in the interview, may have useful implications in the avenue of antigen-based testing.
This type of testing focuses on detecting proteins found in SARS-CoV-2 and alongside typical nucleic acid testing, can be used to test for current infection of the coronavirus.
As of now, the Food and Drug Administration has granted Emergency Use Authorizations for two antigen diagnostic tests, both of which detect viral nucleocapsid proteins. These proteins are multifunctional structural proteins that hold the virus’s genetic information.
But as to the future of PEARL itself, Acosta-Alvear has weighed up some more exciting possibilities.
“Testing PEARL with other sample types will be important to determine the breadth of its applicability. For example, one could envision using PEARL with urine or blood samples…”
“PEARL also offers a first step towards field-deployability of pathogen testing as it can be performed in the field, and it can be coupled to our recently developed CRISPR-based SARS-CoV-2 detection method we call CREST.”
“We are exploring this direction currently and are hopeful we might be able to come up with a fully field-ready test for SARS-CoV-2.”
And the field will be waiting to shake hands with this promising gem — a PEARL, that is.
