Developing New RNA Extraction Strategies for SARS-CoV-2 PCR
Proteinase K has been in short supply, but researchers are finding solutions
Since the SARS-CoV-2 virus first emerged in late 2019, researchers worldwide have scrambled to devise sensitive methods for testing. Accurate testing in real-time not only enables people with the virus to self-isolate and protect others, but taking “snapshots” of what’s happening across a region is also an essential part of the reopening process.
Research testing has come a long way in the last six months. Two primary methods are being used to detect the novel coronavirus: serology and polymerase chain reaction (PCR). The high sensitivity and specificity of PCR has made it the gold standard for identification of numerous bacteria and viruses, the SARS-CoV-2 virus now among them.
Extraction—an essential part of the overall workflow—isolates the viral RNA from the many other components in the biological sample taken via nasal swab. During extraction, the sample is treated with a proteinase, often Proteinase K, which digests any proteins present and protects the nucleic acids from degradation via nuclease. After extraction from the sample, the collected RNA is used in qRT-PCR kits: reverse transcription converts the RNA to DNA, which is then amplified by specific primer pairs, and a fluorescent signal can be detected if the probe is able to bind to a designated sequence.
Given the pressures of the current crisis and the need to scale quickly across the globe, there is unprecedented demand for resources routinely used in viral research—extraction kits among them. Proteinase K has suffered short supply in recent months, which significantly impedes the scaling process. Shortage of components of extraction kits has pushed life sciences companies that manufacture RNA extraction kits to literally work around the clock to upscale manufacturing.
To overcome some of the shortages of Proteinase K, which is a key component of most RNA extraction kits, researchers began to investigate and leverage workflows without the RNA extraction phase. However, this strategy is not advised, since protocols without the extraction step can have higher failure rates since contaminating compounds, known as PCR inhibitors, are not removed. Leaving out extraction generally results in a greater number of false negatives, which is detrimental to tracking the spread of the virus. Additionally, although costs might be saved in methods without RNA extraction, the re-testing that might also be required would eliminate any net savings. Finally, re-testing would require qPCR components, which may have their own supply risks.
It is much preferred to keep the extraction step and devise new methods that use alternatives to those in short supply. Our team discovered that a supplemental protocol for one of our existing research genomics extraction kits could be used in the extraction of the novel coronavirus, allowing the extraction step to remain, but without the use of Proteinase K. In short, following lysis of the capsid, the RNA is immobilized onto magnetic particles, which allows for the separation from contaminants using a magnetic field. The contaminants are then rinsed away using a simple wash procedure. This is essentially another strategy for extraction that obviates the need for Proteinase K.
iRepertoire in Huntsville, Alabama independently utilized the method for research, performing 100 RNA extractions and more than 500 qPCR reactions over a three-day period. The results were extremely encouraging.
We have been able to upscale our efforts and dramatically increase our manufacturing capabilities for RNA extraction kits by redistributing resources, both our workforce and raw materials. This resulted in a 10-fold increase in our RNA extraction production in March 2020. The kit is now available to high-throughput laboratories worldwide, in the effort to continue to ramp up RNA extraction capabilities.
The Food and Drug Administration recently included the extraction method on its FAQ page for use with the Centers for Disease Control and Prevention Emergency Use Authorization-authorized COVID-19 test.
Developing the technology to expand availability of extraction solutions around the world will be an ongoing aim for researchers. Collaboration with other organizations can be an important part of pushing forward R&D, especially in such unprecedented times as these. Identifying the presence of the novel coronavirus accurately and swiftly will be a collective need for some time—and will play a key role in moving out of the pandemic as quickly as possible.