Faster and affordable alternatives to RT-PCR

Many biotech companies and researchers have developed new testing methods to detect SARS-CoV-2 in order to cut backlogs and increase the accessibility of testing.

At the moment, nucleic acid amplification testing (PCR/RT-PCR) is the most widely used platform to detect SARS-CoV-2. It is a gold standard test with high levels of specificity and sensitivity but it is also time-consuming, requiring specific laboratory setting and trained technicians. And speed being important to beat this pandemic, researchers have directed their focus on point-of-care-testing to develop diagnostic kits that are fast, affordable, feasible on global scale, and have similar accuracy as the PCR tests.

CRISPR

One of such tests is CRISPR (clustered regularly interspaced short palindromic repeats). Previously known as a gene-editing tool, researchers have used CRISPR-Cas technology to develop SARS-CoV-2 assays that have as less as 5 minutes turnaround time and as accurate as PCR genetic tests.

Two American biotech firms, Sherlock Biosciences and Mammoth Biosciences, have received an emergency use authorization (EUA) from the US Food and Drug Administration (FDA) for their respective CRISPR-based testing kits for COVID-19.

Sherlock CRISPR SARS-CoV-2 kit uses the company’s patented technology that amplifies genetic sequences and programs a CRISPR molecule to detect unique signatures from the novel coronavirus. According to the company’s website, the CRISPR enzyme is activated when the signature is found and it cuts reporter RNAs provided as part of the kit to release a detectable signal, yielding results in about an hour.

A pilot study of 20 clinical samples to evaluate the accuracy and performance of Sherlock’s CRISPR SARS-CoV-2 reported to have 100% concordance with results from Dartmouth-Hitchcock Medical Center’s lab-based polymerase chain reaction (PCR) test method. While this test is conducted in the lab, the company is further developing its INSPECTR platform to create an instrument-free, handheld test to provide rapid detection of a genetic match of the SARS-CoV-2 virus.

Another CRISPR-based test kit created by researchers led by Jennifer Doudna, co-founder of Mammoth Biosciences and who also won a share of this year’s Nobel Prize in Chemistry for her co-discovery of CRISPR, however, doesn’t amplify coronavirus RNA but uses multiple RNA guides to increase the sensitivity of the test. Without the need for expensive lab equipment this test can be deployed at doctor’s offices, schools and office buildings.

Melanie Ott, a senior investigator at the Gladstone Institutes says that the kit does not track the virus down to one virus per microliter as the conventional coronavirus diagnostic setup, but it was able to accurately identify a batch of five positive clinical samples with perfect accuracy in just 5 minutes per test. And apart from revealing whether a sample is positive, the test can also detect the amount of virus in the sample.

In India, the Council of Scientific and Industrial Research’s (CSIRs) Institute of Genomics and Integrative Biology (IGIB), Delhi has also developed Feluda paper strip test for diagnosis of SARS-CoV-2 based on CRISPR-Cas9 technology. The Indian Council of Medical Research (ICMR) has issued an advisory for the use of test by laboratories. The cost of the test is expected to be around 500 rupees ($6.80). Co-founder and IGIB scientist Debojyoti Chakraborty says the current prototype requires a PCR machine for processing, but they are working on saliva or self-swabbing version that can be used at home.

2020 Nobel Laureates in Chemistry

Two female scientists, Emmanuelle Charpentier and Jennifer A. Doudna were jointly awarded this year’s Nobel Prize in Chemistry for discovering a revolutionary gene-editing tool called CRISPR/Cas9 that allows making precise edits to the genome of animal, plants and microorganisms.

CRISPR is an adapted microbial ‘immune system’ that allows the prokaryotes — bacteria and archaeal —to detect the viral DNA and destroy it. Part of the CRISPR system is a protein called Cas-9 that is able to seek out, cut and eventually degrade a viral DNA in a specific way, says Doudna (TED Talk, September 2015)

Earlier studies had identified the CRISPR-associated proteins (Cas), including Cas9, but Charpentier, now director of the Max Planck Unit for the Science of Pathogens in Berlin, discovered another key component of the CRISPR system, an RNA molecule that is involved in recognizing phage sequences, in the bacterium Streptococcus pyogenes. She published her findings in 2011 and in the same year she met Doudna of the University of California, Berkeley, during a conference in Puerto Rico and they teamed up to begin a research on the activity of Cas-9.

During their research the pair realized they could harness the function of Cas-9 for genome engineering to delete or insert specific bits of DNA into cells. They recreated the bacteria’s genetic scissors in a test tube and showed that the system could be programmed to recognize particular DNA sequences and cut the DNA in specific sites.

CRISPER/Cas9 also dubbed as ‘genetic scissors’ can now easily edit genomes that were time-consuming, difficult and even impossible before.

The technology has enormous power and raises hopes in many areas from editing crops, treating genetic conditions to making powerful antibiotics and antivirals and creating gene drives. Jennifer Doudna and many other scientists have, however, called for a worldwide pause in using the CRISPR tool on any experiment with the human genome.

Bioluminescence Testing

Many life forms, including sea creatures, fireflies, and glowworm, use an enzyme called luciferase to produce their own glow. Scientists are exploiting this light-yielding chemical reaction found in these bioluminescent creatures to make SARS-CoV-2 tests that are simple, faster, and more affordable than the current assays.

Researchers at Eindhoven University of Technology (TU/e) are creating a bioluminescent testing kit to detect the presence of antibodies in response to SARC-CoV-2 virus. The kit, which uses luciferase, generates a luminescent light when SARS-CoV-2 antibodies are present in a patient’s serum or plasma sample.

According to Maarten Merkx, professor of protein engineering at TU/e, the technology is based on fusing two protein domains to the sensor protein complex: a blue light-emitting luciferase enzyme and a green fluorescent protein.

“In the absence of the target antibody, these two domains are in close proximity, resulting in the emission of green light. But, if the antibody is present, and it binds to the sensor protein, the two domains become separated and the sensor protein predominantly emits blue light.” The ratio between the two colors can also determine the antibody concentration. The bluer the color, the higher the antibody levels, Prof. Merkx says.

His team is now developing two variations of the technology, one to detect the antibodies produced in response to SARS-CoV2, and the other to indicate the presence of the virus itself, by detecting traces of viral proteins. The test is expected to give results in 30 minutes. Prof. Merkx says that this test could be useful for quick triaging; if one is in doubt, one can quickly confirm for infection.

Lab-on-chip testing

Lab-on-a-chip (LOC) integrates several laboratory functions, such as PCR and DNA sequencing, into a single chip on a very small scale. Singapore was one of the first countries to use this technology. They used VereCoV diagnosis kit from Veredus Laboratories for screening people at its land, sea, and air checkpoints in March, 2020. The test claimed an accuracy of more than 99 percent, and gave results in three hours.

Since then, many LOC rapid test kits have been developed. Th e University of California, Irvine (UCI), has created one called TinyArray, developed by a research group headed by Weian Zhao, PhD, and professor of pharmaceutical sciences at UCI.

According to labpulse, the kit uses blood from a finger-prick to probe hundreds of antibody responses to 14 respiratory viruses, including SARS-CoV-2, in two to four hours. The results are then printed on the TinyArray imager, which combines a 3D-printed prototype with an off -the-shelf light-emitting diode (LED) and small 5-MP camera to find markers for many antibodies simultaneously.

The device costs only $200, but it is portable and has accuracy of expensive lab equipment. It can process the results of both nose swab and antibody tests on a single platform. Th e kit is expected to be available through the United States by the end of the year.

CovidNudge is another test based on lab-on-a-chip system. Developed by UK startup company DnaNudge, it uses an analyzer that has successfully miniaturized the gold-standard lab-based PCR test to detect the genetic material of COVID-19 virus, while delivering results in just over an hour.

According to Med-Tech Innovation News, the NudgeBox analyzer is about the size of a shoebox, and the test cartridge itself fi ts into the palm of a hand. “The cartridge has 72 tiny wells in its assay, and it can extract RNA from a naso-pharyngeal, nasal, or buccal swab (inner cheek swab) sample, which is then inserted into the NudgeBox for analysis and reverse transcribing to DNA.”

The Lancet Microbe reported that the test delivered highly accurate results, with an average sensitivity of 94.4 percent, compared against standard NHS lab-based tests, and a specifi city of 100 percent. This test can detect the presence of both human RNA as well as viral RNA to eliminate ‘false negatives’ by control testing for inadequate swab specimen collection technique, and also off ers the capability to test for FluA, FluB, and respiratory syncytial virus (RSV), the journal said.

The United Kingdom is currently using CovidNudge solution in NHS urgent patient care and elective surgery settings and out-of-hospital locations.

References:

1 Sherlock Biosciences Receives FDA Emergency Use Authorization for CRISPR SARS-CoV-2 Rapid Diagnostic, https://sherlock.bio

2. Toward COVID-19 Testing Any Time, Anywhere, https://www.the-scientist.com/

3. Industry News: Sherlock Biosciences announces clinical data from study of Sherlock CRISPR SARS-CoV-2 Kit, https://www.selectscience.net/

4. New test detects coronavirus in just 5 minutes, https://www.sciencemag.org/

5. Th is paper-strip test with gene-editing technology can detect Covid-19 in less than an hour, https://economictimes.indiatimes.com/

6. Using Protein Engineering For Easy and Rapid Testing of Coronavirus Antibodies, https://www.tue.nl/en/tue-campus/

7. Lab-on-a-chip array could provide low-cost SARS-CoV-2 antibody test, https://www.labpulse.com/

8. Assessing a novel, lab-free, point-of-care test for SARS-CoV-2 (CovidNudge): a diagnostic accuracy study, https://www.thelancet.com/

9. The Nobel Prize, https://www.nobelprize.org/prizes/chemistry/2020/advanced-information/

One comment

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