Nucleic-acid-based assays are the gold standard for detecting the presence of the SARS-CoV-2 virus, but they eventually will be replaced with simpler and more advanced techniques, such as CRISPR-Cas, LAMP, and other faster and less-expensive methods.
Nucleic-acid-based assays are the gold standard for detecting the presence of the SARS-CoV-2 virus, but they eventually will be replaced with simpler and more advanced techniques, such as CRISPR-Cas, LAMP, and other faster and less-expensive methods, according to a paper published in APL Bioengineering (a publication of the American Institute of Physics).
In that paper, “Evaluation of current diagnostic methods for COVID-19,” corresponding author Oguzhan Gunduz at Marmara University in Istanbul and colleagues evaluated the strengths and weaknesses of multiple diagnostic assays in use or in development for the current pandemic.
Significant differences among COVID-19 diagnostic tests are resulting in inaccurate results that sometimes put populations at risk or quarantine people needlessly, contributing to skepticism about pronouncements from scientists and public health authorities.
There is a great need for point-of-care diagnostic testing that performs with sensitivity and selectivity without the limitations of real-time reverse transcription-polymerase chain reaction (RT-PCR), they noted.
Additionally, “There is not any available single test for the entire stage of the disease,” Gunduz said in a statement. For example, “Viral antigens and antibodies (typically IgM and IgG) become detectable at different periods during infection…and the detection time…depends on several parameters, such as viral features, individual patient variability and the applied test.” Consequently, performing an immunoassay in the early stages of an infection will be negative because the antibodies cannot be produced yet, even though the disease is present. Likewise, performing a PCR test at the end of the disease also will return a negative result because of the lack of – or a very low – viral load.
The widespread availability, therefore, of accurate assays for each stage of disease is important in curbing the spread of COVID-19 and subsequent pandemics.
PCR, while the gold standard for detecting viral load, is not always the best answer. Other techniques are primed to replace it.
Compared to PCR:
- Reverse transcription loop-mediated isothermal amplification (RT-LAMP), is relatively inexpensive and easy to perform. “The amount of DNA produced is much higher, and a positive test result can be viewed visually without the need for an additional analysis step,” the study reported. Sensitivity has been recorded as the same as, or 97% that of, RT-PCR, depending on the study. Because RT-LAMP uses six to eight primers, it is considered highly specific.
- CRISPR-cas-based analysis is another less costly, simple procedure. The methods developed by Mammoth Biosciences uses Cas12a to “detect the viral RNA sequences of the envelope (E) and nucleocapsid protein (N) genes, followed by isothermal amplification of the target, causing a visual reading with a fluorophore.” Sherlock Biosciences uses Cas13 to cut reporter RNA sequences after activation by guide RNA.
- Antigen-based assays target the spike (S) protein of other structural proteins of the virus. “The S protein has an amino acid sequence variation among coronaviruses, enabling the specific diagnosis of the novel virus,” the study authors wrote. Specificity and sensitivity can be enhanced by using multiple antigens. Serological methods are faster than PCR and can be used at the point-of-care, but only if their accuracy can equal or exceed molecular techniques, the researchers said. As either antigen- or antibody-based assays, “They will become increasingly important to understand the history of pandemics and predict the future (outbreaks).”
The researchers also identified several promising next-generation diagnostic technologies that are still in development for COVID-19 applications.
“Whole-genome sequencing is the most promising method for COVID-19 characterization, genomic surveillance, understanding viral transmission and pathogenicity, identifying viral mutations, and developing new therap(ies). However, this costly and time-consuming method limits its practical application,” the authors wrote.
Electrochemical sensors seem auspicious for selective and sensitive detection, identification, and quantification of viruses. They enable portable DNA sensors that detect viral cDNA or RNA (or any sequence unique to them) using a probe. Advantages include sensitivity, better limit of detection, and miniaturization.
Biosensor-based systems using nanotechnology, microfluidics, and advanced instrumentation “are predicted to be among the most promising technologies in pandemic situations like COVID-19,” they said.
For example, “I think it may be possible to detect the (viral) attack at any stage of the disease with nano-based sensor technologies,” Gunduz added. Already, one transistor-based biosensor designed by researchers in Korea has detected SARS-CoV-2 at the femtogram level in milliliters without sample preparation. “In the future…easier and more mature biosensor platforms will replace RT-PCR,” the authors wrote.
Currently available diagnostic techniques vary greatly in reliability and specificity, so understanding which type of test is most appropriate for a given circumstance is necessary to avoid false reports. Therefore, “It is vital to select the appropriate diagnostic test within the correct timing for an accurate diagnosis. Further studies are needed to compare existing methods in terms of robustness, reproducibility, reliability, and sensitivity,” the researchers concluded.
Until a vaccine is available, accurate diagnostic tests are will form a bulwark against the spread of infection. Curbing the coronavirus pandemic relies heavily on how quickly a potentially exposed individual can be tested and quarantined. “Rapid diagnosis and rapid isolation are the key factors for prevention of the pandemic,” Gunduz stressed.
