SARS-CoV-2 test in 20 minutes with FISH

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 181 million confirmed infections and over 3.9 million deaths globally. A pandemic of this magnitude has not been seen since the 1918 Spanish flu.

The challenges which have arisen from the coronavirus 2019 (COVID-19) pandemic have illustrated how the dependence of normality relies heavily on effective virus detection through efficient testing.

As the current gold standard RT-PCR test has drawbacks such as being time-consuming, the high volume of demand can be overwhelming for COVID-19 laboratories. This illustrates the requirement for rapid, affordable, and sensitive methods for virus detection.

A collaboration from the University of Oxford and the University of Warwick has led to research into a novel method of viral detection through rapid fluorescence in situ hybridization (FISH) in order to effectively detect the SARS-CoV-2 virus within 20 minutes.

A pre-print version of the paper is available on the medRxiv* server, while the article undergoes peer review.

Current SARS-CoV-2 tests

The most common diagnostic test for the COVID-19 virus that is used currently consists of nucleic acid-based amplification, antigen detection, and serology tests. The reverse transcriptase-mediated polymerase chain reaction (RT-PCR) test is considered to be the gold standard for SARS-CoV-2 testing.

However, an RT-PCR test is time-consuming and only performed in a laboratory setting since viral lysis, and RNA purification procedures are required and sample collection, transportation, and reception. Commonly, these routines delay test results by 24 hours or more.

The COVID-19 pandemic emphasizes the limitations of RT-PCR testing

Alternatives to RT-PCR tests

Loop-mediated isothermal amplification (LAMP) is an alternative to RT-PCR since it allows results to be provided after only one hour.

This is a significant difference when compared to what is required of RT-PCR tests. However, both RT-PCR and LAMP tests are still limited by supply chain issues resulting from the expensive production and storage requirements of the reagents used.

Other alternatives which have been developed for rapid antigen-detection tests are based on immunoassays and lateral flow formats in order for the fast detection of SARS-CoV-2. However, these tests are limited by their sensitivity levels, decreasing their accuracy in detection.

Novel testing method

Fluorescent in situ hybridization, or FISH, is a cytological technique that utilizes selective binding of fluorescently labeled oligo- and polynucleotides to complementary DNA and RNA sections. This can be within fixed cells and tissues for detection, quantification, and spatial localization by fluorescent microscopy.

Efficiency of virus detection in a throat swab. A) Combined nasal and throat swabs were taken according to WHO instructions [40]. Subsequently, the throat swabs were diluted in saline and spiked with the same concentration of IBV (104 PFU/mL) in each sample. After a centrifugation step, virus particles were immobilized and FISH stained as described. B) Efficiency of virus detection in throat swabs compared to virus diluted with saline, dependent on the dilution factor. [+]: IBV probes, [-]: SARS-Cov-2 probes, U: undiluted, error bars: SD of three independent experiments.
Efficiency of virus detection in a throat swab. A) Combined nasal and throat swabs were taken according to WHO instructions [40]. Subsequently, the throat swabs were diluted in saline and spiked with the same concentration of IBV (104 PFU/mL) in each sample. After a centrifugation step, virus particles were immobilized and FISH stained as described. B) Efficiency of virus detection in throat swabs compared to virus diluted with saline, dependent on the dilution factor. [+]: IBV probes, [-]: SARS-Cov-2 probes, U: undiluted, error bars: SD of three independent experiments.

Usually, this technique is used to detect chromosomal abnormalities and rearrangements within fixed cells, such as translations, insertions, and deletions. It can also be used to detect pathogens within cells, such as bacterial and viral infections like HIV or the Epstein-Barr virus.

RNA FISH can also be used in order to visualize gene transcription in situ; through increasing the number of fluorophores that bind one RNA transcript, it is possible to detect single transcripts – this process led to the single-molecule FISH (smFISH) technique. Through the use of smFISH, single viral RNA molecules can be detected and identified without requiring enzymatic amplification.

The researchers of this study have introduced a rapid viral FISH (rvFISH) technique for the detection of viral particles, which depended on systemic analysis of hybridization reaction; the researchers were able to analyze the efficiency of hybridization reactions while reducing the steps of the procedure as well as the time taken. This technique has enabled the detection of influenza particles and an avian coronavirus, infectious bronchitis virus (IBV), within a 20-minute assay.

This technique was also able to detect inactivated SARS-CoV-2 particles in the same manner as well as detecting virus particles from nasopharyngeal swabs. This is important as it illustrates the technique's sensitivity, which would be useful for detecting SARS-CoV-2 efficiently and more effectively.

Through using a dual-labeling strategy, the background resulting from misidentified bright fluorescent spots was able to be suppressed. This further confirmed the application of smFISH as a possible technique to rapidly and easily detect and quantify virus strains, which would benefit the COVID-19 pandemic.

A limitation explored in the study included the requirement of a fluorescence microscope, with the researchers using a commercial instrument to detect fluorescence from single molecules to calibrate the detection limit for virus particles. However, due to brighter particles such as fluorescent beads being used for this calibration, single-molecule detection is not needed. The viral particles should be able to be detected by less sensitive microscopes.

The research from this pre-print paper provides a beneficial and efficient basis for developing a novel testing method for SARS-CoV-2, which could be more dependable and effective due to the heavy reliance on testing during this pandemic.

rvFISH can be a versatile tool for investigating hybridization of oligonucleotides to viral particles and possibly be an alternative or addition to the use of RT-PCR tests; this may relieve the burden on this current test and speed up the resulting process, which may aid with a semblance of normalcy during the COVID-19 pandemic.

*Important notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Marzia Khan

Written by

Marzia Khan

Marzia Khan is a lover of scientific research and innovation. She immerses herself in literature and novel therapeutics which she does through her position on the Royal Free Ethical Review Board. Marzia has a MSc in Nanotechnology and Regenerative Medicine as well as a BSc in Biomedical Sciences. She is currently working in the NHS and is engaging in a scientific innovation program.

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