A recent study shows how a new, integrated lab-on-a-chip platform can represent a rapid, affordable and accurate molecular diagnostic tool for detecting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The document is currently available free of charge on the medRxiv * preprint server while it is peer reviewed.
In response to the current coronavirus disease (COVID-19) pandemic and the obvious disparities in vaccine coverage in low- and middle-income countries, it is essential to adhere to a widespread screening and screening program for surveillance and infection control in areas with limited medical resources.
The gold standard for diagnosing SARS-CoV-2 infection is the use of real-time reverse transcription-polymerase chain reaction (RT-qPCR) to detect ribonucleic acid (RNA) viral in respiratory tract samples. But although the method is very sensitive and specific, it requires expensive equipment and highly trained personnel.
Several point-of-care RNA detection technologies avoid the need for expensive instruments and simultaneously use reverse transcription and isothermal amplification; a notable example is isothermal loop-mediated amplification (LAMP) which is slowly gaining importance for many different infectious diseases.
And then there are the CRISPR-Cas assisted SARS-CoV-2 tests, which are seen as transformative methods for point-of-care COVID-19 diagnostics. However, they currently lack streamlined sample preparation and integration into the automated and portable system.
This new manuscript, first written by Dr Bongkot Ngamso of the University of Hull in the UK, demonstrated the manual operation of a microfluidic CRISPR device as a COVID-19 molecular diagnostic tool by an operator semi-trained in limited laboratory resources in sub-Saharan Africa (such as Kenya).
A combination of microfluidics and CRISPR-Cas
Researchers combined a microfluidic technique known as Surface Tension Assisted Immiscible Filtration (IFAST) with recent developments in CRISPR-Cas12-based detection, resulting in a sensitive, cost-effective, target-specific and completely Integrated for COVID-19 Diagnostic.
The device was nicknamed ‘IFAST-CRISPR’. It streamlined sample preparation to allow rapid isolation and concentration of RNA directly from nasopharyngeal swab specimens or saliva samples, followed by CRISPR-Cas assisted detection with reading of the lateral flow.
In summary, the use of suitable functionalized magnetic particles enables the isolation and purification of a magnetically reactive analyte directly from complex matrices, a technology that could be used in the future for other infectious diseases.
IFAST-CRISPR device for the detection of SARS-CoV-2. (A) Design and (B) photography of the IFAST-CRISPR device. Chamber 1 = sample + GuHCl + paramagnetic silica beads; chambers 2, 4, 6, 8 = mineral oil; chamber 7 = RT-LAMP reagent; chamber 9 = CRISPR-Cas12 reagent. (C) The IFAST-CRISPR device detects SARS-CoV-2 viral RNA from an untreated nasopharyngeal (NP) swab or saliva sample in a one-hour sample-response workflow . Step 1: RNA is extracted from a sample via paramagnetic silica beads and 5 M GuHCl. Step 2: RNA isolated by MB is transcribed in vitro and amplified into DNA amplicons via RT-LAMP. Step 3: Hybridization of the targeted DNA sequence activates the gRNA-Cas12a complex to digest the ssDNA probe, thereby producing a test line (T) on the lateral flow band that can be visualized with the naked eye. (D) Principle of lateral flow readings for the detection of SARS-CoV-2. The control line (C) appears from the intact biotinylated FAM ssDNA reporter. The test line (T) is present from the cleaved ssDNA reporter after the target dsDNA-gRNA hybridization.
High sensitivity and specificity
By combining the aforementioned LAMP with the CRISPR-Cas12 assays targeting the SARS-CoV-2 nucleoprotein gene, the researchers achieved a visual identification of over 470 viral copies per ml in 45 minutes – without any cross-reactivity to coronaviruses. seasonal or flu.
In addition, on-chip tests revealed the ability to detect and isolate SARS-CoV-2 from a thousand copies of the genome of replication-deficient viral particles within an hour, with the potential to be in further optimized and refined.
In short, this affordable, simple yet highly integrated platform showed sensitivity and specificity characteristics comparable to the much more expensive gold standard of RT-qPCR – requiring only a single source of heating. .
Analytical validation of individual and combined on-chip tests. (A) RNA on-chip extraction followed by RT-LAMP assays; gel electrophoresis results showing that target dsDNA is amplified from RNA extracted by MB from initial concentrations 470 mL-1 copies, confirmed by test lines on lateral flow test strips (n = 2). (B) CRISPR-Cas12 on-chip assays of amplicons from tube-based RNA extraction and RT-LAMP – collateral cleavage of side-flow ssDNA reporters after hybridization between gRNA target and dsDNA showing a test line in a positive sample of MB-extract RNA (n = 1). (C) Integrated steps on RNA extraction chip, detection assisted by RT-LAMP and CRISPR-Cas from samples containing free genomic RNA of SARS-CoV-2 (in a mixture containing HCoV RNAs -OC43 and H1N1, n = 2), and from viral particles containing the SARS-CoV-2 genome (SARS-CoV-2 verification panel, n = 1).
A future of diagnostics
The adaptability of this platform can certainly be implemented for CRISPR-Cas-based detections of other pathogens, which holds great promise as a future addition to the on-site diagnostic arsenal. care, especially in resource-limited and decentralized regions and middle-income countries.
“The platform only required a basic heating source such as simple incubators or hotplates which are typically available in most laboratories in low-resource environments, without the need for expensive or specialized instruments.” , emphasize the authors of the study in this document. medRxiv paper.
Further research into the multiplexing and direct interfacing of the easily accessible Swan brand cigarette filter for saliva sample collection could provide a constant workflow for COVID-19 diagnostics from saliva samples suitable for low resource environments.
medRxiv publishes preliminary scientific reports that are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or treated as established information.