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A rapid CRISPR/Cas12a-based assay for the detection of HIV-1 Clade C in infants
Anjli Gaur1, Harsh Bhakhri1, Nitesh Mishra1
1Department of Biochemistry, All India Institute of Medical Sciences, New Delhi 110029, India.
Insights
A new CRISPR/Cas12a assay offers sensitive and specific detection of HIV-1 Clade C, aiding early diagnosis in infants. This molecular diagnostic tool provides rapid results for improved antiretroviral therapy initiation.
Area of Science:
- Molecular Diagnostics
- Viral Immunology
- CRISPR Technology
Background:
- Early detection of Human Immunodeficiency Virus type 1 (HIV-1) infection is crucial for timely antiretroviral therapy (ART) initiation.
- Standard serology tests are unreliable in infants due to maternal antibodies, necessitating alternative diagnostic methods.
Purpose of the Study:
- To develop and evaluate a CRISPR/Cas12a-based assay for the sensitive and specific detection of HIV-1 Clade C.
- To assess the assay's performance as a potential point-of-care diagnostic tool, particularly for infants.
Main Methods:
- A CRISPR/Cas12a assay was designed targeting conserved HIV-1 pol gene regions, coupled with isothermal preamplification via recombinase polymerase amplification (RPA).
- Detection utilized CRISPR/Cas12a's collateral cleavage activity on a DNA reporter, visualized via fluorescence, visual readout, or lateral flow dipsticks.
- Assay performance was validated using clinical samples, including infant plasma, HIV-1 plasmids, and control viral samples.
Main Results:
- The assay demonstrated high sensitivity (96%) and specificity (92.65%) for HIV-1 Clade C detection.
- Results were obtainable through fluorescence, visual inspection, and lateral flow dipstick formats, indicating ease of use.
- The assay successfully differentiated HIV-1C from other viral pathogens and healthy donor samples.
Conclusions:
- The developed CRISPR/Cas12a assay is a sensitive and specific molecular diagnostic tool for HIV-1 Clade C.
- It shows significant potential as a point-of-care test for early HIV-1 detection in infants, addressing limitations of antibody-based tests.
- This assay can facilitate earlier ART initiation, improving patient outcomes and disease management.
Background:
Early detection of HIV-1 infection is essential for initiating antiretroviral therapy (ART) to suppress viremia and prevent disease progression. Timely diagnosis, especially in infants, is critical as rapid antibody-based serology tests are ineffective due to the presence of maternal antibodies.
Methods:
We developed a CRISPR/Cas12a-based HIV-1 detection assay by optimizing components for coupled isothermal preamplification using recombinase polymerase amplification (RPA). The assay targeted the conserved region in the pol gene specific to HIV-1 with the designed CRISPR RNA (crRNA). CRISPR/Cas12a-mediated cleavage of viral cDNA was visualized through the collateral cleavage of a single-stranded DNA-FAM-BQ reporter, enabling rapid and visually detectable outcomes. The performance of the assay was evaluated using plasma from 41 HIV-1 Clade C (HIV-1C) seropositive individuals, including 28 HIV-1C infected infant samples, HIV-1 Indian Clade C and Clade B genome plasmids, viral disease control DNA/RNA samples (Influenza, RSV, Parvovirus, HPIV, CMV, and HBV), and 31 healthy donor plasma samples. Sensitivity and specificity were assessed, and detection was performed using fluorescence, visual readout, and lateral flow dipsticks.
Results:
The CRISPR/Cas12a-based HIV-1 Clade C detection assay achieved a sensitivity of 96 % and a specificity of 92.65 %. The assay successfully provided results through both fluorescence and visual readouts and was compatible with lateral flow dipstick formats, facilitating easy and rapid detection.
Conclusions:
The developed CRISPR/Cas12a-based HIV-1C detection assay demonstrates high sensitivity and specificity for Clade C, indicating its potential as a robust point-of-care molecular diagnostic tool for HIV-1C. Additionally, it may serve as a rapid nucleic acid test alternative for detecting mother-to-child transmission of HIV-1C in infants under two years of age, where traditional antibody-based tests are ineffective. This assay holds promise for improving early HIV-1 diagnosis and timely initiation of ART, ultimately contributing to better disease management and outcomes.
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