{"title":"A rapid CRISPR/Cas12a-based assay for the detection of HIV-1 Clade C in infants","authors":"Anjli Gaur , Harsh Bhakhri , Nitesh Mishra , Shaifali Sharma , Tanu Bansal , Mani Kalaivani , Megha Brijwal , Bimal Kumar Das , Rakesh Lodha , Subrata Sinha , Kalpana Luthra","doi":"10.1016/j.cca.2025.120518","DOIUrl":null,"url":null,"abstract":"<div><h3>Background</h3><div>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.</div></div><div><h3>Methods</h3><div>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.</div></div><div><h3>Results</h3><div>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.</div></div><div><h3>Conclusions</h3><div>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.</div></div>","PeriodicalId":10205,"journal":{"name":"Clinica Chimica Acta","volume":"578 ","pages":"Article 120518"},"PeriodicalIF":2.9000,"publicationDate":"2025-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Clinica Chimica Acta","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0009898125003973","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MEDICAL LABORATORY TECHNOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
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.
期刊介绍:
The Official Journal of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)
Clinica Chimica Acta is a high-quality journal which publishes original Research Communications in the field of clinical chemistry and laboratory medicine, defined as the diagnostic application of chemistry, biochemistry, immunochemistry, biochemical aspects of hematology, toxicology, and molecular biology to the study of human disease in body fluids and cells.
The objective of the journal is to publish novel information leading to a better understanding of biological mechanisms of human diseases, their prevention, diagnosis, and patient management. Reports of an applied clinical character are also welcome. Papers concerned with normal metabolic processes or with constituents of normal cells or body fluids, such as reports of experimental or clinical studies in animals, are only considered when they are clearly and directly relevant to human disease. Evaluation of commercial products have a low priority for publication, unless they are novel or represent a technological breakthrough. Studies dealing with effects of drugs and natural products and studies dealing with the redox status in various diseases are not within the journal''s scope. Development and evaluation of novel analytical methodologies where applicable to diagnostic clinical chemistry and laboratory medicine, including point-of-care testing, and topics on laboratory management and informatics will also be considered. Studies focused on emerging diagnostic technologies and (big) data analysis procedures including digitalization, mobile Health, and artificial Intelligence applied to Laboratory Medicine are also of interest.