Anwar Saleem, Khan Sufia, Azmi Iqbal, Islam Khursheed Ul, Ahmad Tanveer, Iqbal Jawed
Multidisciplinary Centre for Advanced Research and Studies, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India.
Multidisciplinary Centre for Advanced Research and Studies, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India.
Diagn Microbiol Infect Dis. 2025 Dec;113(4):117062. doi: 10.1016/j.diagmicrobio.2025.117062. Epub 2025 Aug 12.
Pathogenic viruses such as SARS-CoV-2 (SCoV-2), continue to pose a significant threat to human civilization. The lessons learnt from SCoV-2 infections have highlighted the requirement for robust and readily available diagnostic tools in order to limit the virus transmission and prevent future pandemics such as COVID-19. RT-qPCR-based detection is routinely used for sensitive and accurate diagnosis, which requires a sophisticated instrument, laboratory setup, and technical expertise. Though RT-qPCR is highly reliable and considered the gold standard for pathogen detection, it is costly, time-consuming, and unaffordable for the masses. Therefore, other reliable methods for nucleic acid-based detection with sensitivity, specificity, and accuracy on-par with RT-qPCR are required. Recent advancement in CRISPR technology promises its development as a POC testing device, providing a high-end, instrument-free, portable, and cost-effective workflow. Further, COVID-19 pandemic has encouraged the development of next-generation CRISPR-based diagnostics with a provision for home-testing which has resulted in the development of portable and smart-phone integrated hand-held devices which can detect various pathogenic infections in a shorter time frame than RT-qPCR. For diagnosing the presence of SCoV-2, CRISPR-based diagnostics (SHERLOCK/DETECTR) are quicker (30-60 min), less expensive ($5-15/test), and portable than RT-qPCR (90-180 min; $10-50/test) demonstrating equivalent specificity (100%) and near-equivalent sensitivity (93-100% for CRISPR-based diagnostics vs 95-100% for RT-qPCR). For high-sensitivity centralized testing, RT-qPCR is still the gold standard, but CRISPR works well in point-of-care settings because it requires little equipment (like lateral flow strips or heating blocks) and allows multiplexing. CRISPR-based diagnostics breakthrough platform like CARMEN leverages microfluidic technology to test 5,000 plus samples in a single run, unlike RT-qPCR, which requires separate reactions for each target.In this review, the advancement in CRISPR technology such as SHERLOCK, DETECTR, and other Cas-9-based diagnostics are highlighted which exclusively focuses on the CRISPR-based diagnostics to detect SCoV-2 and its emerging VOCs, highlighting their advantages and limitations compared to the gold-standard RT-qPCR.
诸如严重急性呼吸综合征冠状病毒2（SCoV-2）等致病性病毒，继续对人类文明构成重大威胁。从SCoV-2感染中吸取的教训凸显了对强大且易于获得的诊断工具的需求，以便限制病毒传播并预防未来的大流行，如2019冠状病毒病（COVID-19）。基于逆转录定量聚合酶链反应（RT-qPCR）的检测通常用于灵敏且准确的诊断，这需要精密的仪器、实验室设施和专业技术知识。尽管RT-qPCR高度可靠且被视为病原体检测的金标准，但它成本高昂、耗时，且大众难以承受。因此，需要其他基于核酸检测的可靠方法，其灵敏度、特异性和准确性与RT-qPCR相当。CRISPR技术的最新进展有望将其开发为一种即时检测（POC）设备，提供一种高端、无需仪器、便携且经济高效的工作流程。此外，COVID-19大流行推动了下一代基于CRISPR的诊断方法的发展，这种方法可进行家庭检测，从而促成了便携式且集成智能手机的手持设备的开发，这些设备能够在比RT-qPCR更短的时间内检测出各种致病性感染。对于诊断SCoV-2的存在，基于CRISPR的诊断方法（SHERLOCK/DETECTR）比RT-qPCR更快（30 - 60分钟）、成本更低（每次检测5 - 15美元）且更便携（RT-qPCR为90 - 180分钟；每次检测10 - 50美元），显示出同等的特异性（100%）和近乎同等的灵敏度（基于CRISPR的诊断方法为93 - 100%，而RT-qPCR为95 - 100%）。对于高灵敏度的集中检测，RT-qPCR仍然是金标准，但CRISPR在即时检测环境中表现良好，因为它所需设备很少（如侧向流动试纸条或加热块）且允许进行多重检测。像CARMEN这样基于CRISPR的诊断突破平台利用微流控技术一次运行可检测5000多个样本，这与RT-qPCR不同，RT-qPCR对每个靶标都需要单独的反应。在本综述中，重点介绍了CRISPR技术的进展，如SHERLOCK、DETECTR和其他基于Cas-9的诊断方法，这些方法专门聚焦于基于CRISPR的诊断以检测SCoV-2及其新出现的变异株，突出了它们与金标准RT-qPCR相比的优势和局限性。
