Bhardwaj Pooja, Dhangur Preeti, Kalichamy Alagarasu, Singh Rajeev
JE-AES Apex Laboratory, ICMR-Regional Medical Research Centre, BRD Medical College Campus, Gorakhpur, India.
ICMR-National Institute of Virology, Pune, Maharashtra, India.
J Med Virol. 2025 Feb;97(2):e70219. doi: 10.1002/jmv.70219.
Globally ≤ 4 billion of the population are at potential risk of contracting dengue virus (DENV) infection. Seasonal outbreaks of dengue are frequently reported causing a high healthcare burden. Undiagnosed DENV can lead to severe morbidity and mortality. Early diagnosis of DENV relies on molecular methods, which are impractical in resource-constrained settings (RCSs). Dengue can be caused by any of the four distinct DENV serotypes. Therefore, a simple method for rapid diagnosis of Pan-DENV serotypes is of utmost importance at RCSs. A fluorescence detection platform for Pan-DENV using RT-RPA and CRISPR/Cas12a was developed targeting nonstructural 1 (NS1) gene for DENV-1, 2, and 3, and envelope (E) gene for DENV-2. Further, crRNA specific to DENV serotypes were designed to facilitate CRISPR/Cas12a detection. Analytical sensitivity was determined using synthetic RNA and DENV serotypes genome. Clinical validation of the assay was performed using RNA extracted from AES/AFI clinical samples. The developed CRISPR/Cas12a-based detection platform can detect all four serotypes of DENV viz 1-4 in a single pot using fluorescence detection. This assay showed the limit of detection ≥ 781 zg reaction , ≥ 1.81 ag reaction, ≥ 62.5 fg reaction, and ≥ 2.5 pg reaction for synthetic DENV-1, DENV-2, DENV-3, and DENV-4 template, respectively. Our assay demonstrated the analytic sensitivity of ≥ 10 ng reaction for DENV-1 and DENV-4, and ≥ 0.5 ng reaction for DENV-3 and DENV-4 genomes. This assay showed no cross-reactivity with other related etiologies tested causing AFI/AES. With 76 clinical samples (DENV PCR positive = 16, DENV PCR negative = 60), the assay demonstrated 93.7% sensitivity and 100% specificity with an overall accuracy of 98.7% for detection of the Pan-DENV serotypes. Our assay displayed comparable results to that of RT-PCR. The ease of interpretation and rapid detection of the Pan-DENV, represents the potential of the developed assay as an ideal point-of-care test. This assay upon field-deployment could help in reducing healthcare burden, provide differential diagnosis and support initiating early and prompt treatment to patients at RCS.
全球范围内，有40亿人口面临感染登革病毒（DENV）的潜在风险。登革热季节性疫情频发，造成了沉重的医疗负担。未确诊的登革病毒感染可导致严重的发病和死亡。登革病毒的早期诊断依赖分子方法，但在资源有限的环境中并不实用。登革热可由四种不同的登革病毒血清型中的任何一种引起。因此，在资源有限的环境中，一种快速诊断泛登革病毒血清型的简单方法至关重要。我们开发了一种基于RT-RPA和CRISPR/Cas12a的泛登革病毒荧光检测平台，该平台针对登革病毒1型、2型和3型的非结构蛋白1（NS1）基因以及登革病毒2型的包膜（E）基因。此外，还设计了针对登革病毒血清型的crRNA，以促进CRISPR/Cas12a检测。使用合成RNA和登革病毒血清型基因组确定分析灵敏度。使用从登革热/登革出血热临床样本中提取的RNA对该检测方法进行临床验证。所开发的基于CRISPR/Cas12a的检测平台可以通过荧光检测在一个反应管中检测所有四种登革病毒血清型，即1-4型。该检测方法对合成的登革病毒1型、2型、3型和4型模板的检测限分别为≥781 zg/反应、≥1.81 ag/反应、≥62.5 fg/反应和≥2.5 pg/反应。我们的检测方法对登革病毒1型和4型基因组的分析灵敏度≥10 ng/反应，对登革病毒3型和4型基因组的分析灵敏度≥0.5 ng/反应。该检测方法与其他导致登革热/登革出血热的相关病原体无交叉反应。在76份临床样本（登革病毒PCR阳性=16份，登革病毒PCR阴性=60份）中，该检测方法检测泛登革病毒血清型的灵敏度为93.7%，特异性为100%，总准确率为98.7%。我们的检测方法与RT-PCR的结果相当。泛登革病毒检测方法易于解读且检测快速，显示出其作为理想的即时检测方法的潜力。该检测方法在现场部署后有助于减轻医疗负担，提供鉴别诊断，并支持在资源有限的环境中对患者进行早期和及时的治疗。
