Yang Xuan, Wang Yue, Xu Chengming, Liu Zhiyi, Guan Yuanqi, Wang Fei, Chen Shuliang, Wang Yuan, Cheng Yibin, Dong Yanming
State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, Hubei University, Wuhan 430062, Hubei, China.
State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, Institute of Medical Virology, TaiKang Medical School (School of Basic Medical Sciences), Wuhan University, Wuhan 430072, Hubei, China.
ACS Synth Biol. 2024 Dec 20;13(12):4119-4130. doi: 10.1021/acssynbio.4c00545. Epub 2024 Dec 5.
Hand, foot, and mouth disease (HFMD), caused by enteroviruses, mostly including EV71, CVA6, CVA10, and CVA16, is an acute infectious disease commonly found in children. Due to no approved antiviral therapies and available vaccines, except for EV71, developing accurate diagnostic methods of HFMD is essential for controlling its spread and mitigating its impact on public health. Here, we create a MIRA-HEV-PAND multiple nucleic acid typing method that utilizes Ago to identify enterovirus type A pathogens (EV71, CVA6, CVA10, and CVA16) and universal type EVU. The MDC (minimum detection concentration) level of MIRA-HEV-PAND is within the range of 1.66 aM (1.0 copy/μL), which was matched to that of qPCR assays and even more sensitive up to 10%. Importantly, the MIRA-HEV-PAND method exhibits higher sensitivity and less time-consuming efficiency compared to the approach that combines PCR amplification instead of MIRA amplification. Meanwhile, though the quintuple and single-tube multiple MIRA-HEV-PAND detection system can be used for one viral target or multiple viral target detection, the single-tube detection system detects more efficiently and rapidly than the quintuple-tube multiple detection system. Moreover, the diagnostic results obtained by evaluating clinical samples using MIRA-HEV-PAND show a complete consistency of 100% with qPCR assays. The MIRA-HEV-PAND method can screen a wider range of target regions using low-cost guide DNA without being limited to PAM sequences, compared to the MARPLES based on the CRISPR-Cas12a. The utilization of this correlation can be beneficial for the application of molecular testing for clinical diagnoses and the study of human enteroviruses A infection and virus typing on an epidemiological scale.
手足口病（HFMD）是一种常见于儿童的急性传染病，由肠道病毒引起，主要包括肠道病毒71型（EV71）、柯萨奇病毒A6型（CVA6）、柯萨奇病毒A10型（CVA10）和柯萨奇病毒A16型（CVA16）。由于除EV71外尚无获批的抗病毒疗法和可用疫苗，因此开发准确的手足口病诊断方法对于控制其传播和减轻其对公共卫生的影响至关重要。在此，我们创建了一种MIRA-HEV-PAND多重核酸分型方法，该方法利用Ago来鉴定甲型肠道病毒病原体（EV71、CVA6、CVA10和CVA16）以及通用型EVU。MIRA-HEV-PAND的最低检测浓度（MDC）水平在1.66 aM（1.0拷贝/μL）范围内，与定量聚合酶链反应（qPCR）检测方法相当，甚至灵敏度高出10%。重要的是，与采用PCR扩增而非MIRA扩增的方法相比，MIRA-HEV-PAND方法具有更高的灵敏度和更低的耗时效率。同时，尽管五重管和单管多重MIRA-HEV-PAND检测系统均可用于一种病毒靶标或多种病毒靶标的检测，但单管检测系统比五重管多重检测系统检测效率更高、速度更快。此外，使用MIRA-HEV-PAND评估临床样本获得的诊断结果与qPCR检测方法的完全一致性为100%。与基于CRISPR-Cas12a的MARPLES相比，MIRA-HEV-PAND方法可以使用低成本的引导DNA筛选更广泛的靶标区域，而不受原间隔序列邻近基序（PAM）序列的限制。这种关联的利用有助于分子检测在临床诊断中的应用以及在流行病学规模上对人类甲型肠道病毒感染和病毒分型的研究。
