基于CRISPR的单管快速高效登革热病毒检测

Zhong Jiaye, Li Juezhuo, Chen Shiyu, Xu Yue, Mao Xiaolei, Xu Minghui, Luo Shuyin, Yang Yi, Zhou Jiawei, Yuan Jinghua, Su Lan, Wang Gang, Zhang Xinling, Li Xiaoping

Key Laboratory of Artificial Organs and Computational Medicine in Zhejiang Province, Shulan International Medical College, Zhejiang Shuren University, Hangzhou, Zhejiang, China.

Wycombe Abbey School Hangzhou, Hangzhou 311261, P. R. China.

J Appl Microbiol. 2025 Apr 1;136(4). doi: 10.1093/jambio/lxaf070.

BACKGROUND

Dengue Virus (DENV) is prevalent in tropical and subtropical regions. With the projected climate change, traditional detection methods face limitations, and there is an urgent need for more accurate and efficient diagnostic techniques.

OBJECTIVE

The aim is to integrate Recombinase-aided Amplification (recombinase-aided amplification, RAA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to establish a special CRISPR-Cas system for rapid, convenient, high sensitivity, and high specificity typing detection of DENV.

METHODS

CRISPR RNA (crRNA) and RAA primers were designed based on the whole genome of four DENV serotypes. A single-tube assay combining RAA with CRISPR-Cas13a technology was developed after optimizing reagent concentrations.

RESULTS

The limit of detection (LoD) of DENV types 1-4 was 10³ copies·mL -1. No cross-reaction was observed between any of the DENV serotypes and the other three flaviviruses (Zika, West Nile, and Murray Valley encephalitis). The average sensitivity of one-step method was 95.8%, and the average specificity was 96.6%. Fluorescent signal intensities demonstrated a clear dose-dependent response, with the signal increasing as the sample concentration rose. This system can effectively distinguish non-target substances. Among them, one-step method has advantages in timeliness, ease of operation, and contamination control because it runs efficiently inside a tube and does not require the lid to be removed, but its sensitivity is relatively low. The two-steps method performs well in sensitivity.

CONCLUSION

In this study, we developed a novel method for rapid typing and detection of DENV using RAA and CRISPR-Cas13a in a single-tube homogeneous system.

背景

登革病毒（DENV）在热带和亚热带地区流行。随着预计的气候变化，传统检测方法面临局限性，迫切需要更准确、高效的诊断技术。

目的

整合重组酶辅助扩增（RAA）和规律成簇间隔短回文重复序列（CRISPR），建立一种用于登革病毒快速、便捷、高灵敏度和高特异性分型检测的特殊CRISPR-Cas系统。

方法

基于4种登革病毒血清型的全基因组设计CRISPR RNA（crRNA）和RAA引物。优化试剂浓度后，开发了一种将RAA与CRISPR-Cas13a技术相结合的单管检测方法。

结果

1-4型登革病毒的检测限（LoD）为10³拷贝·mL-1。任何登革病毒血清型与其他三种黄病毒（寨卡病毒、西尼罗河病毒和墨累谷脑炎病毒）之间均未观察到交叉反应。一步法的平均灵敏度为95.8%，平均特异性为96.6%。荧光信号强度呈现明显的剂量依赖性反应，信号随样品浓度升高而增加。该系统能有效区分非靶物质。其中，一步法在及时性、操作简便性和污染控制方面具有优势，因为它在管内高效运行，无需打开盖子，但其灵敏度相对较低。两步法在灵敏度方面表现良好。