Zhang Yanli, Chen Xuhan, Ouyang Guifang, Wang Jiaping, Sun Yongcheng, Lai Yanli, Zhang Ping, Guo Fei, Yang Shujun, Mao Rui
Department of Hematology, The First Affiliated Hospital of Ningbo University, Ningbo, China.
Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences, Ningbo, China.
Front Microbiol. 2024 Aug 7;15:1435010. doi: 10.3389/fmicb.2024.1435010. eCollection 2024.
() is the most common pathogen causing hospital respiratory tract infection and epidemic. Gold standard procedures of microscopic examination and biochemical identification are widely used in clinical diagnosis with disadvantages of low sensitivity, time-consuming and sophisticated equipment requiring. An efficient, nucleic acid amplification-based sensitive and specific on-site identification of in clinical is necessary to facilitate clinical medication and disease control.
We developed a closed dumbbell mediated isothermal amplification (CDA) assay for the rapid and sensitive detection of conserved A gene in by real-time fluorescence monitoring and end-point colorimetric judgement. We designed and selected a pair of inner primers of CDA to detect . Then outer and loop primers were designed and verified to accelerate CDA reaction to achieve more efficient detection of .
The results showed the detection limit of CDA assay was 1.2 × 10 ng/μL (approximately 1 copy of the target gene) within 60 min, which was 100-fold more sensitive than real-time quantitative PCR (qPCR). Several pathogen genomic DNAs (, , and ) were used to evaluate the sensitivity and specificity of the established CDA assay. Total 224 batches of samples from other strains tested were negative and 296 batches of extracted DNA samples were positive by the developed CDA amplification approach, revealing high specificity and specificity of the diagnostic assay. In addition, the results of real-time fluorescence amplification of the CDA were in consistent with those of end-point colorimetric results.
The established real-time fluorescence and visual CDA assays of with merits of rapid, sensitive and specificity could be helpful for on-site diagnosis and clinical screening in rural areas.
（某病原体）是引起医院呼吸道感染和流行的最常见病原体。显微镜检查和生化鉴定的金标准程序在临床诊断中广泛应用，但存在灵敏度低、耗时且需要精密设备的缺点。临床上需要一种高效、基于核酸扩增的灵敏且特异的现场鉴定方法，以促进临床用药和疾病控制。
我们开发了一种封闭哑铃介导的等温扩增（CDA）检测方法，通过实时荧光监测和终点比色判断，快速灵敏地检测（某病原体）中保守的A基因。我们设计并选择了一对CDA内引物来检测（该病原体）。然后设计并验证了外引物和环引物，以加速CDA反应，实现对（该病原体）更高效的检测。
结果表明，CDA检测方法在60分钟内的检测限为1.2×10 ng/μL（约1个目标基因拷贝），比实时定量PCR（qPCR）灵敏100倍。使用几种病原体基因组DNA（其他几种病原体）来评估所建立的（该病原体）CDA检测方法的灵敏度和特异性。通过开发的CDA扩增方法，对来自其他菌株的总共224批样本检测均为阴性，对296批提取的（该病原体）DNA样本检测为阳性，揭示了该诊断检测方法具有高特异性。此外，（该病原体）CDA的实时荧光扩增结果与终点比色结果一致。
所建立的（该病原体）实时荧光和可视化CDA检测方法具有快速、灵敏和特异的优点，有助于农村地区的现场诊断和临床筛查。
