Hu Hui, Zhang Xiaomei, Cai Mengting, Song Xiaojun, Cheng Hongrui, Zhai Fangya, Tai Yulei, Xu Zheng, Lin Shan, Chen Yu, Luo Yun, Jin Dazhi
School of Laboratory Medicine, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China.
Key Laboratory of Biomarkers and In Vitro Diagnosis Translation of Zhejiang Province, Hangzhou, Zhejiang, China.
Microbiol Spectr. 2025 Jul 22:e0303024. doi: 10.1128/spectrum.03030-24.
Toxigenic is the cause of infection, highlighting the critical need for rapid and easy-to-use detection. In this study, a multiple cross displacement amplification (MCDA) assay was developed to detect toxigenic and evaluated against real-time PCR and VIDAS toxin A & B assay (CDAB). The results showed that MCDA targeting the gene had a limit of detection (LOD) of 12.5 fg (2.5 copies of genomic DNA) per reaction with no non-specific amplification from other pathogens. The LOD for MCDA was the same as real-time PCR but had significantly faster detection time, detecting 500 pg and 12.5 fg per reaction in 5.7 ± 0.5 and 21.35 ± 2.2 min, respectively. Among 201 stool samples, MCDA identified 40 positive for toxigenic with a shorter average detection time of 16.08 ± 4.62 min compared to real-time PCR (39.82 ± 4.44 min). Using cell cytotoxicity neutralization assays as reference, MCDA demonstrated a significantly higher specificity (92.4%) and a positive predictive value (67.5%) than real-time PCR (80.8 and 43.2%, respectively; < 0.05). MCDA also had a significantly higher sensitivity (93.1%) and a negative predictive value (98.8%) compared to VIDAS CDAB (34.5 and 90.1%, respectively; ≤ 0.001). This was also higher compared to real-time PCR (86.2 and 97.2%, > 0.05). MCDA offers a promising screening alternative for point-of-care testing to detect toxigenic and effectively rule out non-toxigenic . It is rapid, easy to use, and has a balanced performance, making it applicable in clinical and community settings, particularly in resource-limited settings.IMPORTANCERapid detection of toxigenic is important for early intervention and outbreak control of infection in primary healthcare facilities. This study developed an MCDA assay and showed that it was rapid, sensitive, user-friendly, and a suitable point-of-care screening alternative for accurately detecting toxigenic . MCDA had the same sensitivity as real-time PCR while providing significantly faster turnaround times and improved specificity along with positive and negative predictive values. Its balanced performance and rapid detection capability make it well-suited for clinical detection of toxigenic and point-of-care testing in communities, particularly in resource-limited settings, and for potential self-testing.
产毒[病原体名称]是感染的病因，这凸显了对快速且易于使用的检测方法的迫切需求。在本研究中，开发了一种多重交叉置换扩增（MCDA）检测方法来检测产毒[病原体名称]，并与实时荧光定量PCR和VIDAS毒素A和B检测（CDAB）进行了比较评估。结果表明，针对[基因名称]基因的MCDA检测每个反应的检测限（LOD）为12.5 fg（2.5个基因组DNA拷贝），未出现来自其他病原体的非特异性扩增。MCDA的检测限与实时荧光定量PCR相同，但检测时间明显更快，每个反应分别在5.7±0.5分钟和21.35±2.2分钟内检测到500 pg和12.5 fg。在201份粪便样本中，MCDA鉴定出40份产毒[病原体名称]阳性样本，其平均检测时间（16.08±4.62分钟）比实时荧光定量PCR（39.82±4.44分钟）短。以细胞毒性中和试验为参考，MCDA显示出比实时荧光定量PCR更高的特异性（92.4%）和阳性预测值（67.5%）（实时荧光定量PCR分别为80.8%和43.2%；P<0.05）。与VIDAS CDAB相比，MCDA的敏感性（93.1%）和阴性预测值（98.8%）也显著更高（VIDAS CDAB分别为34.5%和90.1%；P≤0.001）。与实时荧光定量PCR相比也更高（实时荧光定量PCR分别为86.2%和97.2%，P>0.05）。MCDA为即时检测提供了一种有前景 的筛查方法，可用于检测产毒[病原体名称]并有效排除非产毒[病原体名称]。它快速、易于使用且性能平衡，适用于临床和社区环境，特别是在资源有限的环境中。重要性快速检测产毒[病原体名称]对于基层医疗设施中[病原体名称]感染的早期干预和疫情控制至关重要。本研究开发了一种MCDA检测方法，并表明它快速、灵敏、用户友好，是准确检测产毒[病原体名称]的合适即时筛查方法。MCDA与实时荧光定量PCR具有相同的敏感性，同时提供了显著更快的周转时间，并提高了特异性以及阳性和阴性预测值。其平衡的性能和快速检测能力使其非常适合临床检测产毒[病原体名称]以及社区中的即时检测，特别是在资源有限的环境中，也适用于潜在的自我检测。
