Zhang Binzhe, Qiu Yulie, Shi Chenxi, Zhang Jian
School of Ocean, Yantai University, Yantai 264005, China.
Shandong Engineering Research Center of Healthy Land-Sea Relay Farming of Economic Fish, Yantai 264005, China.
Vet Sci. 2025 Apr 2;12(4):327. doi: 10.3390/vetsci12040327.
The genus represents a critical group of bacterial pathogens in the marine environment globally, leading to massive mortality in the aquaculture industry. Diagnosing vibriosis, an infection caused by species, in clinical samples poses challenges due to its non-specific clinical manifestations. In this study, we developed a TaqMan probe-based multiplex real-time PCR method for the simultaneous detection and quantification of four pathogens: (Va), (Val), (Vh), and (Vsc). The assay targets conserved intra-species regions and specific inter-species regions using specific primers and TaqMan probes to ensure specificity. Sensitivity analysis demonstrated that the multiplex real-time PCR assay could simultaneously detect the four different bacteria, with detection limits of 26-60 copies per reaction, making it 100 times more sensitive than conventional PCR assays. Additionally, the assay exhibited high reproducibility, with intra- and inter-group coefficients of variation below 1.4%. A total of 63 clinical samples was analyzed using this established assay, which successfully detected both single and mixed infections. These results demonstrate that the multiplex quantitative PCR assay is a rapid, specific, and sensitive diagnostic tool for the detection of Va, Val, Vh, and Vsc, making it suitable for monitoring these bacteria in both single- and co-infected clinical samples.
该属是全球海洋环境中一类关键的细菌病原体，会导致水产养殖业大量死亡。由于其临床表现不具特异性，在临床样本中诊断弧菌病（一种由该属物种引起的感染）存在挑战。在本研究中，我们开发了一种基于TaqMan探针的多重实时PCR方法，用于同时检测和定量四种弧菌病原体：哈维氏弧菌（Va）、溶藻弧菌（Val）、副溶血弧菌（Vh）和创伤弧菌（Vsc）。该检测方法使用特异性引物和TaqMan探针靶向种内保守区域和种间特异性区域，以确保特异性。敏感性分析表明，多重实时PCR检测方法能够同时检测这四种不同的细菌，每个反应的检测限为26 - 60个拷贝，比传统PCR检测方法灵敏100倍。此外，该检测方法具有高重复性，组内和组间变异系数均低于1.4%。使用此既定检测方法共分析了63份临床样本，成功检测出单一感染和混合感染情况。这些结果表明，多重定量PCR检测方法是一种快速、特异且灵敏的诊断工具，可用于检测Va、Val、Vh和Vsc，适用于监测单一感染和共同感染的临床样本中的这些细菌。
