Wang Haojie, Sun Yue, Chen Jianxing, Wang Wei, Yu Haibo, Gao Caixia, An Tongqing, Wang Yue, Chen Hongyan, Zhu Liangquan, Jin Zhimin, Yu Changqing, Xia Changyou, Zhang He
State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
College of Life Sciences and Technology, Mudanjiang Normal University, Mudanjiang, China.
Front Cell Infect Microbiol. 2024 Nov 26;14:1468783. doi: 10.3389/fcimb.2024.1468783. eCollection 2024.
, , , and are the primary pathogens responsible for gastrointestinal diseases in pigs, posing a significant threat to the health and productivity of pig production systems. Pathogen detection is a crucial tool for monitoring and managing these infections.
We designed primers and probes targeting the gene of , the 23S gene of , the gene of , and the gene of . We developed a quadruplex TaqMan real-time quantitative PCR assay capable of simultaneously detecting these four pathogens.
This assay demonstrated high sensitivity, with detection limits of 100 copies/μL for the recombinant plasmid standards pEASY-23S rRNA, pEASY-aspA, and pEASY-, and 10 copies/μL for pEASY-. The standard curves exhibited excellent linearity (R values of 0.999, 0.999, 1, and 0.998, respectively) and high amplification efficiencies (93.57%, 94.84%, 85.15%, and 81.81%, respectively). The assay showed high specificity, with no cross-reactivity detected against nucleic acids from Streptococcus suis, porcine epidemic diarrhoea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), , , , porcine delta coronavirus (PDCoV), porcine group A rotavirus (GARV), and porcine teschovirus (PTV). The assay also exhibited excellent repeatability, with inter- and intra-assay coefficient of variation (CV) ranging from 0.15% to 1.12%. High concentrations of nucleic acids did not interfere with the detection of low concentrations, ensuring robust performance in complex samples. Among 263 diarrhoeic samples, the assay detected in 23.95%, in 26.24%, in 33.84%, and in 22.43%.
This quadruplex TaqMan qPCR assay offers a rapid, sensitive, and specific tool for the simultaneous detection of , , , and in pigs.
[病原体名称1]、[病原体名称2]、[病原体名称3]和[病原体名称4]是导致猪胃肠道疾病的主要病原体，对养猪生产系统的健康和生产力构成重大威胁。病原体检测是监测和管理这些感染的关键工具。
我们设计了针对[病原体名称1]的[基因名称1]基因、[病原体名称2]的23S rRNA基因、[病原体名称3]的[基因名称2]基因和[病原体名称4]的[基因名称3]基因的引物和探针。我们开发了一种能够同时检测这四种病原体的四重TaqMan实时定量PCR检测方法。
该检测方法显示出高灵敏度，重组质粒标准品pEASY - 23S rRNA、pEASY - aspA和pEASY - [病原体名称3]的检测限为100拷贝/μL，pEASY - [病原体名称4]的检测限为10拷贝/μL。标准曲线表现出良好的线性（R值分别为0.999、0.999、1和0.998）和高扩增效率（分别为93.57%、9,4.84%、85.15%和81.81%）。该检测方法具有高特异性，未检测到与猪链球菌、猪流行性腹泻病毒（PEDV）、猪传染性胃肠炎病毒（TGEV）、[其他病原体名称1]、[其他病原体名称2]、[其他病原体名称3]、猪德尔塔冠状病毒（PDCoV）、猪A组轮状病毒（GARV）和猪捷申病毒（PTV）核酸的交叉反应。该检测方法还具有出色的重复性，批间和批内变异系数（CV）范围为0.15%至1.12%。高浓度核酸不干扰低浓度核酸的检测，确保在复杂样品中具有稳健的性能。在263份腹泻样品中，该检测方法检测到[病原体名称1]的占23.95%，[病原体名称2]的占26.24%，[病原体名称3]的占33.84%，[病原体名称4]的占22.43%。
这种四重TaqMan qPCR检测方法为同时检测猪体内的[病原体名称1]、[病原体名称2]、[病原体名称3]和[病原体名称4]提供了一种快速、灵敏且特异的工具。
