Ren Jing, Li Fujun, Yu Xue, Li Yang, Li Meng, Sha Yujie, Li Xiaowen
Shandong Engineering Research Center of Swine Health Data and Intelligent Monitoring, Dezhou University, Dezhou, China.
Shandong Provincial Key Laboratory of Biophysics, Institute of Biophysics, Dezhou University, Dezhou, China.
Front Vet Sci. 2024 Aug 14;11:1450066. doi: 10.3389/fvets.2024.1450066. eCollection 2024.
PEDV, and , are highly contagious diarrheal pathogens that have caused significant harm to the global swine industry. Co-infections with multiple pathogens are common, making it challenging to identify the actual causative agents depending only on clinical information. It is crucial to develop a reliable method to simultaneously detect and differentiate these pathogens.
Based on the conserved regions of the M gene of PEDV, NADH oxidase gene of , and the 16S rDNA gene of , specific probes and primers for the multiplex real-time PCR assay were designed. The concentrations of primers and probes were optimized using a matrix method.
The approach demonstrated high specificity and no cross-reactivity with major pathogens related to diarrheal diseases. It showed high sensitivity with a detection limit of 10 copies/μL for and , and 100 copies/μL for PEDV, respectively. It also demonstrated high reproducibility and stability with low coefficients of variation. Results from the multiplex real-time PCR method were in complete agreement with the commercial singleplex real-time PCR kit for detecting PEDV, and . Clinical data revealed single infection rates of 31.46% for PEDV, 58.43% for , and 98.6% for . The co-infection rates were 16.85% for PEDV + , 31.46% for PEDV + , 57.86% for + , and 16.85% for PEDV + + , respectively.
The new multiplex real-time PCR method can simultaneously differentiate PEDV, and , making it a valuable diagnostic tool for preventing and controlling infectious diseases, as well as aiding in epidemiological investigations.
猪流行性腹泻病毒（PEDV）、[此处原文缺失一种病原体名称]和[此处原文缺失另一种病原体名称]是高度传染性的腹泻病原体，已对全球养猪业造成重大危害。多种病原体的共同感染很常见，仅依靠临床信息来确定实际病原体具有挑战性。开发一种可靠的方法来同时检测和区分这些病原体至关重要。
基于PEDV的M基因保守区、[此处原文缺失一种病原体名称]的NADH氧化酶基因以及[此处原文缺失另一种病原体名称]的16S rDNA基因，设计了多重实时荧光定量PCR检测的特异性探针和引物。采用矩阵法优化引物和探针浓度。
该方法具有高度特异性，与腹泻相关主要病原体无交叉反应。对[此处原文缺失一种病原体名称]和[此处原文缺失另一种病原体名称]的检测限分别为10拷贝/μL，对PEDV的检测限为100拷贝/μL，显示出高灵敏度。同时具有高重复性和稳定性，变异系数低。多重实时荧光定量PCR方法的结果与检测PEDV、[此处原文缺失一种病原体名称]和[此处原文缺失另一种病原体名称]的商业单重实时荧光定量PCR试剂盒完全一致。临床数据显示，PEDV的单感染率为31.46%，[此处原文缺失一种病原体名称]为58.43%，[此处原文缺失另一种病原体名称]为98.6%。PEDV与[此处原文缺失一种病原体名称]的共同感染率为16.85%，PEDV与[此处原文缺失另一种病原体名称]为31.46%，[此处原文缺失一种病原体名称]与[此处原文缺失另一种病原体名称]为57.86%，PEDV与[此处原文缺失一种病原体名称]与[此处原文缺失另一种病原体名称]为16.85%。
新的多重实时荧光定量PCR方法可同时区分PEDV、[此处原文缺失一种病原体名称]和[此处原文缺失另一种病原体名称]，使其成为预防和控制传染病以及辅助流行病学调查的有价值诊断工具。
