基于Taqman探针的多重实时逆转录定量聚合酶链反应法同时检测鸭坦布苏病毒、新型鸭呼肠孤病毒、鸭甲型肝炎病毒1型和鸭甲型肝炎病毒3型

Taqman-probe-based multiplex real-time RT-QPCR for simultaneous detection of duck tembusu virus, novel duck reovirus, duck hepatitis a type 1 virus and duck hepatitis a type 3 virus.

作者信息

Qiu Qian, Hu Ruiming, Liu ZiRui, Yan Linjie, Dai Xueyan, Xing Chenghong, Yang Fan, Cao Huabin

机构信息

Jiangxi Provincial Key Laboratory for Animal Health, Institute of Animal Population Health, College of Animal Science and Technology, Jiangxi Agricultural University, No. 1101 Zhimin Avenue, Economic and Technological Development District, Nanchang 330045, Jiangxi, PR China.

Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, No.22, Xinong Road, Yangling District, Xianyang 712000, Shaanxi PR China.

出版信息

Poult Sci. 2025 Jul 3;104(10):105508. doi: 10.1016/j.psj.2025.105508.

DOI:10.1016/j.psj.2025.105508

PMID:40644908

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12275695/

Abstract

Novel duck reovirus (NDRV), duck hepatitis A type 1 virus (DHAV-1), duck Tembusu virus (DTMUV), and duck hepatitis A type 3 virus (DHAV-3) caused substantial economic losses within the duck industry. In our research, we aimed to develop a TapMan probe-based multiplex quantitative reverse transcription polymerase chain reaction (RT-qPCR) method to enable the early detection of these four viruses in suspicious duck populations. The detection limits for DTMUV, NDRV, DHAV-1, and DHAV-3 were 8.08 × 10¹, 1.24 × 10², 6.03 × 10¹, and 1.88 × 10² copies/μL, respectively. Additionally, the DTMUV, NDRV, DHAV-1, and DHAV-3 were successfully detected. No positive signals were detected for duck enteritis virus (DEV), muscovy duck parvovirus (MDPV), goose parvovirus (GPV), avian influenza virus (AIV), and newcastle disease virus (NDV). The correlation coefficient is greater than 0.99, and the amplification efficiency is 80-100 %. In the reproducibility tests conducted with standard plasmid concentrations at 10, 10, and 10 copies/μL, both the intra-assay and inter-assay coefficients of variation were below 10 %. The multiplex RT-qPCR method was used to detect 215 clinical samples. The positivity rates for NDRV, DHAV-3, DHAV-1, and DTMUV were 2.32 %, 6.27 %, 5.58 %, and 19.30 %. The positivity rates for DHAV-3 and DTMUV co-infection were 5.58 % for DHAV-3, DTMUV, and NDRV co-infection were 0.05 %.

摘要

新型鸭呼肠孤病毒（NDRV）、鸭甲型肝炎病毒1型（DHAV-1）、鸭坦布苏病毒（DTMUV）和鸭甲型肝炎病毒3型（DHAV-3）给养鸭业造成了巨大经济损失。在我们的研究中，旨在开发一种基于TaqMan探针的多重定量逆转录聚合酶链反应（RT-qPCR）方法，以便能够在可疑鸭群中早期检测这四种病毒。DTMUV、NDRV、DHAV-1和DHAV-3的检测限分别为8.08×10¹、1.24×10²、6.03×10¹和1.88×10²拷贝/微升。此外，成功检测到了DTMUV、NDRV、DHAV-1和DHAV-3。未检测到鸭肠炎病毒（DEV）、番鸭细小病毒（MDPV）、鹅细小病毒（GPV）、禽流感病毒（AIV）和新城疫病毒（NDV）的阳性信号。相关系数大于0.99，扩增效率为80%-100%。在用10³、10⁴和10⁵拷贝/微升的标准质粒浓度进行的重复性试验中，批内和批间变异系数均低于10%。使用多重RT-qPCR方法检测了215份临床样本。NDRV、DHAV-3、DHAV-1和DTMUV的阳性率分别为2.32%、6.27%、5.58%和19.30%。DHAV-3和DTMUV共感染的阳性率为5.58%，DHAV-3、DTMUV和NDRV共感染的阳性率为0.05%。