[1]陈萌萌,王冠萱,仇汝龙,等.兔出血症病毒RHDV1和RHDV2一步法双重TaqMan探针荧光定量RT-PCR体系的建立和应用[J].江苏农业学报,2025,(05):937-942.[doi:doi:10.3969/j.issn.1000-4440.2025.05.012]
　CHEN Mengmeng,WANG Guanxuan,QIU Rulong,et al.Establishment and application of one-step dual TaqMan probe quantitative RT-PCR system for rabbit hemorrhagic disease viruses RHDV1 and RHDV2[J].,2025,(05):937-942.[doi:doi:10.3969/j.issn.1000-4440.2025.05.012]
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兔出血症病毒RHDV1和RHDV2一步法双重TaqMan探针荧光定量RT-PCR体系的建立和应用()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2025年05期
页码:
937-942
栏目:
畜牧兽医·水产养殖·益虫饲养
出版日期:
2025-05-31

文章信息/Info

Title:
Establishment and application of one-step dual TaqMan probe quantitative RT-PCR system for rabbit hemorrhagic disease viruses RHDV1 and RHDV2
作者:
陈萌萌1; 2; 3; 王冠萱1; 2; 仇汝龙2; 3; 范志宇2; 3; 胡波2; 3; 宋艳华1; 2; 3; 魏后军2; 3; 徐为中2; 3
(1.南京农业大学动物医学院,江苏南京210095；2.江苏省农业科学院兽医研究所,江苏南京210014；3.兽用生物制品<泰州>国泰技术创新中心,江苏泰州225300)
Author(s):
CHEN Mengmeng1; 2; 3; WANG Guanxuan1; 2; QIU Rulong2; 3; FAN Zhiyu2; 3; HU Bo2; 3; SONG Yanhua1; 2; 3; WEI Houjun2; 3; XU Weizhong2; 3
(1.College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;2.Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;3.Guotai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China)
关键词:
兔出血症病毒; 荧光定量RT-PCR; TaqMan探针
Keywords:
rabbit hemorrhagic disease virus; fluorescence quantitative RT-PCR; TaqMan probe
分类号:
S855.3
DOI:
doi:10.3969/j.issn.1000-4440.2025.05.012
文献标志码:
A
摘要:
兔出血症病毒(RHDV)引起的兔出血症是一种急性、高度致死性传染病,对养兔业造成严重危害。本研究基于RHDV衣壳蛋白(VP60)基因序列设计引物和TaqMan探针,建立可同时检测RHDV1和RHDV2两种毒株的双重TaqMan荧光定量RT-PCR检测体系。反应体系:2× One Step RT-PCR Buffer Ⅲ 10.0 μL,上下游引物 (10 μmol/L)各0.6 μL,ROX Reference Dye Ⅱ (50×) 0.4 μL,两种荧光探针(10 μmol/L)各0.8 μL,待测样本2.0 μL,ddH2O 4.8 μL。反应程序:42 ℃ 5 min，95 ℃ 10 s，95 ℃ 5 s，60 ℃ 30 s,40个循环。试验结果表明,该体系特异性强,与轮状病毒、兔多杀性巴氏杆菌、兔支气管败血波氏菌、绿脓杆菌、沙门氏菌无交叉反应；灵敏度高,最低检测限为1 μL 100拷贝。通过组内重复和组间重复进行重复性分析,Ct值变异系数为0.2%～2.9%,表明该体系稳定性较好。使用该体系对112份临床样本(60份肝脏组织、52份鼻肛拭子)进行检测,RHDV总检出率为69%,而常规RT-PCR体系的检出率仅为51%。本研究建立的双重荧光定量RT-PCR方法具有高灵敏度、强特异性和良好的重复性,可为RHDV的临床快速诊断及定量分析提供可靠技术支持。
Abstract:
Rabbit hemorrhagic disease (RHD) caused by rabbit hemorrhagic disease virus (RHDV) is an acute and highly lethal infectious disease that poses a serious threat to the rabbit breeding industry. In this study, primers and TaqMan probes were designed based on the capsid protein (VP60) gene sequences of RHDV to establish a dual TaqMan quantitative RT-PCR detection system capable of simultaneously detecting both RHDV1 and RHDV2 strains. The reaction system included 10.0 μL of 2×One Step RT-PCR Buffer Ⅲ, 0.6 μL of forward primer (10 μmol/L), 0.6 μL of reverse primer (10 μmol/L), 0.4 μL of ROX Reference Dye Ⅱ (50×), 0.8 μL of each fluorescent probe (10 μmol/L), 2.0 μL of test sample, and 4.8 μL ddH2O. The reaction program was as follows: 42 ℃ for 5 min, 95 ℃ for 10 s, 95 ℃ for 5 s, 60 ℃ for 30 s, and 40 cycles. Results showed that the system had strong specificity and no cross-reactions with rotavirus, Pasteurella multocida, Bordetella bronchiseptica, Pseudomonas aeruginosa, and Salmonella spp., and high sensitivity with a minimum detection limit of 100 copies per microliter. Repeatability analysis through intra-group and inter-group repetitions showed that the coefficient of variation of Ct values was between 0.2% and 2.9%, indicating good stability of the system. When testing 112 clinical samples (60 liver tissues and 52 nasal and anal swabs) using this system, the overall detection rate of RHDV reached 69%, while the detection rate of the conventional RT-PCR system was only 51%. The dual quantitative RT-PCR method established in this study has high sensitivity, strong specificity, and good repeatability, providing a reliable technical support for the rapid clinical diagnosis and quantitative analysis of RHDV.

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备注/Memo

备注/Memo:
收稿日期：2023-12-28基金项目：现代农业产业技术体系建设专项(CARS-43-C-1)作者简介：陈萌萌(1987-),女,连云港人,博士,副研究员,主要从事家兔疾病防治与兽医生物技术研究。(E-mail)moonchen2010@yeah.net通讯作者：王芳,(E-mail)rwangfang@126.com
更新日期/Last Update: 2025-06-24