| 林志雄 1
，鱼海琼 1
，王 莹 1
，佟铁铸 1
，温肖会 2，张 利 3，翟建新 3.马鼻肺炎双重荧光定量 PCR 检测方法的建立与应用[J].广东农业科学,2019,46(8):123-128 |
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马鼻肺炎双重荧光定量 PCR 检测方法的建立与应用 |
| Establishment and Application of Duplex Quantitative Real-TimePCR Method for Detection of Equine Rhinopneumonitis |
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| DOI：10.16768/j.issn.1004-874X.2019.08.017 |
| 中文关键词: 马鼻肺炎 马疱疹病毒 1 型 马疱疹病毒 4 型 实时荧光 PCR 特异性 敏感性 分型检测 |
| 英文关键词: equine rhinopneumonitis EHV1 EHV4 real-time PCR specificity sensitivity detection for distinction |
| 基金项目:广东省动植物与食品进出口技术措施研究重点实验室开放课题（IQTC201801）；国家质检总局科研计划项目（2012IK006） |
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| 中文摘要: |
| 【目的】建立快速、准确检测马疱疹病毒 1 型（EHV1）和 4 型（EHV4）的双重荧光定量 PCR 检
测方法。【方法】以 EHV1、EHV4 的全基因组序列为基础，针对糖蛋白 B（gB）基因的保守序列，分别设计
EHV1 与 EHV4 特异性引物和探针，筛选引物，优化反应条件，分型检测马疱疹病毒（EHV1、EHV4）。【结果】
检测到 EHV1、EHV4、EIV、EAV、EVJ 和 EIAV 标准毒株，EHV1、EHV4 为阳性结果，其余病毒为阴性结果；
EHV1、EHV4 DNA 模板的最低检出限为 1.47×102 copies/μL；检测 64 份临床样本，阳性检出率为 14.06%，病
毒分离证实 100% 符合。【结论】双重荧光定量 PCR 检测方法可直接应用于检测并区分 EHV1、EHV4, 并可在
短时间内获得对 EHV1/EHV4 特异性及敏感性的检测结果。 |
| 英文摘要: |
| 【Objective】 The study was to establish of a duplex quantitative real-time PCR method for rapid and
accurate detection of equine herpesvirus 1 (EHV1) and equine herpesvirus 4 (EHV4). 【Method】 Based on the complete
genome sequence of EHV1 and EHV4, according to the conserved sequences of glycoprotein B (gB) gene in GenBank, a
series of specific primers and probes were designed. By selecting suitable primers, optimizing reaction conditions, two kinds
of equine herpesviruses (EHV1, EHV4) were detected. 【Result】 The standard strains such as EHV1, EHV4, EIV, EAV,
EVJ and EIAV were detected, among which EHV1 and EHV4 was positive and other stains were negative. The minimum
detection limit was 1.47×102copies/μL. The positive detection rate in 64 clinical samples was 14.06%, which was 100%
consistent with virus isolation. 【Conclusion】 The duplex quantitative real-time PCR detection method can be directly
applied to detect and distinguish EHV1 and EHV4, and the test results of specificity and sensitivity of EHV1/EHV4 can be
obtained in a short time. |
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