山羊地方性鼻内肿瘤病毒（ENTV-2）SYBR-Green Ⅰ  实时荧光定量PCR检测方法的建立与应用

张靖鹏; 江锦秀; 林裕胜; 游伟; 刘道泉; 毛坤明; 江斌; 胡奇林

doi:10.19303/j.issn.1008-0384.2021.07.007

山羊地方性鼻内肿瘤病毒（ENTV-2）SYBR-Green Ⅰ 实时荧光定量PCR检测方法的建立与应用

doi: 10.19303/j.issn.1008-0384.2021.07.007

1.
福建省农业科学院畜牧兽医研究所，福建　福州　350013
2.
福州市动物疫病预防控制中心，福建　福州　350002
3.
福建省福清市畜牧兽医技术服务中心，福建　福清　350300

基金项目: 福建省科技重大专项（2019NZ09007）；福建省科技计划公益类专项（2018R1023-1）

详细信息

作者简介:
张靖鹏（1988−），男，硕士，研究实习员，主要从事动物传染病研究（E-mail：870063543@qq.com）

通讯作者:
胡奇林（1963−），男，研究员，主要从事动物传染病学和免疫学研究（E-mail：hql562713@163.com）

中图分类号: S 852.62
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出版历程
- 收稿日期: 2020-12-04
- 修回日期: 2021-03-25
- 网络出版日期: 2021-07-13
- 刊出日期: 2021-07-28

A SYBR-Green Ⅰ RT-qPCR Assay for Detecting Enzootic Nasal Tumor Virus in Goats

1.
Institute of Animal Husbandry and Veterinary Medicine, Fuzhou, Fujian　350013, China
2.
Fuzhou Animal Disease Prevention and Control Center, Fuzhou, Fujian　350002, China
3.
Fuqing Technical Service Center of Animal Husbandry and Veterinary Medicine, Fuzhou, Fujian　350300, China

摘要

摘要: 目的建立一种快速、敏感的ENTV-2检测方法，用于ENT的早期诊断与流行病学调查。方法通过生物信息学的方法将ENTV-2与ENTV-1、ERVs、JSRV进行比对，寻找ENTV-2的保守序列并设计1对特异性引物，建立SYBR-Green Ⅰ 实时荧光定量PCR检测方法，对所建立的PCR反应条件进行优化，将PCR扩增产物连接T载体构建的阳性质粒作为标准品，对SYBR-Green Ⅰ 实时荧光定量PCR检测方法的特异性、敏感性和重复性进行验证。结果建立的检测ENTV-2 qPCR 方法标准曲线呈现良好的线性关系（R² ＝0.992）；该方法的特异性良好，对ENTV-2可以产生特异性扩增曲线，与ENTV-2高度同源的ERVs没有交叉反应，也无法扩增羊口疮病毒（ORFV）、绵羊肺炎支原体（Mo）、丝状支原体山羊亚种（Mmc）等常见病原；敏感性良好，最低检测限度为7.5×10² copies·μL⁻¹，敏感性可达常规PCR检测方法的100倍；批内、批间的变异系数CV＜1%，重复性良好；对81份临床样品的阳性检出率为17.3%。结论建立的SYBR-Green Ⅰ 实时荧光定量PCR方法特异性良好，敏感性较高，重复性良好，为山羊地方性鼻内肿瘤的早期、快速检测提供了技术支持。
- 山羊地方性鼻内肿瘤病毒 /
- SYBR-Green Ⅰ /
- 荧光定量 PCR
Abstract: Objective A rapid, sensitive detection method for early diagnosis and epidemiological survey on enzootic nasal tumor (ENT) in goats was established. Method Bioinformatics methods were employed for sequence alignment of ENTV-2 with ENTV-1, ERVs, and JSRV in search for the conserved sequence of the virus. Primers for qPCR were designed to establish a SYBR-Green I RT-qPCR methodology for its detection. Reaction conditions were optimized, and a standard positive plasmid used to determine the specificity, sensitivity, and reproducibility of the newly developed assay. Result A linear standard curve was found for the detection with a R²=0.992. The method specifically detected only ENTV-2, not the highly homologous ERVs, nor amplified ORFV, MO, or Mmc. It showed a detection sensitivity of up to 7.51×10² copies·μL⁻¹, which was 100 times greater than the conventional PCR can deliver, a repeatability with a coefficient variations (CV) of less than 1% on intra- and inter-batch tests, and a positive detection rate of 17.3% on 81 clinical samples. Conclusion The newly established SYBR-Green I RT-qPCR was specific, sensitive, repeatable, and considered adequate for early and rapid detection of ENTV-2 in goats.
- Enzootic nasal tumor virus of goats(ENTV-2) /
- SYBR-Green Ⅰ /
- RT-qPCR

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图 1 ENTV-2 荧光定量PCR标准曲线

Figure 1. Standard curve of ENTV-2 detected by SYBR-Green Ⅰ RT-qPCR

下载: 全尺寸图片幻灯片

图 2 ENTV-2荧光定量PCR熔解曲线

Figure 2. Melting curve of ENTV-2 detected by SYBR-Green Ⅰ RT-qPCR

下载: 全尺寸图片幻灯片

图 3 ENTV-2 SYBR Green Ⅰ qPCR的特异性

注：1：ENTV-2; 2：羊肾1；3：羊肾2；4：羊肾3；5：羊肾4；6：羊肾5；7：山羊皮细胞；8：绵羊肺细胞；9：ORFV； 10：BCV；11：Mo；12：Mmc；13：Mccp；14：H₂O。

Figure 3. Specificity of SYBR Green Ⅰ RT-qPCR in detecting ENTV-2

Note: 1: ENTV-2; 2: kidney cell of goat No. 1; 3: kidney cell of goat No. 2; 4: kidney cell of goat No. 3; 5: kidney cell of goat No. 4; 6: kidney cell of goat No. 5; 7: goat skin cells; 8: sheep lung cells; 9: ORFV; 10: BCV; 11: MO; 12: Mmc; 13: Mccp; 14: H₂O.

下载: 全尺寸图片幻灯片

图 4 部分临床样品检测结果

注： 1为阳性样品，2～17均为临床样品，18为H₂O。

Figure 4. Partial results of detection by SYBR-Green Ⅰ RT-qPCR on clinical samples

Note: 1: positive sample; 2-7: clinical samples; 18: H₂O.

下载: 全尺寸图片幻灯片

表 1 ENTV-2 荧光定量PCR的重复性

Table 1. Repeatability of SYBR-Green I RT-qPCR in detecting ENTV-2

Entv阳性质粒含量/ （copies·μL⁻¹）	批内重复 Intra-assay reproducibility		批间重复 Inter-assay reproducibility
Entv阳性质粒含量/ （copies·μL⁻¹）	Ct±SD	CV/%	Ct±SD	CV/%
7.51×10³	25.25±0.15	0.59	25.53±0.16	0.63
7.51×10⁵	18.78±0.11	0.58	19.11±0.17	0.89
7.51×10⁷	12.44±0.12	0.96	12.89±0.12	0.93

下载: 导出CSV

表 2 SYBR Green I荧光定量PCR和传统PCR法临床样品检测ENTV-2结果比较

Table 2. Detections by SYBR-Green Ⅰ RT-qPCR and conventional PCR on clinical samples

检测方法 Detecting method		荧光定量PCR SYBR-Green Ⅰ qPCR		总计 Sum
检测方法 Detecting method		阳性数量 Positive number	阴性数量 Negative number	总计 Sum
常规PCR Convenional PCR	阳性数量 Positive number	10	0	10
	阴性数量 Negative number	4	67	71
总计 Sum		14	67	81

下载: 导出CSV

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