牛病毒性腹泻/黏膜病病毒荧光定量PCR检测方法的建立

张康; 郭志廷; 仇正英; 张景艳; 王磊; 张凯; 王贵波; 梁芬芬; 马倩; 李建喜

doi:10.19303/j.issn.1008-0384.2021.09.007

牛病毒性腹泻/黏膜病病毒荧光定量PCR检测方法的建立

doi: 10.19303/j.issn.1008-0384.2021.09.007

张康^{1, 2,},
郭志廷¹,
仇正英¹,
张景艳¹,
王磊¹,
张凯¹,
王贵波¹,
梁芬芬¹,
马倩^1, ,,
李建喜^1, ,

1.
中国农业科学院兰州畜牧与兽药研究所/农业农村部兽用药物创新重点实验室/甘肃省新兽药工程重点实验室，甘肃　兰州　730050
2.
甘肃农业大学动物医学院，甘肃　兰州　730050

基金项目: 国家现代农业产业技术体系建设项目（CARS36）；甘肃省青年科技基金计划（21JR7RA030）；中国农业科学院科技创新工程协同创新项目（CAAS-XTCX2016011-01-09）；中兽医与临床科技创新工程项目（CAAS-ASTIP-2015-LIHPS）

详细信息

作者简介:
张康（1987−），男，助理研究员，主要从事奶牛疾病诊断研究（E-mail：467863181@qq.com）

通讯作者:
马倩（1991−），女，助理研究员，主要从事兽医临床诊断研究（E-mail：499462890@qq.com）

李建喜（1971−），男，研究员，主要从事奶牛疾病免疫学研究（E-mail：Lzjianxil@163.com）

中图分类号: S 852.62
计量
- 文章访问数: 424
- HTML全文浏览量: 125
- PDF下载量: 18
- 被引次数: 0
出版历程
- 收稿日期: 2020-11-02
- 修回日期: 2021-08-30
- 网络出版日期: 2021-10-23
- 刊出日期: 2021-09-28

A RT-PCR Assay for Quantitative Detection of Bovine Viral Diarrhea Virus

1.
Key laboratory of New Animal Drug Project of Gansu Province, Key Laboratory of Veterinary Drug Innovation and Ministry of Agriculture, Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS of Ministry of Agriculture, Lanzhou, Gansu　730050, China
2.
College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, Gansu　730070, China

摘要

摘要: 目的设计一种能够快速检测出牛病毒性腹泻病毒（bovine viral diarrhea virus, BVDV）的方法。方法依据GenBank公布的BVDV 5′UTR基因属于特异性保守区域的前提，构建具有特异性的探针和引物，以体外转录病毒RNA作为绝对定量标准品。对荧光定量RT-PCR方法的各反应条件进行优化，创建BVDV荧光定量PCR检测方法。结果 5.026 7 copies·μL⁻¹是此检测方法的最低下限值，该方法拥有良好的重复性，变异系数在组内、组间均＜1%。特异性较高，对其他病毒核酸的扩增均为阴性，如猪瘟病毒、口蹄疫病毒等。结论建立的BVDV荧光定量PCR方法敏感性高、特异性强、重复性好，为牛病毒性腹泻的早期诊断提供了重要的技术支撑。
- 牛病毒性腹泻病毒 /
- 5′UTR /
- 实时荧光定量PCR /
- RNA标准品
Abstract: Objective A rapid detection and quantification method for bovine viral diarrhea virus (BVDV) was established. Method For the methodology development, specific primers and probes were designed based on the target regions of the BVDV 5′UTR gene published by GenBank. The RNA of in vitro transcription viruses was used as the absolute quantitative standard. Reaction conditions of the fluorescent quantitative RT-PCR were optimized. Result The newly developed assay had a minimum detection limit of 5.0267copies/μL and an intragroup variation coefficient of less than 1% with high repeatability and specificity. Other than BVDV, it amplified no viral nucleic acids of viruses such as swine fever and foot-and-mouth diseases. Conclusion The established fluorescence quantitative RT-PCR method was highly sensitive, specific, and repeatable in detecting and quantifying BVDV. It appeared appropriate for early diagnosis of bovine viral diarrhea.
- Bovine viral diarrhea virus /
- 5′UTR /
- RT-PCR /
- standard RNA

HTML全文

图 1 标准品引物扩增产物

注：M为DNA marker；1为PCR产物；2为阴性对照。

Figure 1. Amplification products of RNA standard primers

Note: M: DNA marker; 1: PCR product; 2: negative control.

下载: 全尺寸图片幻灯片

图 2 荧光定量PCR方法特异性试验

注：A为阳性样品（BVDV NADL标准毒株）；B为FMDV；C为IBRV；D为水（阴性对照）；E为CSFV；F为PRRS。

Figure 2. Specificity of RT-PCR assay

Note: A: BVDV positive control; B: FMDV; C: IBRV; D: negative control; E: CSFV; F: PRRS.

下载: 全尺寸图片幻灯片

图 3 荧光定量PCR敏感性试验

Figure 3. Sensitivity of RT-PCR assay

下载: 全尺寸图片幻灯片

图 4 荧光定量PCR标准曲线

Figure 4. Standard curve for RT-PCR assay

下载: 全尺寸图片幻灯片

图 5 荧光定量PCR重复性

Figure 5. Reproducibility of RT-PCR assay

下载: 全尺寸图片幻灯片

图 6 荧光定量PCR方法的应用

注：1为阳性样品（BVDV NADL标准毒株）；2～5为临床样品检出BVDV阳性。

Figure 6. Application of RT-PCR assay

Note: 1: BVDV positive control, 2-5: BVDV positive clinical samples were detected.

下载: 全尺寸图片幻灯片

表 1 引物和探针

Table 1. Primers and probes applied

引物名称 Primers	引物序列（5′→3′） Primer sequences（5′-3′）	目的片段大小/bp Product size/bp
A-F	TAATACGACTCACTATAGGG-CATGCCCATAGTAGGAC	325
B-R	TTTTTTTTTTTTTTTTTTTTTTT CCATGTGCCATGTACAG
Q-F	GAGGGTAGCAACAGTGGTGA	76
Q-R	AGGCGTCGAACCACTGACG
探针	FAM-GTGGTGAGTTCGTTGGATGGCT-BHQ1

下载: 导出CSV

表 2 核酸浓度

Table 2. Nucleic acid concentration

扩增曲线 Amplification curve	核酸浓度 Nucleic acid concentration（copies ·μL⁻¹）
1	5.026 7×10⁸
2	5.0267×10⁷
3	5.026 7×10⁶
4	5.026 7×10⁵
5	5.026 7×10⁴
6	5.026 7×10³
7	5.026 7×10²
8	5.026 7×10¹
9	5.026 7
10	0.502 67

下载: 导出CSV

表 3 实时荧光定量PCR方法的变异系数

Table 3. Coefficients of variation between inter- and intra-groups of RT-PCR assay

稀释度 Dilution rate	组内重复性 Reproducibility intra-batch		组间重复性 Reproducibility inter-batch
稀释度 Dilution rate	Ct平均值 Ct average values	组内变异系数 Coefficient of variation/%	Ct平均值 Ct average values	组间变异系数 Coefficient of variation/%
浓度1	21.82±0.12	0.55	21.79±0.08	0.37
浓度2	25.31±0.19	0.75	24.93±0.49	1.97
浓度3	28.63±0.27	0.94	28.45±0.16	0.56
浓度4	31.21±0.27	0.87	31.27±0.07	0.22
浓度5	35.65±0.27	0.76	35. 14±0.48	1.37

下载: 导出CSV

参考文献(20)

[1]	BECHER P, THIEL H J. Pestivirus[M]//The Springer Index of Viruses. New York, NY: Springer New York, 2011: 483-488.
[2]	VILČEK Š, PATON D J, DURKOVIC B, et al. Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups [J]. Archives of Virology, 2001, 146(1): 99−115. doi: 10.1007/s007050170194
[3]	RIDPATH J F, BOLIN S R, DUBOVI E J. Segregation of bovine viral diarrhea virus into genotypes [J]. Virology, 1994, 205(1): 66−74. doi: 10.1006/viro.1994.1620
[4]	PETERHANS E, BACHOFEN C, STALDER H, et al. Cytopathic bovine viral diarrhea viruses (BVDV): Emerging pestiviruses doomed to extinction [J]. Veterinary Research, 2010, 41(6): 44. doi: 10.1051/vetres/2010016
[5]	MOENNIG V, LIESS B. Pathogenesis of intrauterine infections with bovine viral diarrhea virus [J]. Veterinary Clinics of North America:Food Animal Practice, 1995, 11(3): 477−487. doi: 10.1016/S0749-0720(15)30462-X
[6]	BAKER J C. The clinical manifestations of bovine viral diarrhea infection [J]. The Veterinary Clinics of North America Food Animal Practice, 1995, 11(3): 425−445. doi: 10.1016/S0749-0720(15)30460-6
[7]	HOUE H. Economic impact of BVDV infection in dairies [J]. Biologicals, 2003, 31(2): 137−143. doi: 10.1016/S1045-1056(03)00030-7
[8]	REICHEL M, HILL F, VOGES H. Does control of bovine viral diarrhoea infection make economic sense? [J]. New Zealand Veterinary Journal, 2008, 56(2): 60−66. doi: 10.1080/00480169.2008.36809
[9]	LANYON S R, SIMS S K, COCKCROFT P D, et al. Comparison of serum, ear notches, and nasal and saliva swabs for Bovine viral diarrhea virus antigen detection in colostrum-fed persistently infected (PI) calves and non-PI calves [J]. Journal of Veterinary Diagnostic Investigation, 2014, 26(6): 783−787. doi: 10.1177/1040638714550181
[10]	DRISKELL E A, RIDPATH J F. A survey of bovine viral diarrhea virus testing in diagnostic laboratories in the United States from 2004 to 2005 [J]. Journal of Veterinary Diagnostic Investigation, 2006, 18: 600−605. doi: 10.1177/104063870601800615
[11]	李家伟, 郭利, 杨勇, 等. 牛病毒性腹泻病毒LAMP检测方法的建立与应用 [J]. 中国畜牧兽医, 2015, 42(12):3111−3118. LI J W, GUO L, YANG Y, et al. Establishment and application of LAMP detection method of bovine viral diarrhea virus [J]. China Animal Husbandry & Veterinary Medicine, 2015, 42(12): 3111−3118.(in Chinese)
[12]	范晴, 谢芝勋, 刘加波, 等. 牛病毒性腹泻病毒实时荧光定量RT-PCR检测方法的建立 [J]. 动物医学进展, 2010, 31(10):10−14. doi: 10.3969/j.issn.1007-5038.2010.10.003 FAN Q, XIE Z X, LIU J B, et al. Establishment of real-time flourescent quantitative PCR for detection of bovine viral diarrhea virus [J]. Progress in Veterinary Medicine, 2010, 31(10): 10−14.(in Chinese) doi: 10.3969/j.issn.1007-5038.2010.10.003
[13]	胡俊英, 鲁海冰, 常晓冉, 等. 基于单抗捕获牛病毒性腹泻病毒(BVDV)抗原ELISA方法的建立及应用 [J]. 中国兽医学报, 2019, 39(3):401−407. HU J Y, LU H B, CHANG X R, et al. Development of a sandwich ELISA for capture of bovine viral diarrhea virus antigen and epidemiological investigation [J]. Chinese Journal of Veterinary Science, 2019, 39(3): 401−407.(in Chinese)
[14]	王荣, 李文文, 王研, 等. 牛病毒性腹泻病毒实时荧光定量RT-PCR方法的建立及应用 [J]. 中国畜牧兽医, 2014, 41(2):35−39. WANG R, LI W W, WANG Y, et al. Establishment and application of real-time fluorescent quantitative RT-PCR method for detection of bovine viral diarrhea virus [J]. China Animal Husbandry & Veterinary Medicine, 2014, 41(2): 35−39.(in Chinese)
[15]	国家市场监督管理总局, 国家标准化管理委员会. 牛病毒性腹泻/粘膜病诊断技术规范: GB/T 18637—2018[S]. 北京: 中国标准出版社, 2018.
[16]	MACKAY I M, ARDEN K E, NITSCHE A. Real-time PCR in virology [J]. Nucleic Acids Research, 2002, 30(6): 1292−1305. doi: 10.1093/nar/30.6.1292
[17]	ZIMMER G M, VAN MAANEN C, DE GOEY I, et al. The effect of maternal antibodies on the detection of bovine virus diarrhoea virus in peripheral blood samples [J]. Veterinary Microbiology, 2004, 100(3/4): 145−149.
[18]	DENG M L, JI S K, FEI W T, et al. Prevalence study and genetic typing of bovine viral diarrhea virus (BVDV) in four bovine species in China [J]. PLoS One, 2015, 10(4): e0121718. doi: 10.1371/journal.pone.0121718
[19]	付运星, 吴永丽, 徐道修, 等. 犬瘟热病毒一步法荧光定量PCR检测方法的建立 [J]. 中国兽医学报, 2016, 36(3):407−411. FU Y X, WU Y L, XU D X, et al. Development of real-time PCR assay for detection and quantification of the canine distemper virus [J]. Chinese Journal of Veterinary Science, 2016, 36(3): 407−411.(in Chinese)
[20]	李占鸿, 廖德芳, 杨振兴, 等. 帕利亚姆血清群病毒一步法RT-PCR检测技术的建立 [J]. 中国预防兽医学报, 2020, 42(1):40−45. LI Z H, LIAO D F, YANG Z X, et al. The development of a one-step RT-PCR assay for the detection of PALV [J]. Chinese Journal of Preventive Veterinary Medicine, 2020, 42(1): 40−45.(in Chinese)