牛嵴病毒<i>Taq</i>Man荧光定量RT-PCR检测方法的建立及应用

赵润涛; 特木尔巴根; 郭宇; 武娅楠; 王旭芬; 侯琳; 张贺; 赵洋; 张志丹; 周伟光

doi:10.19303/j.issn.1008-0384.2023.07.011

牛嵴病毒TaqMan荧光定量RT-PCR检测方法的建立及应用

doi: 10.19303/j.issn.1008-0384.2023.07.011

1.
内蒙古农业大学兽医学院/农业农村部动物疾病临床诊疗技术重点实验室，内蒙古　呼和浩特　010018
2.
内蒙古自治区动物疫病预防控制中心，内蒙古　呼和浩特　010020

基金项目: 内蒙古自治区科技重大专项（2020ZD0006）

详细信息

作者简介:
赵润涛（1995 —），女，硕士研究生，主要从事动物传染病诊断与防控研究，E-mail：854963174@qq.com

通讯作者:
周伟光（1971 —），男，博士，教授，主要从事动物传染病诊断与防控研究，E-mail：zhouwg2001@aliyun.com

中图分类号: S852.62
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出版历程
- 收稿日期: 2022-10-31
- 修回日期: 2023-04-10
- 网络出版日期: 2023-08-16
- 刊出日期: 2023-07-28

TaqMan qRT-PCR Assay for Detecting Bovine Kobuvirus

1.
College of Veterinary Medicine, Inner Mongolia Agricultural University/Key Laboratory of Clinical Diagnosis and Treatment of Animal Diseases, Ministry of Agriculture and Rural Affairs, Hohhot, Inner Mongolia　010018, China
2.
Inner Mongolia Animal Disease Control Center, Hohhot, Inner Mongolia　010020, China

摘要

摘要: 目的建立一种快速、准确且能定量分析牛嵴病毒（Bovine kobuvirus，BKoV）的检测方法。方法根据GenBank上发布的牛嵴病毒3D基因序列设计合成一对特异性引物和一条探针，通过优化反应体系建立牛嵴病毒TaqMan荧光定量RT-PCR检测方法，并对临床样品进行检测。结果该方法最佳上下游引物浓度均为300 nmol·L⁻¹，探针浓度为400 nmol·L⁻¹，在1×10¹～1×10⁸ copies·µL⁻¹呈现良好的线性关系，线性相关系数R²=0.999，扩增效率为103%；特异性较强，在多个犊牛腹泻相关病原中，只检测出牛嵴病毒；敏感性较高，对BKoV质粒标准品最低检测下限为1×10¹ copies·µL⁻¹，而普通RT-PCR对BKoV质粒标准品最低检测下限为1×10² copies·µL⁻¹；重复性较好，组内变异系数和组间变异系数均小于3%。2021年3～5月采自内蒙古地区牧场的37份犊牛粪样中BKoV的检出率为24.3%，通过标准曲线计算其病毒载量，其中腹泻粪样平均病毒载量为3.7×10⁵ copies·µL⁻¹，健康犊牛粪样平均病毒载量为8.65×10³ copies·µL⁻¹。结论建立的牛嵴病毒TaqMan荧光定量RT-PCR检测方法特异性强、稳定性好、灵敏度高，为BKoV的检测和分子流行病学调查提供了有力的手段。
- 牛嵴病毒 /
- 荧光定量RT-PCR /
- 犊牛腹泻 /
- 特异性 /
- 敏感性 /
- 重复性
Abstract: Objective A rapid, accurate qRT-PCR method for detecting bovine kobuvirus (BKoV) was established and clinically tested for application. Method A pair of specific primers and a probe were designed and synthesized according to the 3D gene sequence of BKoV published on GenBank for the establishment of a TaqMan fluorescence qRT-PCR assay. The reaction system of the analytic was optimized prior to verification on clinical specimens. Result The optimal upstream and downstream primers were 300 nmol·L⁻¹ in concentration, and the applied probe 400 nmol·L⁻¹. A linearity with a correlation coefficient of R²= 0.999 was achieved in the range of 1×10¹−1×10⁸ copies·μL⁻¹. The amplification efficiency reached 103%. The assay demonstrated a high specificity among the pathogens related to diarrhea in calves, a high sensitivity with the minimum detection limit of 1×10¹ copies·μL⁻¹ on BKoV plasmid standard, in comparison to that of 1×10² copies·μL⁻¹ by conventional RT-PCR, and a high repeatability with a coefficient of variation of less than 3% within and between groups. In 37 bovine fecal samples collected from different pastures in Inner Mongolia from March to May 2021, the assay showed a positive detection rate of 24.3%. The calculated viral loads using the standard curve had an average on the feces from diarrheal animals at 3.7×10⁵ copies·μL⁻¹ and at 8.65×10³ copies·μL⁻¹ on the specimens from healthy calves. Conclusion The newly developed TaqMan fluorescent qRT-PCR assay was specific, repeatable, and sensitive in detecting BKoV. It could be applied for clinic diagnosis and epidemiological investigation on the disease.
- Bovine kobuvirus /
- fluorescence qRT-PCR /
- calf diarrhea /
- specificity /
- sensitivity /
- repeatability

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图 1 BKoV荧光定量RT-PCR引物优化结果

A～E：引物浓度分别为500、400、300、200、100 nmol·L⁻¹。

Figure 1. Optimization on primer concentration for detecting BKoV by qRT-PCR

A–E: Concentrations of primers at 500, 400, 300, 200, and 100 nmol·L⁻¹, respectively.

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图 2 BKoV荧光定量RT-PCR探针优化结果

A～E：探针浓度分别为500、400、300、200、100 nmol·L⁻¹。

Figure 2. Optimization on probe concentration for detecting BKoV by qRT-PCR

A–E: Concentrations of probe at 500, 400, 300, 200, and 100 nmol·L⁻¹, respectively.

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图 3 BKoV荧光定量RT-PCR标准曲线

Figure 3. Standard curve of qRT-PCR for BKoV detection

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图 4 BKoV荧光定量RT-PCR特异性试验结果

A：牛嵴病毒；B：其他病原和阴性对照。

Figure 4. Specificity of qRT-PCR assay for BKoV detection

A: BKoV; B: other pathogens and negative control.

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图 5 BKoV荧光定量RT-PCR敏感性试验结果

A～H分别为质粒DNA 含量1.0×10⁸～1.0×10¹ copies·µL⁻¹；I：阴性对照；J：1.0×10⁰ copies·µL⁻¹质粒DNA。

Figure 5. Sensitivity of qRT-PCR assay for BKoV detection

A–H: plasmid DNA at 1.0×10⁸−1.0×10¹ copies·μL⁻¹, respectively; I: negative control ; J:1.0×10⁰ copies·μL⁻¹ plasmid DNA.

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图 6 BKoV普通RT-PCR敏感性试验结果

M：500 bp Ladder DNA Marker；1～9：质粒DNA用量分别为 1.0×10⁸～1.0×10⁰ copies·µL⁻¹；10：阴性对照。

Figure 6. Sensitivity of qRT-PCR assay for BKoV detection

M: 500 bp ladder DNA marker; 1–9: plasmid DNA concentrations at 1.0×10⁸−1.0×10⁰ copies·μL⁻¹, respectively; 10: negative control.

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表 1 牛嵴病毒引物及探针序列信息

Table 1. Sequences of BKoV primers and probe

项目 Items	名称 Names	序列(5'-3') Sequences(5'-3')	扩增长度 Amplification length/bp
引物 Primers	BKoV-F-221	TYTCACCYGCTTGGCTCTC	200
引物 Primers	S-BKoV-R-421	AGTGTAGGTGCCRCGCAT+A	200
探针 Probe	BKV-TZ	FAM-CTTCACACAGTGGCGTGGTGAACT-BHQ1

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表 2 牛嵴病毒荧光定量RT-PCR组内及组间重复性

Table 2. Intra- and inter-group repeatability of qRT-PCR assay for BKoV detection

标准品 Plasmid standard concentration/ (copies·µL⁻¹)	组内重复性 Reproducibility intra-group		组间重复性 Reproducibility intra-group
标准品 Plasmid standard concentration/ (copies·µL⁻¹)	平均值（M±SD）/%	变异系数 CV/%	平均值（M±SD）/%	变异系数 CV/%
1×10⁶	21.99±0.23	1.0	22.02±0.04	0.2
1×10⁵	25.30±0.03	0.1	25.31±0.09	0.4
1×10⁴	28.76±0.24	0.8	28.91±0.13	0.4
1×10³	32.04±0.03	0.1	32.19±0.29	0.9
1×10²	34.82±0.49	1.4	34.77±0.63	1.8

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表 3 犊牛粪样牛嵴病毒载量检测

Table 3. Detection of BKoV load on calf fecal samples

粪样 Fecal samples	数量 Number	阳性数量 Positive number	最低拷贝数 Minimum copies/ （copies·µL⁻¹）	最高拷贝数 Maximum copies/ （copies·µL⁻¹）	平均拷贝数 Average copies/ （copies·µL⁻¹）
腹泻 Diarrhea	30	7	2.33×10³	1.90×10⁶	3.70×10⁵
健康 Healthy	7	2	3.54×10³	1.38×10³	8.65×10³

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