新型鹅星状病毒RT-PCR检测方法的建立及初步应用

余明兴; 林裕胜

doi:10.19303/j.issn.1008-0384.2021.06.007

新型鹅星状病毒RT-PCR检测方法的建立及初步应用

doi: 10.19303/j.issn.1008-0384.2021.06.007

余明兴^1,,
林裕胜²

1.
福建省浦城县动物疫病预防控制中心，福建　浦城　353400
2.
福建省农业科学院畜牧兽医研究所，福建　福州　350013

基金项目: 福建省科技计划公益类专项（2018R1101014-17）；福建省农业科学院自由探索科技创新项目（ZYTS2019024）

详细信息

作者简介:
余明兴（1966−），男，高级兽医师，主要从事动物传染病防控及监测（E-mail：fjpcymx@126.com）

中图分类号: S 852.62
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- 被引次数: 0
出版历程
- 收稿日期: 2021-01-20
- 修回日期: 2021-03-05
- 网络出版日期: 2021-06-06
- 刊出日期: 2021-06-28

A RT-PCR Method for Detecting New Astrovirus in Geese

YU Mingxing^1
,,
LIN Yusheng²

1.
Animal Disease Prevention and Control Center, Pucheng County, Fujian　353400, China
2.
Institute of Animal Husbandry & Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China

摘要

摘要: 目的建立一种新型鹅星状病毒（New goose astrovirus，NGAstV）的RT-PCR检测方法。方法根据ORF2基因序列，利用Oligo 7软件设计并筛选出特异性扩增引物，通过优化扩增条件，建立检测新型鹅星状病毒的RT-PCR方法，对其进行特异性及敏感性检验，并在临床上进行初步应用。结果 RT-PCR最佳反应体系为：Premix 10 μL，ORF2上下游引物浓度10 μmol·L⁻¹各1 μL，模板3 μL，无菌去离子水补足至20 μL。最佳反应程序为：94 ℃ 2 min；94 ℃ 30 s、55 ℃ 15 s、72 ℃ 15 s，30 个循环；72 ℃ 10 min。该方法对新型鹅星状病毒能扩增出特异性片段，对其他鹅常见病原无特异性扩增；检测灵敏度为62 fg·μL⁻¹。对45份临床样品进行检测，结果检测出15份阳性样品，阳性率为33.33%。结论建立的新型鹅星状病毒RT-PCR方法特异性强、稳定性好，可作为新型鹅星状病毒快速检测和流行病学调查的实用技术。
- 新型鹅星状病毒 /
- RT-PCR /
- 检测
Abstract: Objective A RT-PCR method for detecting new goose astrovirus (NGAstV) was developed. Method According to the ORF2 sequence, specific primers were designed according to Oligo 7, and the amplification conditions optimized for the new methodology. Result The optimized reaction system applied 10 μL premix, 1 μL each of ORF2 upstream and downstream primer in the concentration of 10 μmol·L⁻¹, 3 μL template, and sterile deionized water to make up 20 μL with the following testing steps: 94 ℃ for 2 min, 30 cycles of 94 ℃ for 30 s, 55 ℃ for 15 s, and 72 ℃ for 15 s, and kept at 72 ℃ for 10 min. The method amplified the specific fragment of NGAstV but no other common disease viruses of geese and delivered a detection sensitivity of 62 fg·μL⁻¹ on the NGAstV nucleic acid. Out of 45 clinical samples, 15 were positively detected at a rate of 33.33% by the newly developed method. Conclusion The new RT-PCR method exhibited the specificity and repeatability that could satisfactorily be used for detecting and/or epidemiological studies relating to NGAstV.
- new goose astrovirus /
- RT-PCR /
- detection

HTML全文

图 1 新型鹅星状病毒ORF2基因的RT-PCR扩增结果

注：M为2000标注；1：新型鹅星状病毒ORF2基因；2：阴性对照。

Figure 1. Amplification on ORF2 of NGAstV by RT-PCR

Note：M: DNA marker DL2000; 1: NGAstV ORF2 gene; 2: negative control.

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图 2 新型鹅星状病毒RT-PCR特异性试验结果

注：M为2000标注；1～3：新型鹅星状病毒分离株；4～8：分别为新城疫病毒、禽呼肠孤病毒、鹅细小病毒、鹅多瘤病毒、鹅圆环病毒；9：空白对照。

Figure 2. Specificity of NGAstV detection by RT-PCR

Note：M: DNA marker DL2000; 1-3: templates of NGAstV strain; 4-8: templates of NDV, ARV, GPV, GPoV, and GoCV, respectively; 9: blank control.

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图 3 RT-PCR敏感性试验

注：M为2000标注；1～9：新型鹅星状病毒cDNA模板质量浓度分别为6.2 ng·μL⁻¹、620 pg·μL⁻¹、62 pg·μL⁻¹、6.2 pg·μL⁻¹、620 fg·μL⁻¹、62 fg·μL⁻¹、6.2 fg·μL⁻¹、620 ag·μL⁻¹、62 ag·μL⁻¹。

Figure 3. Sensitivity of RT-PCR assay

Note：M: DNA marker DL2000; 1–9: concentrations of template DNA of NGAstV at 6.2 ng·μL⁻¹, 620 pg·μL⁻¹, 62 pg·μL⁻¹, 6.2 pg·μL⁻¹, 620 fg·μL⁻¹, 62 fg·μL⁻¹, 6.2 fg·μL⁻¹, 620 ag·μL⁻¹, and 62 ag·μL⁻¹, respectively.

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图 4 部分临床样品RT-PCR检测结果

注：M为2000标注;1～17: 临床样品。

Figure 4. Detection of NGAstV on clinical samples by RT-PCR assay

Note：M: DNA marker DL2000; 1-17: clinical samples.

下载: 全尺寸图片幻灯片

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