| 陈平亚，张亮亮，吴少荣，黄纪徽，王绥家，吴山楠.致对虾肝胰腺坏死病副溶血弧菌荧光定量PCR 检测方法的建立[J].广东农业科学,2021,48(11):119-125 |
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致对虾肝胰腺坏死病副溶血弧菌荧光定量PCR 检测方法的建立 |
| Establishment of Real-time PCR for Detection of Vibrio parahaemolyticus Causing Acute Hepatopancreas Necrosis Disease in Shrimps |
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| DOI：10.16768/j.issn.1004-874X.2021.11.015 |
| 中文关键词: 对虾 急性肝胰腺坏死病 副溶血弧菌 pirB 基因 荧光定量 PCR |
| 英文关键词: shrimp acute hepatopancreas necrosis disease Vibrio parahaemolyticus pirB gene real-time PCR |
| 基金项目:海南省重点研发计划项目（ZDYF2020088） |
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| 中文摘要: |
| 【目的】建立一种对虾急性肝胰腺坏死病（Acute Hepatopancreatic Necrosis Disease）病原菌副溶血弧菌（Vibrio parahaemolyticus, VpAHPND）荧光定量 PCR 检测方法，对该病进行快速检测。【方法】根据副溶血弧菌基因保守序列设计特异性引物和探针，经优化反应体系及条件，建立检测 VpAHPND 的荧光定量 PCR 方法；以重组质粒为标准品制作标准曲线，并进行特异性、敏感性、重复性及临床应用试验。【结果】该方法定量范围宽，起始模板浓度范围为 2.3×101
~2.3×107 拷贝 /μL 时，标准曲线具有良好的线性关系；最低检测限为 2.3 拷贝/μL，与对虾其他病原菌及病毒无交叉反应，重复试验变异系数为 0.45%~0.69%，可在 1 h 内完成样品检测；对临床对虾样品的检测结果表明，该方法与世界动物卫生组织（OIE）推荐的套式 PCR 法检测结果一致。【结论】应用 TaqMan 探针建立的检测 VpAHPND 的荧光定量 PCR 方法具有灵敏度高、特异性强、重复性好的特点，可有效用于临床对虾样品的 VpAHPND 检测。 |
| 英文摘要: |
| 【Objective】A real-time PCR method was developed to detect Acute Hepatopancreas Necrosis Disease
（AHPND）caused by Vibrio parahaemolyticus（VpAHPND）for the rapid detection of such disease.【Method】A pair of
specific primers and fluorogenic-labeled TaqMan probe were designed according to the conservative sequence of VpAHPND,
and a real-time PCR method for the detection of VpAHPND was established by optimizing the reaction system and conditions.
The standard curve was made with recombinant plasmids as standard products, and the specificity, sensitivity, repeatability
and clinical application test were carried out.【Result】The method had a wide quantitative range from 2.3×101
to 2.3×107
copies/μL and had linear relationship in its standard curve. The real-time PCR method had a high sensitivity with the
detection limit as low as to 2.3 copies/μL for the purified recombinant plasmids of PMD-18T-pirB, and the entire detection
could be completed within 1 h for a single sample. It also had a high specificity in detecting DNA of VpAHPND. The variation
coefficients among cycle thresholds（Ct）of the repeatability test were 0.45%-0.69%. Comparing with the nested PCR recommended by OIE, the TaqMan probe real-time PCR had a same test rate of VpAHPND.【Conclusion】The TaqMan probe
real-time PCR method was highly specific, sensitive and repeatable, which could be used for the early diagnosis of acute and
latent infections of VpAHPND in shrimp samples. |
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