童桂香,黎小正,吴祥庆,黄鸾玉,黄光华,韦信贤.TaqMan–MGB探针荧光定量PCR检测致对虾急性肝胰腺坏死病副溶血弧菌[J].湖南农业大学学报:自然科学版,2019,45(4):.
TaqMan–MGB探针荧光定量PCR检测致对虾急性肝胰腺坏死病副溶血弧菌
  
DOI:
中文关键词:  对虾  急性肝胰腺坏死病  副溶血弧菌  TaqMan–MGB探针  荧光定量PCR方法
英文关键词:shrimp  acute hepatopancreas necrosis disease  Vibrio parahaemolyticus  TaqMan-MGB probe  real-time PCR
基金项目:广西水产畜牧科技推广应用项目(桂渔牧科201633022);广西创新驱动发展专项(桂科AA17204081–4);广西自然科学基金项目(2015GXNSFBA139069);广西壮族自治区直属公益性科研院所基本科研业务费专项(GXIF–2016–10);广西水产遗传育种与健康养殖重点实验室项目(17–259–29)
作者单位
童桂香,黎小正,吴祥庆,黄鸾玉,黄光华,韦信贤 1.广西水产科学研究院广西 南宁 5300212.广西水产遗传育种与健康养殖重点实验室广西 南宁 530021 
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中文摘要:
      根据pirAVp基因保守序列设计特异性引物和TaqMan–MGB探针,经优化反应体系及条件,建立检测致对虾急性肝胰腺坏死病副溶血弧菌(VpAHPND)的TaqMan–MGB探针荧光定量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。结果表明:该方法定量范围宽,起始模板浓度范围为1×101~1×109拷贝/反应时,标准曲线具良好的线性关系;最低检测限约为10拷贝/反应,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0.37%~0.47%,可在1 h 内完成单个样品检测;对临床对虾样品及水样的检测结果表明,与世界动物卫生组织(OIE)推荐的套式PCR法相比,可提高VpAHPND的阳性检出率,且检测为阳性的样品包含全部套式PCR法检测为阳性的样品。可见,应用TaqMan–MGB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、能快速定量等优点,可用于临床对虾样品及水样的VpAHPND检测。
英文摘要:
      In this study, a pair of specific primers and fluorogenic-labeled TaqMan-Minor Groove Binder(TaqMan-MGB) probe were designed and synthesized based on the conserved region of pirAVp gene, then a real-time PCR method was developed to detect AHPND-causing Vibrio parahaemolyticus(VpAHPND). The results showed that the method had a wide quantitative range from 1×101 to 1×109 copies per reaction, with a good linear relationship in its standard curve. The real-time PCR method had a high sensitivity with the detection limit as low as to 10 copies per reaction for the purified recombinant plasmids of pTOPO-T-pirAVp, and the entire detection could be completed within 1 h for a single sample and it also had a high specificity in detecting DNA of VpAHPND. The variation coefficients among cycle thresholds(Cts) of the repeatability test were 0.37%-0.47%. Comparing with the nested PCR recommended by OIE, the TaqMan-MGB probe real-time PCR had better positive detection rate of VpAHPND. In conclusion, the TaqMan-MGB probe real-time PCR assay developed in the study is fast, sensitive, specific, quantitative and of high reproducibility, making it a ideal method for detecting VpAHPND in shrimp and water samples.
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