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2.3×10^(1)~2.3×10^(7)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。
【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 2.3×10^(1) to 2.3×10^(7) 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-18 T-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.
作者
陈平亚
张亮亮
吴少荣
黄纪徽
王绥家
吴山楠
CHEN Pingya;ZHANG Liangliang;WU Shaorong;HUANG Jihui;WANG Suijia;WU Shannan(Haikou Customs Technical Center,Haikou 570311,China)
出处
《广东农业科学》
CAS
2021年第11期119-125,共7页
Guangdong Agricultural Sciences
基金
海南省重点研发计划项目(ZDYF2020088)。
作者简介
陈平亚(1987-),男,硕士,工程师,研究方向为水产品微生物检测,E-mail:ya931892094@163.com;通信作者:吴山楠(1981-),男,博士,高级兽医师,研究方向为动物检疫与防疫,E-mail:wsnhiciq@163.com。
