虾肝肠胞虫TaqMan-MGB荧光定量PCR检测技术的建立

Establishment of TaqMan-MGB Fluorescence Quantitative PCR for Detection of Enterocytozoon hepatopenaei in Shrimp

近年来,虾肝肠胞虫病(Enterocytozoon hepatopenaei, EHP)严重威胁虾类健康成长,为了精准快速检测该病原,本文选取虾肝肠胞虫保守基因片段PTP2为靶序列,设计并合成TaqMan-MGB实时荧光定量PCR引物及探针,同时将扩增获得的特异PCR目的基因片段克隆到pMD18-T载体,得到重组质粒标准品。通过优化反应条件,建立了EHP TaqMan-MGB实时荧光定量PCR检测方法,并对该方法进行了灵敏度、特异性和重复性分析。试验结果证明,该反应体系的最佳退火温度为56℃,上下游引物浓度为10 μmol/L,探针浓度为5 μmol/L;灵敏度检测限最低可达到2.7 ×102拷贝/反应。特异性试验表明,该方法与WSSV、IHHNV和SHIV这3种水生动物病原无交叉反应,只与目标病原有特异性扩增。重复性试验显示,该方法具备良好的重复性及稳定性。采用研究建立的检测方法,初步应用在了实际临床样品检测中,结果显示,该方法与TaqMan荧光定量PCR方法具有类似的敏感性。以上结果显示,研究建立的EHP的TaqMan-MGB实时荧光定量PCR方法具有较高的敏感性、良好的特异性及重复性,适用于虾类及产品中肝肠胞虫的检测与监测。

In recent years, Enterocytozoon hepatopenaei (EHP) has seriously threatened the healthy growth of shrimp. In order to accurately and quickly detect this pathogen, the conserved gene fragment ptp 2 of EHP was selected as the target sequence, and TaqMan-MGB real-time fluorescent quantitative PCR primers and probes were designed and synthesized. At the same time, the amplified specific PCR target gene fragment was cloned into pMD18-T vector to obtain the recombinant plasmid standard. By optimizing the reaction conditions, a real-time fluorescent quantitative PCR method for EHP TaqMan-MGB was established, and the sensitivity, specificity and reproducibility of the method were analyzed. The results showed that the optimal annealing temperature of the reaction system was 56˚C, the concentration of primers in the upstream and downstream was 10 μmol/l and the probe concentration was 5 μmol/L;the sensitivity detection limit can reach at least 2.7 ×102 copies/reaction. The specificity test showed that the method had no cross-reaction with WSSV, IHHNV and SHIV, and only had specific amplification with the target pathogen. The repeatability test shows that the method has good repeatability and stability. The established detection method has been applied to the detection of clinical samples. The results show that this method has a similar sensitivity to TaqMan fluorescence quantitative PCR. The above results show that the TaqMan-MGB real-time fluorescence quantitative PCR method for EHP has high sensitivity, good specificity and reproducibility, and is suitable for the detection and monitoring of Enterocytozoon hepatopenaei in shrimp and products.