石斑鱼肠孢虫(Enterospora epinepheli)环介导等温扩增(LAMP)检测方法的建立及应用

Establishment and application of a loop-mediated isothermal amplification (LAMP) for detection ofSEnterospora epinepheli

中文关键词: 石斑鱼肠孢虫  环介导等温扩增  检测  核糖体小亚基rRNA

英文关键词: Enterospora epinepheli  LAMP  Detection  SSUSrRNA

中文摘要:

      石斑鱼肠孢虫(Enterospora epinepheli)是当前危害石斑鱼养殖产业的重要病原,但该病原自2017年报道以来,仅有2种基于核酸检测方法的报道。本研究以E. epinepheli的核糖体小亚基rRNA(smallSsubunit ribosomalSRNA, SSUSrRNA)基因为靶序列,设计3对环介导等温扩增(Loop-mediated isothermal amplification, LAMP)引物,建立了该病原的LAMP检测方法(LAMP for E. epinepheli, E.ep_LAMP)。添加EvaGreen_?^{核酸染料的检测体系置于定量}PCR仪中,以2.68×10₂^—2.68×10₉ 拷贝/uL的重组质粒标准品pMD18_E.ep为模板,各浓度均可在20 min内完全检测,其检测灵敏度为2.68×10₂^拷贝/反应；分析pMD18_E.ep中目的基因核酸拷贝数的对数值(x)与其对应Ct值(y)的线性关系得到检测方法的标准曲线：y=-1.7195x+23.971(R₂^=0.9962)；重复性检测中,其批内和批间检测的变异系数(Coefficient of Variation, CV)值范围分别为0.22%—3.52%和0.24%—1.70%。特异性检测中,E.ep_LAMP与虾肝肠胞虫(Enterocytozoon hepatopenaei, EHP)和肌孢虫(Ameson portunus),以及其他11种细菌和病毒均无交叉反应。E.ep_LAMP检测体系中使用核酸染料 GeneFinder_?^后,还可在简易恒温条件下进行现场检测,具有诊断试剂产品开发的前景。总体而言,本研究建立的方法快速、灵敏和特异性强,兼顾定量和可现场诊断的优点,可为当前频发的石斑鱼微孢子虫病的诊断及防控提供新的有效检测方法。

英文摘要:

      Enterospora epinepheli has been a major pathogen for cultured groupers (Epinephelus spp.), located on the coast of Fujian, Guangdong, Shandong and Hainan Provinces, and Guangxi Zhuang autonomous region currently, and can cause large economic losses to groupers farming industry. Early and specific diagnosis is essential for the treatment and management of this emerging pathogen. However, since it was reported in 2017, there are only two nucleic acid-based techniques assays including PCR and qPCR assay based on TaqMan probes assay for detection of E. epinepheli . In the present study, a method for quick and accurate detection of E. epinepheli by loop-mediated isothermal amplification (LAMP) with three primer pairs were developed by targeting the small subunit ribosomal RNA gene (SSU rRNA) of E. epinepheli (LAMP for E. epinepheli, E.ep_LAMP) was developed. The sensitivity, specificity, repeatability, and clinical applications of the developed E.ep_LAMP assay were verified. The constructed recombinant plasmid standard pMD18_E.ep, diluted with 10-fold gradient into different concentrations from 2.68×109 to 2.68×101 copies/uL, were used as the template to generate the standard curve and used to evaluate the detection sensitivity for E.ep_LAMP. The results showed that the detection limit of E.ep_LAMP was as low as 2.68×102 copies per reaction. The standard curve, y=-1.7195x+23.971(R2=0.9962), was obtained by plotting the threshold cycle values (y) against the common logarithmic copies (log10 [Copy number] as x) of pMD18_E.ep plasmids template ranging from 2.68×102 to 2.68×109 copies/uL. To assess the repeatability of the E.ep_LAMP, the intra-assay (variance within runs) and inter-assay (variance between runs) were evaluated, the coefficient of variation (CV) , which are equal to the percentage of the standard deviation (SD) to the mean of the Ct, of intra-assay and inter-assay values were ranged from 0.22% to 3.52%, indicating that this assay was highly repeatable and reproducible over a wide range of detection from 2.68×102 to 2.68×109 copies of pMD18_E.ep plasmids template. To test the detection specificity of E.ep_LAMP, different gDNA extracted from different pathogens and diseased groupers tissues were used as templates. The results showed that the developed assay was also highly specific to E. epinepheli and had no cross-reaction with Enterocytozoon hepatopenaei (EHP), Ameson portunus, and another eleven pathogens including Vibrio campbellii, V. parahaemolyticus, V. alginolyticus, V. harveyi, V. rotiferianus, V. natriegens, Spiroplasma eriocheiris, P.damselae subsp.damselae, White spot syndrome virus (WSSV), Decapod iridescent virus 1(DIV1) and Infectious hypodermal and hematopoietic necrosis (IHHNV). In validation of the E.ep_LAMP on clinical samples, a total of 50 gDNA samples extracted from clinical groupers samples were applied to detect the quantitation of E. epinepheli copies by E.ep_LAMP assay, and to detect by the conventional PCR assays described by previous report, respectively. The results showed that 50.0% of the total samples were detected positive using E.ep_LAMP, and loads of E. epinepheli in these positive clinical samples varied within the range from 4.54×102 to 3.23×106 copies per mg tissue. By contrast, 42.0% of the total samples were detected positive using the PCR assay. These results showed that the E.ep_LAMP was qualified with more sensitivity in detecting E. epinepheli in clinical samples. The E.ep_LAMP can be carried out on a simple heating device for incubation. By adding the fluorescent dyes GeneFinder? to the tubes of LAMP products, green fluorescence can be observed clearly with the naked eye in the positive control reaction tubes and E. epinepheli-infected samples, whereas it was orange in the negative control. Taken together, the E.ep_LAMP diagnostic protocol developed in the present study was rapid, specificity and sensitivity, and can be used a diagnostic tool for the detection of microsporidian disease occurred in grouper culture.