肝肠胞虫RPA-LFS快速检测方法的建立及应用

Establishment and Application of Recombinase Polymerase Amplification and Lateral Flow Strip Combined Method for the Rapid Detection of Enterocytozoon hepatopenaei

肝肠胞虫(Enterocytozoon hepatopenaei)感染虾不会出现明显的临床症状,养殖场无法及时发现并进行针对性治疗,最终极易给养殖带来较大的经济损失。目前尚无治疗肝肠胞虫的有效方法,因此,快速、准确地开展早期检测对控制其传播、减少经济损失具有重要意义。针对孢壁蛋白(spore wall protein,swp)基因设计了特异性引物和探针,建立了重组酶聚合酶扩增结合侧向流试纸条(RPA-LFS)的等温检测方法,测试了该方法的特异性及敏感性,并对不同虾养殖场的48份临床样本进行了检测。结果表明,该方法在37℃恒温下反应30 min即可完成扩增,整体检测时间可控制在35 min内;特异性试验中对于其他种类水产致病菌无交叉反应;检测限为10~0 copies;临床样本检测准确度比巢式PCR更高。该方法简单、快速、准确,可解决偏远地区养殖场缺乏专业人员和专业设备的问题,因此有望成为偏远地区养殖场实时检测肝肠孢虫的手段之一。

Enterocytozoon hepatopenaei infected shrimp has no obvious clinical symptoms;farms cannot detect EHP in time and take useful treatment, which will eventually cause economic losses. At present, there is no effective treatment for EHP, so rapid and accurate early detection is of great significance to control its transmission and reduce economic losses. Specific primers and probe were designed according to sequence of the spore wall protein(swp) gene, and an isothermal detection method of recombinase polymerase amplification combined with lateral flow strip(RPA-LFS) was established. 48 clinical samples collected from different shrimp farms were tested to confirm specificity and sensitivity of the method. Results show that: this method can complete the amplification at 37 ℃ constant temperature for 30 min, and the detection time can be controlled within 35 min;there is no cross-reaction to other aquatic pathogens in the specificity test, the limitation of RPA-LFS is 10~0 copies. The detection accuracy of clinical samples is higher than that of nested PCR. This method is simple, fast and accurate. Farms in remote areas often lack professional person and equipment, thus this method is expected to become an important method for the real-time detection of EHP in remote farms.