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以嗜水气单胞菌促旋酶B亚单位(gyrase subunit B,gyrB)基因为检测靶标,设计6条特异性引物和1条异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记探针,建立了嗜水气单胞菌的LAMP-LFD快速检测方法。该方法的流程可简单概括为将生物素标记的环介导等温扩增(loop-mediated isothermal amplification,LAMP)产物与FITC标记的探针进行特异性杂交,并使用横向流动试纸条(lateral flow dipstick,LFD)完成检测。经优化,LAMP最佳反应条件为63℃,反应30min,从LAMP扩增到LFD结果判读仅需40min,比常规PCR检测缩短近2h。试验结果表明,LAMP-LFD可特异性检出嗜水气单胞菌,对鳗弧菌等常见水产或食源性病原菌检测结果为阴性;其针对细菌纯培养物的检测灵敏度达2.03×101 CFU/mL或0.81CFU/反应,是常规PCR(以F3/B3为引物)的100倍;可从人工污染1×102 CFU/mL浓度的嗜水气单胞菌的鲫鱼肝组织匀浆样品中检测到细菌。嗜水气单胞菌(106 CFU)人工感染鲫鱼24h,传统的细菌分离培养方法可获得肝、肾的载菌量分别为1.2×105 CFU/g和6×103 CFU/g;利用LAMP-LFD技术可成功从鲫鱼的肝、肾组织中检测出该病原;以F3/B3为引物的PCR方法仅能从感染鲫鱼的肝组织中检测到该病原菌,表明面对实际感染样品,LAMP-LFD的检测灵敏度优于PCR方法。因此,该方法可快速、灵敏、特异地检测出嗜水气单胞菌,而且操作简单,仪器设备依赖性低,有望成为嗜水气单胞菌现场检测的常规技术手段。
Six specific primers and one fluorescein isothiocyanate (FITC) labeled probe were designed by using gyrB gene of Aeromonas hydrophila, The rapid detection of Aeromonas hydrophila LAMP-LFD was established. The procedure of this method can be briefly summarized as the specific hybridization of a biotinylated loop-mediated isothermal amplification (LAMP) product to a FITC-labeled probe and the use of a lateral flow test strip dipstick, LFD) to complete the test. Optimized conditions, LAMP optimum reaction conditions were 63 ℃, the reaction 30min, LAMP amplification to LFD interpretation of the results only 40min, shorter than conventional PCR detection nearly 2h. The results showed that LAMP-LFD could detect Aeromonas hydrophila specifically and negative for the common aquatic or foodborne pathogens such as Vibrio anguillarum. The detection sensitivity of LAMP-LFD against pure cultures of bacteria was 2.03 × 101 CFU / mL, or 0.81 CFU / reaction, which is 100 times of that of conventional PCR (using F3 / B3 as primer); it can be detected from artificial liver tissue homogenate samples of Aeromonas hydrophila at a concentration of 1 × 102 CFU / mL To bacteria. Crucian carp (106 CFU) were infected artificially with crucian carp for 24 h. The bacterial and cell culture methods of traditional bacterial isolation and culture were 1.2 × 105 CFU / g and 6 × 103 CFU / g, respectively. Using LAMP-LFD Technology can successfully detect the pathogen from liver and kidney tissues of crucian carp. The PCR method using F3 / B3 primer can only detect the pathogen from the liver tissue of infected crucian carp, indicating that in the actual infected samples, LAMP-LFD Detection sensitivity is better than PCR method. Therefore, the method can detect Aeromonas hydrophila rapidly, sensitively and specifically, and has the advantages of simple operation, low dependence on equipment, and is expected to be the routine technical measure of the field test of Aeromonas hydrophila.