Tapia-Cammas D, Yañez A, Arancibia G, Toranzo A E, Avendaño-Herrera R
Laboratorio de Patología de Organismos Acuáticos y Biotecnología Acuícola, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Viña del Mar, Chile.
Dis Aquat Organ. 2011 Dec 6;97(2):135-42. doi: 10.3354/dao02395.
A multiplex (m-)PCR-based protocol was designed for the simultaneous detection of the main marine bacterial pathogens in Chilean salmon farms: Streptococcus phocae, Aeromonas salmonicida, Vibrio anguillarum and Piscirickettsia salmonis. Each of the 4 oligonucleotide primer pairs exclusively amplified the target gene of the specific bacterial pathogen. The detection limit of the m-PCR using purified total bacterial DNA was 50 pg microl(-1) for V anguillarum, 500 fg microl(-1) for P. salmonis, and 5 pg microl(-1) for S. phocae and A. salmonicida. This corresponded to average limits in the m-PCR sensitivity of 3.69 x 10(5) CFU ml(-1) of V anguillarum, 1.26 x 10(4) CFU m(-1) of S. phocae, and 5.33 x 10(4) CFU ml(-1) of A. salmonicida, while the detection limits for the spiked fish tissues, regardless of the sample (spleen, kidney, liver or muscle) were 2.64 +/- 0.54 x 10(7) CFU g(-1) for V. anguillarum, 9.03 +/- 1.84 x 10(5) CFU g(-1) for S. phocae, 3.8 +/- 0.78 x 10(3) CFU mg(-1) for A. salmonicida and 100 P. salmonis cells. However, high amounts of DNA from 3 bacterial species had a reduction of -1 log-unit on the amplification sensitivity of S. phocae or A. salmonicida when these were present in lower concentration in the multiplex reaction. The assay described in this study is a rapid, sensitive and efficient tool to detect the presence of S. phocae, A. salmonicida, V. anguillarum and P. salmonis simultaneously from pure cultures and tissues from clinically diseased fish. Therefore, it may be a useful alternative to culture-based methods for the diagnosis of infections in fish obtained from Chilean salmon farms.
设计了一种基于多重（m-）PCR的方案，用于同时检测智利鲑鱼养殖场中的主要海洋细菌病原体：海豹链球菌、杀鲑气单胞菌、鳗弧菌和鲑鱼立克次氏体。4对寡核苷酸引物中的每一对都专门扩增特定细菌病原体的靶基因。使用纯化的总细菌DNA进行m-PCR的检测限，鳗弧菌为50 pg μl（-1），鲑鱼立克次氏体为500 fg μl（-1），海豹链球菌和杀鲑气单胞菌为5 pg μl（-1）。这相当于m-PCR敏感性的平均检测限，鳗弧菌为3.69×10（5）CFU ml（-1），海豹链球菌为1.26×10（4）CFU m（-1），杀鲑气单胞菌为5.33×10（4）CFU ml（-1），而对于加标的鱼组织，无论样本（脾脏、肾脏、肝脏或肌肉）如何，鳗弧菌的检测限为2.64±0.54×10（7）CFU g（-1），海豹链球菌为9.03±1.84×10（5）CFU g（-1），杀鲑气单胞菌为3.8±0.78×10（3）CFU mg（-1），鲑鱼立克次氏体为100个细胞。然而，当多重反应中3种细菌的DNA浓度较低时，来自这3种细菌的大量DNA会使海豹链球菌或杀鲑气单胞菌的扩增敏感性降低-1个对数单位。本研究中描述的检测方法是一种快速、灵敏且高效的工具，可同时从纯培养物和临床患病鱼的组织中检测出海豹链球菌、杀鲑气单胞菌、鳗弧菌和鲑鱼立克次氏体的存在。因此，它可能是基于培养的方法用于诊断从智利鲑鱼养殖场获得的鱼类感染的一种有用替代方法。
