Abstract：
Both Klebsiella pneumoniae and Chryseobacterium cause an increasing number of diseases in fish, resulting in great economic losses in aquaculture. In addition, the disease infected with Klebsiella pneumoniae or Chryseobacterium exhibited the similar clinical symptoms in aquatic animals. However, there is no effective means for the simultaneous detection of co-infection and discrimination them for these two pathogens. Here, we developed a duplex polymerase chain reaction (PCR) method based on the outer membrane protein A (ompA) gene of Klebsiella pneumoniae and Chryseobacterium. The specificity and validity of the designed primers were confirmed experimentally using simplex PCR. The expected amplicons for Klebsiella pneumoniae and Chryseobacterium had a size of 663 and 1404 bp, respectively. The optimal condition for duplex PCR were determined to encompass a primer concentration of 0.5 μM and annealing temperature of 57°C. This method was analytical specific with no amplification being observed from the genomic DNA of Escherichia coli, Vibrio harveyi, Pseudomonas plecoglossicida, Aeromonas hydrophila and Acinetobacter johnsonii. The limit of detection was estimated to be 20 fg of genomic DNA for Chryseobacterium and 200 fg for Klebsiella pneumoniae, or 100 colony-forming units (CFU) of bacterial cells in both cases. The duplex PCR was capable of simultaneously amplifying target fragments from genomic DNA extracted from the bacteria and fish liver. For practical validation of the method, 20 diseased fish were collected from farms, among which 4 samples were PCR-positive for Klebsiella pneumoniae and Chryseobacterium. The duplex PCR method developed here is time-saving, specific, convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of Klebsiella pneumoniae and Chryseobacterium in the field of aquaculture.
摘要：
肺炎克雷伯菌和金黄杆菌在鱼类中引起越来越多的疾病，给水产养殖造成了巨大的经济损失。此外,感染肺炎克雷伯菌或金杆菌的疾病在水生动物中表现出相似的临床症状。然而,没有有效的方法可以同时检测合并感染并区分这两种病原体。这里,我们开发了一种基于肺炎克雷伯菌和金黄杆菌的外膜蛋白A（ompA）基因的双重聚合酶链反应（PCR）方法。所设计的引物的特异性和有效性用单纯性PCR实验证实。肺炎克雷伯菌和金杆菌的预期扩增子的大小为663和1404bp，分别。双重PCR的最佳条件被确定为包含0.5μM的引物浓度和57°C的退火温度。该方法具有分析特异性，未从大肠杆菌的基因组DNA中观察到扩增，哈维氏弧菌,假单胞菌，嗜水气单胞菌和乔氏不动杆菌。估计检测限是金杆菌的基因组DNA为20fg，肺炎克雷伯菌的检测限为200fg，或100个菌落形成单位（CFU）的细菌细胞在这两种情况下。双重PCR能够同时扩增从细菌和鱼肝提取的基因组DNA的靶片段。为了对该方法进行实际验证，从农场收集了20条患病的鱼，其中4份样本的肺炎克雷伯菌和金黄杆菌PCR阳性.这里开发的双重PCR方法节省时间，具体,方便,并可能被证明是水产养殖领域中肺炎克雷伯菌和黄杆菌的分子检测和流行病学调查的宝贵工具。
