| The tilapia was come from Africa and become a worldwide cultured aquatic species. The production of global tilapia had reached to 390 tons per year now. The Chinese production was 150 tons in 2014, and half of them used for export processing,which is the biggest part among the freshwater fish kinds.In recent years, the tilapia bacterial diseases broke out frequently, it cause of a serous damage to the interests of the farmers. The Streptococcus agalactiae disease was the most serious disease among them, for the high death rates and hard to cure.After tilapia suffer the streptococcus disease, the farmers typically use antimicrobial drugs for the treatment. But this method achieve a bad effect, for the speed of streptococcus is very fast, and when tilapia have obvious symptoms,and the farmers recognize the tilapia was sick, the infection was in the late. According to this, the key to preventing the Streptococcus disease is to diagnose the infrction successfully and make the appropriate measures in the early stage of infection, before the tilapia had symptoms.In order to detect Streptococcus agalactiae in tilapia quickly and accurately, we developed a nested PCR detection method which is basing on the Streptococcus agalactiae's cfb gene and the PCR reaction in this article. According to bacteria experiment, blood bacteria mixed experiment, toxic experiment and vivo experiment,we had done some preliminary study. The main contents and result of this work were as follow:1. According to Streptococcus agalactiae gene, we designed and synthesized two pairs of primer, the nested PCR method was established. Withing bacteria liquid as a template, we taken the nested PCR experiment and groped the test condition. The nested PCR condition was 25+25 cycle, of which the Primer annealing at 55?. In this condition, we can fast detect the Srteptococcus agalactiae at the sensitivity of 8.7×100CFU/m L, which was much higher than the traditional PCR detection metond(8.7×105CFU/m L). Ten strains, including Srteptococcus agalactiae, which is association with the tilapia bacterial disease was tested by nested PCR. The results showed that the method has good specificity, for there is no positive results except Streptococcus agalactiae.2. To simulate the diseased tilapia blood, we taken the nested PCR for a betterexperimental conditions with the man-made tilapia blood including bacteria as a template. In this part of the experiment, we extract the heath tilapias blood and mixed with varying amount of Streptococcus agalactiae, to simulate illness tilapia blood with different infection level. The results showed as follow: When the nested cycle condition was set as 35+30 cycle, the test sensitivity can be reached as 8.7×104CFU/m L; In the experiment of using nested PCR method to detect fourteen Streptococcus agalactiae strain which was collected from Liuzhou, all of them could detected by nested PCR and no false positive phenomenon; In the repetitive experments, we had taken 15 trials get 1080 experiment result within three period, the repeatability data of the test result was 1066 and the repetition rate can be reached as98.7%.3. Select healthy tilapias with the weight of about 100 g and inject Streptococcus agalactiae for artificial infection experiments, while using saline as a control. The tilapia death cycle and various stages of the disease characteristics were recorded after artificial infection. Extracting blood from various stages of diseased fish and taking plate count method, we can count the Streptococcus agalactiae in tilapia blood, and find out the connection between the Streptococcus agalactiae in blood and the tilapia's onset phase. Meanwhile, using the blood extracting from tilapias as a template, we took the nested PCR test. By taking plate count method and nested PCR test, we can determine in which stage of infection the nested PCR test can detect the Streptococcus agalactiae from tilapia blood. The results showed as follow: After the Streptococcus agalactiae infected the tilapia, the tilapia showed obvious symptoms when the concentration of Streptococcus agalactiae in blood had reached 105CFU/m L;While the nested PCR test could detect the Streptococcus agalactiae when the concentration of Streptococcus agalactiae in blood had reached 104CFU/m L.4. We had separated and figure out the pathogenic bacteria from diseased tilapia in guangxi province, and then the used the the nested PCR method to rapidly detect the treptococcus agalactiae disease. In 2014, we answered out call more than 50 times and got 46 strains of pathogenic bacteria which included 24 strains of Streptococcus agalactiae. All of Streptococcus agalactiae strains, could rapidly detect by the nested PCR method. On the other hand, others srains which is not Streptococcus agalactiae could not gain a positive result by the nested PCR method. On the drug sensitivity experiment, we got a large amount of date, this can provide data base for preventionand treatment the Streptococcus agalactiae disease. |
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