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本研究根据鲤春病毒血症病毒的核酸保守区序列,开发、设计多条引物和探针序列,从中筛选敏感、特异的引物、探针,在循环参数、病毒核酸提取方法及逆转录条件优化的基础上,建立了快速的检测鲤春病病毒的荧光RT-PCR检测技术。同时,又将该方法与一步法RT-PCR方法和细胞查毒方法进行了互相验证和对比研究,结果表明,荧光RT-PCR方法检测病毒悬液的灵敏度最高,病毒悬液10-6稀释仍然可以检出,而细胞分离病毒方法测得的TCID50为10-4。本文还对北京地区出口渔场送检样品、人工实验感染该病毒鱼的病料进行了组织脏器中病毒分布和病毒含量的研究。结果显示,北京地区出口渔场送检样品未见细胞病变,所有检测样品均为阴性;病毒致死鱼的肠道病毒含量最高,肠组织研磨稀释10-4倍仍然可以检出;其次是肾和鳃组织,检出极限为稀释10-3倍组织悬液;肉和脑组织中病毒含量较低,肉最高能检出10-2倍组织悬液,而脑组织只能检出10-1倍组织稀释液。
In this study, primers and probes were designed and synthesized based on the nucleotide sequence of the conserved region of VPHV. Sensitive and specific primers and probes were screened out. The parameters of loop parameters, viral nucleic acid extraction and reverse transcription conditions were optimized Based on the establishment of a rapid detection of carp disease virus fluorescence RT-PCR detection technology. At the same time, this method and one-step RT-PCR method and cell virus detection methods were verified and compared with each other, the results show that the highest sensitivity of fluorescent RT-PCR detection of the virus suspension, the virus suspension 10-6 dilution is still Can be detected, while the cell isolation virus method measured TCID50 of 10-4. In this paper, samples from export fishery in Beijing area were also tested, and the virus distribution and virus content in tissues and organs were studied by artificial experiment. The results showed that there was no cytopathic effect in the samples from export fishery in Beijing area and all the samples were negative. The virus of lethal fish had the highest enterovirus content, and the intestinal tissue was still detected after 10-4 times dilution. The second was kidney and gill Tissue, the detection limit is diluted 10-3 times the tissue suspension; the meat and brain tissue virus content is low, the meat can detect 10-2 times the maximum tissue suspension, and brain tissue can only detect 10-1 times the organization Diluent.