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应用巢式聚合酶链反应(nested PCR)建立了一种检测异育银鲫武汉单极虫(Thelohanellus wuhanensis)基因组DNA的方法,提取的DNA作为模板进行两次扩增,第一次扩增用单极虫18S rRNA序列通用引物:5’-CTGCGGACGGCTCAG TAAATCAGT-3’和5’-CCAGGACATCTTAGGGCATCACAGA-3’,扩增长度为1 584 bp;第二次依据第一次扩增产物中武汉单极虫特有的高度保守区设计特异性引物:5’-ACCCACTTCTGTGGC CTTTC-3’和5’-AATCCGACCTACAACGCTGG-3’,扩增长度为853 bp。结果表明,通过PCR反应体系中退火温度、dNTP、Mg2+、延伸时间、扩增循环数和模板浓度的优化,优化后的巢式PCR最低DNA检测量达10 fg,优化后比常规PCR检测的灵敏度高104倍。因此,本文所建立的巢式PCR检测方法适合于水环境或水生动物中武汉单极虫的微量检测,也为异育银鲫武汉单极虫病的诊断和流行病调查提供了一种高灵敏的检测技术。
A nested PCR method was used to detect genomic DNA of Thelohanellus wuhanensis in Wuhan allogynogenetic crucian carp (Carassius auratus gibelio). The extracted DNA was amplified twice as a template. The first amplification The universal primer of monosomic 18S rRNA sequence: 5’-CTGCGGACGGCTCAGTAAATCAGT-3 ’and 5’-CCAGGACATCTTAGGGCATCACAGA-3’, with a length of 1 584 bp. The second time was based on the first amplified product The highly conserved regions were designed to be specific primers: 5’-ACCCACTTCTGTGGC CTTTC-3 ’and 5’-AATCCGACCTACAACGCTGG-3’, with a length of 853 bp. The results showed that the optimal detection limit of nested PCR was 10 fg by optimization of annealing temperature, dNTP, Mg2 +, extension time, number of amplification cycles and template concentration in the PCR reaction system. After optimization, the sensitivity High 104 times. Therefore, the nested PCR detection method established in this paper is suitable for the microdeterminations of Wuhan polar waterwaters in aquatic environment or aquatic animals, as well as for the diagnosis and epidemiological investigation of the monosteotyping of Wuhan silver crucian carp The detection technology.