套式PCR在中华绒螯蟹颤抖病病原螺原体检测中的应用

提取人工分离、培养的中华绒螯蟹颤抖病病原螺原体的基因组DNA,并进行梯度稀释作为PCR模板,根据GenBank已有螺原体16S rRNA基因序列设计出套式PCR外部引物,将螺原体特异性引物作为内部引物,利用套式PCR对梯度稀释的模板进行扩增,得到271 bp产物,对照已有的一步PCR检测法,检测灵敏度提高100倍,最小检测达15.5 fg数量级螺原体DNA,从而建立更为灵敏的中华绒螯蟹颤抖病病原螺原体的检测方法。

The Application of Nested PCR for Detection of Tremor Disease Agent in Chinese Mitten Crab (Eriocheir sinensis)

Genomic DNA was extracted by DNA Mini-Prep Kit from spiroplasmas of tremor disease agent which was isolated from Chinese mitten crab(Eriocheir sinensis) and cultured in artificial medium.The genomic DNA was diluted by grads for PCR template.Outter primers of the nested PCR were designed by 16S rRNA sequence of spiroplasma in GenBank,and the spiroplasma-specifc primers according to literatures was utilized as inner primers.The templates of different dilutions were amplified by the nested PCR method and one step PCR method,then produced 271 bp target DNA fragment.The data showed that the nested PCR method allowed the detection of about 15.5 fg DNA of spiroplasma,and the sensitivity of the method was about 100 times higher than that of one step PCR method,so a higher sensitivity detection method for tremor disease agent,spiroplasma in crab was developed.