摘要:
根据微囊藻毒素合成酶基因簇序列,合成了3对引物epF/mb1R,mcF/teR,mcF/umR,通过全细胞PCR的方法检测了19种不同来源微囊藻产毒的情况.3种引物对1株产毒微囊藻中均可扩增到预期大小的片段,测序结果证明这些片段是微囊藻毒素合成酶基因片段.PCR反应结果与HPLC分析所得到的结果有良好的对应性.在此基础上,初步确定了3对引物检测产毒微囊藻对细胞浓度要求的下限.与其它引物相比,3对引物的特异性强,扩增条带大小适中,便于观察.
Abstract:
To discriminate from toxic and non-toxic Microcystis, 3 pairs of primers epF/mb1R,mcF/teR and mcF/umR were designed, based on sequences of microcystin synthetase gene cluster. The primers were applied to test the toxicity of 19 strains of Microcystis, which came from different places, including China, America, Japan and Australia, via whole-cell PCR. All these primers, epF/mb1R, mcF/teR and mcF/umR, were able to amplify the expected bands from 15 toxic strains of Microcystis. The amplified fragments were 2,800bp, 1,800bp, 2,300bp respectively. The bands were proved to be the fragments of microcystin synthetase gene cluster according to blasting the sequences datum of PCR products on NCBI. In addition, we compared the results by two methods of PCR and HPLC. It showed that both PCR and HPLC methods gave a similar result on toxicity of Microcystis strains, and indicated that 3 pairs of primers could distinguish between toxic and non-toxic Microcystis. To detect the sensitivity of the primers, the threshold of primers for cell concentration was also studied. The results showed that the optimal concentrations were from 2?05 cells/mL to 1?07cells/mL. Especially, the primes mb1R/epF, mcF/umR could amplify the expected bands on the cells concentrations of from 2?04 cells/mL. It was concluded the 3 primers were highly sensitive,and able to apply for discrimination of toxic and non-toxic strains of Microcystis.
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