近岸污染指示半知菌灰黄青霉(Penicillium griseo fulvum)的分子检测

Molecular detection of Penicillium griseofulvum as the coastal pollution indicator

【目的】通过分子方法检测近海污染环境优势种灰黄青霉,并为由此而推断污染程度做准备。【方法】根据GenBank中青霉属不同种和相近属种的ITS序列差异和灰黄青霉特有的IAO序列,设计了污染区优势种灰黄青霉的特异性引物AS1/RS4和IAO1/IAO2,建立相应的特异探针检测体系。通过PCR和套式PCR技术,分析比较两对特异序列检测灰黄青霉的差异。【结果】建立的分子检测体系可以排除其它近似或相关菌株干扰,从环境中扩增到目的基因片段。利用引物AS1/RS4作为核酸探针,通过套式PCR菌株DNA的检测灵敏度可达到10fg/μL,当仅有10个数量级分生孢子时即可检测出,从沉积物中检测灵敏度为102个数量级孢子/0.25g。特异酶基因IAO1/IAO2检测灵敏度较前者稍低。【结论】利用特异序列作为探针检测污染环境优势种灰黄青霉的方法可行,在一定范围内,灰黄青霉的出现频率及数量对污染程度有较好的指示作用。

[ Objective] PCR method was used to detect Penicillium griseofulvum, a dominant species in marine contaminated sediments and thereby to deduce the contamination degree. [ Methods] According to differences in internal transcribed space （ITS） sequences of Penicillium genus and specific IAO sequence, we designed species-specific primers ASI/RS4 and IAOI/IAO2 of Penicillium griseofulvum and established the corresponding PCR systems. By using PCR and nested-PCR,the detection sensitivity was compared. [ Results ] The primers could exclusively amplify destined DNA fragment from environment. Using AS1/RS4 as primers, the detection sensitivity could be 10 fg/μL and 10 spores. The detection sensitivity for the sediments was 102 spores/0.25 g sediments. While the detection was unsensitive when using IAO1/IAO2 as primers. [ Conclusion] It is feasible that the species-specific primers be used as probes for the detection of environmental pollution dominant species, PeniciUium griseofulvum, because the frequency of occurrence and amount of this strain could preferably indicate the pollution degree.