DETECTION OF PHANEROCHAETE-CHRYSOSPORIUM IN SOIL BY PCR AND RESTRICTION ENZYME ANALYSIS
DETECTION OF PHANEROCHAETE-CHRYSOSPORIUM IN SOIL BY PCR AND RESTRICTION ENZYME ANALYSIS
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DOI：
10.1128/aem.60.7.2350-2354.1994
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发表时间：
1994-07-01
影响因子：
4.4
通讯作者：
AUST, SD
中科院分区：
文献类型：
--
作者：
JOHNSTON, CG;AUST, SD
建立了一种非放射性检测土壤基质中黄孢原毛平革菌的方法。该方法包括DNA提取、PCR扩增和限制性内切酶分析。从黄孢原毛平革菌（P.chrysosporium）纯培养物中扩增木质素酶H8 DNA的灵敏度不如扩增高度重复的核核糖体DNA的内转录间隔区（ITS）。PCR扩增的ITS DNA的限制性内切酶酶谱与其它无关真菌相比具有独特性。两株黄孢原毛革菌和两株花脸原毛革菌通过限制性内切酶分析无法区分，而第三株黄孢原毛革菌具有独特的模式。这些结果得到了序列信息的证实，并表明刺毛革菌属的物种命名。应该重新审视。从土壤中生长的黄孢原毛平革菌中提取DNA并进行PCR扩增，其酶切图谱与纯培养的黄孢原毛平革菌相同。从1g土壤中提取的100 μ g总DNA中检测到14ng的ITS序列。
A nonradioactive method to detect Phanerochaete chrysosporium grown in a soil matrix was developed. This method involved DNA extraction, PCR amplification, and restriction enzyme analysis. Amplification of ligninase H8 DNA from pure cultures of P. chrysosporium was not as sensitive as amplification of the internal transcribed spacer (ITS) of the highly repetitive nuclear ribosomal DNA, Amplified ITS DNA was digested with restriction enzymes for analysis. The restriction enzyme pattern of PCR-amplified ITS DNA of P. chrysosporium was unique compared with those of unrelated fungi. Two strains of Phanerochaete chrysosporium and two strains of Phanerochaete sordida were indistinguishable by restriction enzyme analysis, while a third strain of P. chrysosporium had an unique pattern. These results were confirmed by sequence information and indicate that species designations of Phanerochaete spp. should be reexamined. The restriction enzyme pattern of DNA extracted and PCR amplified from P. chrysosporium grown in soil was identical to that from P. chrysosporium grown in pure culture. The ITS sequence was detected in 14 ng of the 100 mu g of total DNA extracted from 1 g of soil.