中国农业科学Issue(18)：3675-3683,9.DOI:10.3864/j.issn.0578-1752.2014.18.015
利用PCR-RFLP方法鉴别黄曲霉毒素产毒菌株
Identification of Aflatoxin Producing Strains by Using PCR-RFLP Method
摘要
Abstract
[Objective]The objective of this study is to develop an identification method of aflatoxin-producing strains with the advantages of high accuracy, good sensitivity, and convenience, which provide early warning to aflatoxin contamination of stored grain, provide technical support for the safety control in food industry and provide the non-toxic microbial sources for biological control of aflatoxin contamination.[Method]The key gene of aflatoxin synthesis, aflR, which belongs to Aspergillus flavus, Aspergillus parasitieus, Aspergillus oryzae and Aspergillus sojae, was analyzed by bioinformatics method. The potential strains producing aflatoxin were identified by PCR-RFLP technology. To verify these experimental results above, their toxin-producing ability was tested and analyzed by HPLC after these strains were cultured and fermented with soybean medium.[Results]The sequences of their aflR gene were analyzed in this research and the results showed high homology among their sequences. On the other hand, the partial bases of their aflR and promoter mutated regularly to induce variation of restriction sites NheⅠ, PvuⅡ, and HincⅡ. The genomic DNA were extracted from all these strains, the sequences of their aflR and promoter were amplified by PCR which includes 798 bp and 515 bp respectively. These promoters of aflR gene were digested by NheⅠand produced 176 bp and 339 bp two short fragments from toxin-producing strains. However, there was no restriction site of NheⅠin those of the non-toxin-producing strains. The results showed that the aflR of A. flavus and A. parasitieus were digested into three fragments (387 bp, 250 bp, 161 bp) and two fragments (548 bp, 250 bp) by HincⅡ, and those of A. flavus and A. parasitieus were digested into two fragments (652 bp, 146 bp) and three fragments (413 bp, 239 bp, 146 bp) by PvuⅡ, respectively. The analysis of aflatoxin-producing demonstrated that both A. oryzae and A. sojae could not produce aflatoxin under the same cultural condition. It showed the same results that no aflatoxin produced by the strains in PCR-RFLP analysis. Among toxin-producing Aspergillus strains, the differences of their toxin-producing ability were remarkable. Not only their toxin yields rose a great many times in variation but their compositions were different.[Conclusion]This study illustrated the differences existed in the key gene aflR sequence among the strains. The active toxin-producing A. flavus and non-toxin-producing ones as well as toxin-producing A. flavus and A. parasitieus were identified by PCR-RFLP rapidly. The development of the method for identifing toxin-producing Aspergillus strains is probably useful for safety check and quality control not only in productive process of cereal and oil products but during food storage.关键词
黄曲霉毒素/aflR基因/PCR-RFLP/产毒菌株鉴别Key words
Aflatoxin/aflR gene/PCR-RFLP/identification of produced strains引用本文复制引用
孙长坡,常晓娇,伍松陵,沈晗..利用PCR-RFLP方法鉴别黄曲霉毒素产毒菌株[J].中国农业科学,2014,(18):3675-3683,9.基金项目
国家“863”计划（2012AA101609）、粮食公益性行业科研专项 （）
