摘要
根据黄曲霉毒素生化途径中的关键调控基因aflR、omt-1和ver-1的序列以及真菌共有的5.8S rDNA的ITS序列分别设计ApaF/ApaR、OmtF/OmtR、VerF/VerR及ITS1/ITS4四对引物,研究建立产黄曲霉毒素真菌及其潜在饲料或食品污染的多重PCR快速灵敏检测体系。PCR扩增的4个DNA片段中,1032bp、797bp和600bp与基因库中对应基因或DNA序列的同源性达99%以上,仅452bp片段与对应基因ver-1的同源性为98%。通过优化主要影响因子,建立了快速检测产黄曲霉毒素真菌的单管多重PCR反应体系,并用于6种曲霉和1种青霉DNA样品的检测。结果显示,上述4个片段均平行地清晰出现在2株黄曲霉Aspergillus flavus和1株寄生曲霉A.parasiticus的DNA样品中,而其余菌种只检测到ITS片段,说明检测特异性很好。灵敏性分析表明,多重PCR检测的保守灵敏度为1ng/μL样品DNA,所有目标片段的条带均很清晰;即使DNA浓度降至0.1ng/μL,除aflR之外的所有条带也可分辨。
In an attempt to develop a multiplex PCR system for rapidly detecting aflatoxigenic fungi that often contaminate food and feed, four pairs of primers （i.e., ApaF/ApaR, OmtF/OmtR, VerF/VerR and ITS1/ITS4） were designed based on the sequences of the three crucial genes aflR, ver-1 and omt-1 regulating aftatoxin biosynthesis and the internal transcribed spacer （ITS） of fungal rDNA. Amplified PCR products fell in the sizes of 1032, 452, 797 and 600bp respectively and were well in accordance with the corresponding DNA sequences in GenBank. The detection system was established by optimizing primary factors influential on the reaction system and successfully applied to detecting genomic DNA samples of six Aspergillus species and one Penicillium species. As a result, the four target DNA sequences were readily detected in three strains of Aspergillus flavus and A. parasiticus known as aflatoxigenic fungi while only the ITS sequence was found in the rest fungal species. This shows an excellent specificity for the multiplex PCR system. Further sensitivity analysis indicated that the developed system was featured with a conserved sensitivity of 1ng/μL DNA sample, at which all the bands for the target DNA sequences were very clear. The target sequences except for that of aflR were also distinguishable even at the concentration of 0. 1ng/μL DNA sample.
出处
《菌物学报》
CAS
CSCD
北大核心
2007年第3期448-454,共7页
Mycosystema
基金
国家质量监督检疫总局资助项目(No.2006IK151)
关键词
黄曲霉毒素
曲霉
青霉
快速检测体系
反应条件优化
特异性
灵敏性
Aflatoxin, Aspergillus, Penicillium, rapid detection system, optimized reaction, specificity, sensitivity
