基于毒素编码基因aflp的黄曲霉菌nested-PCR检测

摘要

黄曲霉Aspergillusflavus 产生的黄曲霉毒素是一类毒性极强的物质，严重威胁到人类健康和经济发展，因此建立一套快速、准确、灵敏的黄曲霉检测方法就显得极为迫切。通过以aflP （GenBank：FN398191）为分子靶标，比较黄曲霉菌近缘种aflp的基因序列，以特异性序列为靶标设计2对PCR引物aflP-1-F／aflP-1-R和aflP-2-F／aflP-1-R，由此建立的PCR检测体系对7种黄曲霉菌、5种其他的曲霉菌、21种其他真菌 cDNA 进行扩增，结果只有在7种黄曲霉菌株中扩增出211 bp 的特异性条带，而其余参试菌株均无扩增产物。巢式PCR能使其检测灵敏度达到10 fg，检测灵敏度提高100倍。该检测体系能从人工接种和自然发病的花生和玉米样品中扩增到211 bp的特异片段，实现对黄曲霉菌的快速可靠检测。%Aspergillus flavus is a devastating pathogen for human health and economic development,for the aflatoxins produced by A. flavus has strong toxicity for humans and animals. Therefore,to establish a rapid, accurate and sensitivity detection system for A. flavus,we developed a nested polymerase chain reaction (PCR) based on the aflP (GenBank:FN398191 )gene. In the present study,aflP was used as a molecular target, alignment of the aflP region sequences with other sequences belonging to Aspergillus species closely related to A. flavus and other fungi was used to identify conserved and differing regions,and then two pairs of PCR primers (aflP-1-F/aflP-1-R and aflP-2-F/aflP-1-R)were developed. For specificity testing,DNA extracted from 7 A. flavus,5 different Aspergillus spp. and 21 other fungi were used,and our results showed that a 211bp of specific band can be amplified only in aflatoxins produced A. flavus strains. A nested PCR procedure using aflP-1-F/aflP-1-R as the first-round primers and the followed using aflP-2-F/aflP-1-R increased the detection sensitivity 100- fold to 10 fg. Furthermore,the aflP- based PCR was used for detection analysis of cDNA from artificially and naturally aflatoxins-contaminated corn and peanut samples and our results suggest that the aflP-based PCR detection is a specific,sensitive and rapid diagnostic tool for A. flavus monitoring.