A multiplex RT-PCR approach to detect aflatoxigenic strains of Aspergillus flavus
A multiplex RT-PCR approach to detect aflatoxigenic strains of Aspergillus flavus
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DOI：
10.1111/j.1365-2672.2006.03256.x
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发表时间：
2007-08-01
影响因子：
4
通讯作者：
Restivo, F. M.
中科院分区：
文献类型：
--
作者：
Degola, F.;Berni, E.;Restivo, F. M.
目的：建立一种鉴别产黄曲霉毒素和不产黄曲霉毒素黄曲霉的多重逆转录-聚合酶链反应（RT-PCR）方法。从波河谷（意大利）田间收集的玉米粒中分离的黄曲霉菌株。A.黄曲霉毒素基因簇的5个基因。用特异性引物靶向两种调节（aflR和aflS）和三种结构（aflD、aflO和aflQ），以突出它们在菌丝体中的表达。在诱导条件下培养以产生黄曲霉毒素。48小时龄的培养物表达了这里分析的全套基因，而24小时龄的则没有。基因组PCR还使用从同一组菌株中提取的染色体DNA平行地进行四重PCR（quadruplex PCR），以将基因的完整性与它们的表达相关联。我们发现，通过多重RT-PCR分析的黄曲霉毒素基因的基因表达与黄曲霉毒素的产生之间存在良好的相关性，除了一个菌株明显转录了所有相关基因，但在培养基中不产生黄曲霉毒素。这是第一个应用多重PCR和RT-PCR方法相结合来筛选A.黄曲霉毒素菌株和非黄曲霉毒素菌株的存在。建议的方案将有助于评估A.黄曲霉毒素在自然环境中的存在，也可能是一个有用的工具，以监测其存在过程中的食品和饲料商品的加工步骤。
Aims: To develop a multiplex reverse transciption-polymerase chain reaction (RT-PCR) protocol to discriminate aflatoxin -producing from aflatoxin-nonproclucing strains of Aspergillus flavus.Methods and Results: The protocol was first optimized on a set of strains obtained from laboratory collections and then validated on A. flavus strains isolated from corn grains collected in the fields of the Po Valley (Italy). Five genes of the aflatoxin gene cluster of A. flavus, two regulatory (aflR and aflS) and three structural (aflD, aflO and aflQ), were targeted with specific primers to highlight their expression in mycelia. cultivated under inducing conditions for aflatoxins production. 48-h-old cultures expressed the complete set of the genes analysed here whereas 24-h-old ones did not. Genomic PCR (quadruplex PCR) was also performed in parallel using chromosomal DNA extracted from the same set of strains to correlate the integrity of the genes with their expression.Conclusions: We show that a good correlation exists between gene expression of the aflatoxin genes, here analysed by multipex RT-PCR, and aflatoxin production, except for one strain that apparently transcribed all the relevant genes but did not produce aflatoxin in the medium.Significance and Impact of the Study: This is the first example of the application of a combination of multiplex PCR and RT-PCR approaches to screen a population of A. flavus for the presence of aflatoxigenic and nonaflatoxigenic strains. The proposed protocol will be helpful in evaluating the risk posed by A. flavus in natural environments and might also be a useful tool to monitor its presence during the processing steps of food and feed commodities.