潜藏性产黄曲霉毒素真菌的多重PCR检测体系构建及真菌DNA提取技术的改进
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摘要
由于黄曲霉毒素(aflatoxin)是人类和畜禽重要的致癌致病因子，因此如何严格控制潜藏产黄曲霉毒素真菌的食品及饲料等产品受到人们的极大关注。目前常用的直接检测法包括薄层层析、液相色谱、免疫化学分析等多种方法，其设计基于黄曲霉毒素的理化性质，无法检出已被产毒真菌污染但尚未产毒的高风险样品。多重PCR检测方法，可在单一泳道反应体系中同时检测多个DNA模板或同一模板的不同区域，具有快捷、高效、低成本等优点，被广泛应用于有害微生物检测。本研究以产毒真菌黄曲霉(Aspergillus flavus)的基因组DNA为模板，根据黄曲霉毒素生物合成中关键酶的基因序列设计引物，构建了能够从样品中快速灵敏检测潜藏性产毒真菌的多重PCR检测体系，并改进了真菌DNA样品的提取方法，使之适于大量样品检测的真菌DNA快速提取要求。
多重PCR检测体系的构建根据黄曲霉毒素生物合成中关键酶的调控基因aflR、omt-1和ver-1的序列以及真菌共有的5.8S rDNA的ITS序列分别设计出ApaF／ApaR、OmtF／OmtR、VerF／VerR及ITS1／ITS4等四对引物，用于构建快速灵敏检测饲料或食品中潜藏性产毒真菌的多重PCR反应体系。PCR扩增的四个DNA片断分别为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％。通过优化反应体系的组成因子，建立了单管多重PCR反应体系并应用于6种曲霉和1种青霉的多个菌株DNA样品的检测。结果显示，四个目标片段均清晰地出现在2株黄曲霉和1株寄生曲霉(Aspergillus parasiticus)的DNA样品中，而其余非产毒菌种的DNA样品中只能检出ITS片段，说明检测特异性十分理想。灵敏性分析结果表明，多重PCR检测的保守灵敏度为1 ngμL~(-1)样品DNA，且所有目标片段的条带均很清晰；即使DNA浓度降低至0.1 ngμL~(-1)，除aflR之外的所有条带也可分辨。
样品DNA提取方法改进通过比较研究常用的液氮研磨法、酶解法和试剂盒法，根据破壁原理和操作步骤的特点，开发出基于超声波和化学试剂Tris饱和苯酚协同作用的菌丝细胞壁破碎法，辅以煮沸-冰冻处理，使胞内DNA充分释放于缓冲液中。这一改进的样品基因组DNA提取法具有操作简便、经济快速的特点，所获样品经多重PCR扩增，各电泳条带清晰。从DNA样品制备到多重PCR结果检出仅需5h，符合快速灵敏可靠的检测要求。
Aflatoxins are carcinogenic and/or pathogenic toxins produced by aflatoxigenic fungi that often contaminate small grains, peanuts and feeds, threatening to human and animal health. Food or feed safety prohibits entry of contaminated products into market. However, current inspection methods, such as thin-layer chromatography, liquid chromatography, enzyme-linked immunosorbent assay and immunoaffinity column assay, are based on physical and chemical properties of the toxins and cannot be applied to examining risky products that may carry aflatoxigenic fungi but have not yet contained detectable toxins.
Multiplex PCR reaction is a DNA-based detection method based on a regular PCR technique and can be used to identify simultaneously more DNA templates or different parts of the same DNA template in a single-lane reaction system. This method has been widely used in diagnosis of hazardous pathogens or microbes. In this study, several key enzyme genes involved in biological synthesis of the aflatoxins were used to design paired primers for developing muitiplex PCR detection system. The strain Aspergillus flavus AS 3.4408 was chosen as a positive aflatoxigenic fungus to testify sensitivity, reliability and efficiency of the developed system.
Construction of multiplex PCR detection system. 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 three key genes (aflR, ver-1 and omt-1) involved in aflatoxin biosynthesis and of internal transcribed spacer (ITS) of fungal 5.8s rDNA. Amplified PCR products of the four target sequences fell in the sizes of 1032, 452, 797 and 600 bp, respectively, and were well in accordance with their DNA sequences documented in GenBank. The detection system was optimized for best reaction 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, which are known as aflatoxigenic fungi, whereas only ITS was found in the DNA samples of the rest unaflatoxigenic fungi. This shows that the developed multiplex PCR system had excellent specificity and sensitivity towards aflatoxigenic fungi. Further sensitivity analysis indicates that the developed system was featured with a conserved sensitivity of 1 ngμL~(-1) DNA sample, at which all the bands for the target DNA sequences were very clear. All the target sequences except aflR were also distinguishable even at the concentration of 0.1 ngμL~(-1) DNA sample.
Improved extraction method of fungal DNA samples. Conventional methods of fungal DNA extraction, such as liquid nitrogen grinding, enzymolysis and TIANGEN~(?) )extraction kit, were compared for their extraction efficiency. Based on the principles of the methods for breaking fungal cell walls, a novel rapid method for extracting genomic DNA from aflatoxigenic Aspergillus species was developed by making use of co-action of ultrasonic treatment and Tris-phenol-based lysis, followed by boiling-icing treatment and chloroform separation of the supernatant from residues. The resultant DNA samples suited well to the multiplex PCR detection. When this improved method was applied to extracting fungal DNAs, the PCR detection of samples potentially contaminated with aflatoxigenic fungi was completed with 5 h, meeting a rapid need for processing large samples.
多重PCR检测体系的构建根据黄曲霉毒素生物合成中关键酶的调控基因aflR、omt-1和ver-1的序列以及真菌共有的5.8S rDNA的ITS序列分别设计出ApaF／ApaR、OmtF／OmtR、VerF／VerR及ITS1／ITS4等四对引物，用于构建快速灵敏检测饲料或食品中潜藏性产毒真菌的多重PCR反应体系。PCR扩增的四个DNA片断分别为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％。通过优化反应体系的组成因子，建立了单管多重PCR反应体系并应用于6种曲霉和1种青霉的多个菌株DNA样品的检测。结果显示，四个目标片段均清晰地出现在2株黄曲霉和1株寄生曲霉(Aspergillus parasiticus)的DNA样品中，而其余非产毒菌种的DNA样品中只能检出ITS片段，说明检测特异性十分理想。灵敏性分析结果表明，多重PCR检测的保守灵敏度为1 ngμL~(-1)样品DNA，且所有目标片段的条带均很清晰；即使DNA浓度降低至0.1 ngμL~(-1)，除aflR之外的所有条带也可分辨。
样品DNA提取方法改进通过比较研究常用的液氮研磨法、酶解法和试剂盒法，根据破壁原理和操作步骤的特点，开发出基于超声波和化学试剂Tris饱和苯酚协同作用的菌丝细胞壁破碎法，辅以煮沸-冰冻处理，使胞内DNA充分释放于缓冲液中。这一改进的样品基因组DNA提取法具有操作简便、经济快速的特点，所获样品经多重PCR扩增，各电泳条带清晰。从DNA样品制备到多重PCR结果检出仅需5h，符合快速灵敏可靠的检测要求。
Aflatoxins are carcinogenic and/or pathogenic toxins produced by aflatoxigenic fungi that often contaminate small grains, peanuts and feeds, threatening to human and animal health. Food or feed safety prohibits entry of contaminated products into market. However, current inspection methods, such as thin-layer chromatography, liquid chromatography, enzyme-linked immunosorbent assay and immunoaffinity column assay, are based on physical and chemical properties of the toxins and cannot be applied to examining risky products that may carry aflatoxigenic fungi but have not yet contained detectable toxins.
Multiplex PCR reaction is a DNA-based detection method based on a regular PCR technique and can be used to identify simultaneously more DNA templates or different parts of the same DNA template in a single-lane reaction system. This method has been widely used in diagnosis of hazardous pathogens or microbes. In this study, several key enzyme genes involved in biological synthesis of the aflatoxins were used to design paired primers for developing muitiplex PCR detection system. The strain Aspergillus flavus AS 3.4408 was chosen as a positive aflatoxigenic fungus to testify sensitivity, reliability and efficiency of the developed system.
Construction of multiplex PCR detection system. 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 three key genes (aflR, ver-1 and omt-1) involved in aflatoxin biosynthesis and of internal transcribed spacer (ITS) of fungal 5.8s rDNA. Amplified PCR products of the four target sequences fell in the sizes of 1032, 452, 797 and 600 bp, respectively, and were well in accordance with their DNA sequences documented in GenBank. The detection system was optimized for best reaction 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, which are known as aflatoxigenic fungi, whereas only ITS was found in the DNA samples of the rest unaflatoxigenic fungi. This shows that the developed multiplex PCR system had excellent specificity and sensitivity towards aflatoxigenic fungi. Further sensitivity analysis indicates that the developed system was featured with a conserved sensitivity of 1 ngμL~(-1) DNA sample, at which all the bands for the target DNA sequences were very clear. All the target sequences except aflR were also distinguishable even at the concentration of 0.1 ngμL~(-1) DNA sample.
Improved extraction method of fungal DNA samples. Conventional methods of fungal DNA extraction, such as liquid nitrogen grinding, enzymolysis and TIANGEN~(?) )extraction kit, were compared for their extraction efficiency. Based on the principles of the methods for breaking fungal cell walls, a novel rapid method for extracting genomic DNA from aflatoxigenic Aspergillus species was developed by making use of co-action of ultrasonic treatment and Tris-phenol-based lysis, followed by boiling-icing treatment and chloroform separation of the supernatant from residues. The resultant DNA samples suited well to the multiplex PCR detection. When this improved method was applied to extracting fungal DNAs, the PCR detection of samples potentially contaminated with aflatoxigenic fungi was completed with 5 h, meeting a rapid need for processing large samples.
引文
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