雞舍基质论文鸡舍环境中镰孢菌的种类及真菌毒素检测.docVIP

鸡舍基质论文：鸡舍环境中镰孢菌的种类及真菌毒素检测 【中文摘要】本研究利用tri5特征性引物对分离自包括空气、饲料、土壤、积尘和饮用水在内的鸡舍环境基质中的镰孢菌分离株基因组DNA扩增来快速筛选产毒镰孢菌,并通过正交试验设计方法对tri5阳性菌株进行产毒培养,采用免疫亲和柱净化-高效液相色谱(HPLC)方法对产毒培养产物和鸡舍空气及基质样品进行T-2毒素和HT-2毒素进行定性和定量分析,明确T-2毒素和产毒镰孢菌的数量关系,在建立空气和周围基质之间镰孢菌—产毒镰孢菌—T-2和HT-2毒素量化关系的基础上,初步探明鸡舍气载镰孢菌及T-2毒素的发生规律,为人类和动物气传真菌病的预防和控制提供理论依据。分别在2009年11月和2010年4月采集鸡舍空气样品19份,基质(包括土壤、积尘、饲料、饮用水)样品共164份。对10份空气样品和40份基质样品中的镰孢菌进行分离鉴定,得到来自空气中的59株和来自其他基质中的80株共139株镰孢菌分离株,其中拟轮枝镰孢和半裸镰孢为优势菌种,所占比例分别为23.74%和17.26%。不同类型基质中,土壤中的镰孢菌浓度最高;不同类型鸡舍中,种鸡舍中获得的镰孢菌分离株最多。tri5-PCR对139株镰孢菌分离株的基因组DNA进行特异性扩增,得到42株tri5~+镰孢菌,其中tri5~+菌株数量最多的镰孢菌种类为禾谷镰孢菌,土壤中分离筛选的tri5~+镰孢菌的比例最高。对空气中得到的10株tri5~+镰孢菌按照40号F. semitectum的最佳产毒培养条件进行产毒培养,HPLC检测培养产物,7株镰孢菌产生T-2毒素,浓度范围为1.3ng/mL~5ng/mL,3株产生了HT-2毒素,浓度最高为17.1ng/mL。按照国家检验检疫局质量检测标准,采用免疫亲和柱净化-高效液相色谱方法检测鸡舍环境基质中的T-2毒素(包括HT-2毒素)、黄曲霉毒素(AF)、玉米赤霉烯酮(ZEA)和赭曲霉毒素A(OTA)。检测结果表明,所有环境基质中均检测到T-2毒素,以土壤中的毒素浓度最高,为21μg/kg,除饲料以外的基质中未检测到OTA和ZEA的存在,饲料中的OTA和ZEA水平在冬季和春季有明显差异,冬季与春季饲料样品中的ZEA浓度具有显著差异(P0.05),所有样品中均未检测到AF的检测结果在检测方法定量限以下。 【英文摘要】The specific primers of the gene tri5 were used to amplify genomic DNA in order to rapidly identify the toxogenic Fusarium strains, which were isolated from the air, feed, soil, dust and water sampled from poultry houses. The condition of toxogenic culture was investigated by orthogonal design method. Quantitative detection of T-2 toxin(HT-2 toxin) was conducted in toxogenic culture product and the samples from poultry houses by immunoaffinity column clean and high performance liquid chromatography(HPLC). The quantitative relationship of T-2 toxin and toxogenic Fusarium strains was analyzed, and their relationship with Fusarium strains from poultry houses was set up into a model to prove the occurrence rule of airborne Fusarium and T-2 toxin in poultry houses. The results provide a theoretical basis for the early hazard warning and control of human and animal fungal diseases.Totally 19 airborne samples and 164 substrate samples including soil, grain, feed and water were collected from poultry houses i