2015~2016年河北省贝类水产品中诺如病毒污染状况研究

目的 对2015~2016年河北省6个设区市的市售牡蛎、海虹、血蚶样品中的诺如病毒进行连续一年的检测, 了解河北省贝类水产品中诺如病毒污染状况。方法 分离贝类水产品消化腺, 用蛋白酶K消化后进行前处理, 提取RNA, 用一步法实时荧光逆转录聚合酶链式反应法检测GⅠ型和GⅡ型诺如病毒。对河北省贝类水产品的诺如病毒污染状况进行分析。结果 共采集691份贝类样品进行检测, 其中仅检出GⅠ型阳性样品16份, 阳性率2.32%; 仅检出GⅡ型阳性样品22份, 阳性率3.18%; 同时检出GⅠ和GⅡ型的阳性样品4份, 阳性率0.58%; 总阳性率6.08%。2016年6~7月阳性率最高。沿海地区样品阳性率为内陆地区的2.35倍, 有统计学差异(χ2=8.58, P<0.01)。结论 河北省市售贝类水产品中部分存在诺如病毒污染, 需要加强对贝类水产品的诺如病毒污染检测和控制, 特别是沿海地区, 降低诺如病毒引起的食源性腹泻的疾病负担。     

Quantitative PCR coupled with sodium dodecyl sulfate and propidium monoazide for detection of viable Staphylococcus aureus in milk

Conventional quantitative PCR (qPCR) are unable to differentiate DNA of viable Staphylococcus aureus cells from dead ones. The aim of this study was to use sodium dodecyl sulfate (SDS) and propidium monoazide (PMA) coupled with lysostaphin to detect viable Staph. aureus. The cell suspensions were treated with SDS and PMA before DNA extraction. The SDS is an anionic surfactant, which can increase the permeability of dead cells to PMA without compromising the viability of live cells. The lysostaphin was applied to improve the effectiveness of DNA extraction. The reliability and specificity of this method were further determined by the detection of Staph. aureus in spiked milk. The results showed that there were significant differences between the SDS-PMA-qPCR and qPCR when a final concentration of 200 μg/mL of lysostaphin was added in DNA extraction. The viable Staph. aureus could be effectively detected when SDS and PMA concentrations were 100 µg/mL and 40 μM, respectively. Compared with conventional qPCR, the SDS-PMA-qPCR assay coupled with lysostaphin was more specific and sensitive. Therefore, this method could accurately detect the number of viable Staph. aureus cells.     

食品中空肠弯曲菌荧光重组酶聚合酶扩增检测方法的建立

本研究为了实现空肠弯曲菌的快速和便捷检测,建立了一种基于荧光重组酶聚合酶扩增技术(exo-RPA)快速检测空肠弯曲菌的方法。通过对空肠弯曲菌和对照菌株的exo-RPA检测来判断该方法的特异性。用梯度稀释的空肠弯曲菌作为模板进行检测来分析exo-RPA方法的灵敏度。通过对模拟污染样品检测来分析exo-RPA的应用效果。分别以exo-RPA和荧光PCR检测实际食品样品来分析二者的检测效果。空肠弯曲菌exo-RPA方法可特异检出空肠弯曲菌,检测灵敏度达到6.0×102CFU/m L。在模拟污染试验中,含2.5×101 CFU/m L空肠弯曲菌的增菌液在培养24 h后可以被exo-RPA检测出阳性信号。exo-RPA和荧光PCR对于40份样品的检测结果相同。本研究建立的空肠弯曲菌exo-RPA具有特异、灵敏和抗干扰性强的特点,方法操作简便快速,具有较好的应用前景。     

实时荧光PCR法鉴定野生食用菌中白牛肝菌成分

目的建立野生食用菌中白牛肝菌(Boletus bainiugan Dentinger)成分的荧光PCR检测方法。方法根据白牛肝菌的内源转录间隔区(internally transcribed spacer,ITS)基因序列设计白牛肝菌物种的特异性引物和探针,对样品中的靶标基因片段进行检测,并进行物种特异性检测、稳定性检测、灵敏度检测和实际应用检测。结果通过对供试的24种食用菌、动、植物材料进行检测,只有白牛肝菌出现特异性扩增,说明方法具有物种特异性;方法对白牛肝菌成分的检测灵敏度为1×10~(-4) ng/μL白牛肝菌DNA或0.1%(m/m)白牛肝菌粉。结论该方法简单、灵敏、快速、准确,能应用于野生食用菌和食品中白牛肝菌的成分检测。     

RT-FQPCR法快速检测葡萄酒中醋酸菌群

建立一种快速、灵敏的检测葡萄酒中醋酸菌群的检测技术。通过改进葡萄酒中脱氧核糖核酸（DNA）提取方法,设计醋酸菌通用引物,优化实时荧光定量聚合酶链式反应（RT-FQPCR）条件,确定葡萄酒中醋酸菌的快速检测方法,同时利用7种醋酸菌对该方法的通用性、特异性以及灵敏度进行了验证。结果表明,7种醋酸菌均具有明显扩增曲线;非目的菌在该种条件下未见扩增,综合检测灵敏度为124 CFU/mL。以10种市售葡萄酒为基质,在其中添加醋酸菌均可检测出目标添加菌株。该方法通用性强,可用于葡萄酒中醋酸菌群的检测。     

通用引物多重PCR新技术对番木瓜转基因成分检测的研究

为了满足能够同时对多个靶标进行检测,克服传统多重PCR的引物间排斥、重现性差等缺点的要求,建立通用引物多重PCR新方法对番木瓜中转基因成分进行高效、快速的检测。选用三种转基因番木瓜(Event 55-1、"GM-YK"和"华农一号")和非转基因番木瓜("台农2号")的新鲜叶片为研究对象,根据三种转基因番木瓜的外源基因和内标木瓜蛋白酶基因(papain)序列,设计五种特异性引物。通过比较不加入接头的特异性引物的PCR验证结果,发现复合引物(带通用接头的引物)无论在单重PCR或者多重PCR的检测中的电泳条带均更为明亮清晰。而由灵敏度的对比,可以得出通用引物多重PCR灵敏度比普通引物的灵敏度提高了1个数量级。通用引物多重PCR新技术较普通多重PCR方法检测番木瓜中转基因成分拥有更低的检测限和灵敏度,为转基因成分的快速检测提供一种新技术。     

基于PCR-DGGE技术的四川麸醋固态发酵过程中微生物群落分析

为了解四川麸醋固态发酵过程中微生物群落变化规律,采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术对其固态发酵过程中真菌和细菌的多样性进行分析。结果表明,四川麸醋固态发酵过程中优势真菌主要有酿酒酵母(Sac.cerevisiae)、扣囊复膜孢酵母(Sac.fibuligera)、伊萨酵母(Iss.hanoiensis)、黑曲霉(Asp.niger)、草青霉菌(Pen.oxalicum)以及不可培养真菌(Uncultured fungus);主要细菌有嗜酸乳杆菌(Lac.acidophilus)、葡萄糖醋杆菌(Glu.oboediens)、巴氏醋杆菌(Ace.pasteurianus)、甲醇酸单胞菌(Aci.methanolica)、不可培养细菌(Uncultured bacterium)、不可培养芽孢杆菌(Uncultured Bacillus sp.)和不可培养乳杆菌(Uncultured Lactobacillus sp.)。PCR-DGGE技术可鉴别醋醅中多种不可培养微生物,四川麸醋固态发酵过程优势微生物种类多、丰度高,菌系十分复杂,多种微生物交替生长,但整体群落结构变化不明显。     

Influence of inoculum and climatic factors on the severity of Fusarium head blight in German spring and winter barley

Fusarium head blight (FHB) of small cereals is a disease of global importance with regard to economic losses and mycotoxin contamination harmful to human and animal health. In Germany, FHB is predominantly associated with wheat and F. graminearum is recognised as the major causal agent of the disease, but little is known about FHB of barley. Monitoring of the natural occurrence of FHB on Bavarian barley revealed differences for individual Fusarium spp. in incidence and severity of grain infection between years and between spring and winter barley. Parallel measurement of fungal DNA content in grain and mycotoxin content suggested the importance of F. graminearum in winter barley and of F. langsethiae in spring barley for FHB. The infection success of these two species was associated with certain weather conditions and barley flowering time. Inoculation experiments in the field revealed different effects of five Fusarium spp. on symptom formation, grain yield and mycotoxin production. A significant association between fungal infection of grain and mycotoxin content was observed following natural or artificial infection with the type B trichothecene producer F. culmorum, but not with the type A trichothecene-producing species F. langsethiae and F. sporotrichioides. Trichothecene type A toxin contamination also occurred in the absence of significant damage to grain and did not necessarily promote fungal colonisation.