桃拟茎点霉对孕酮的生物转化研究

研究了一株桃拟茎点霉（Phomopsis amygdale）对孕酮的生物转化,并对该转化菌株的形态学和分子生物学进行鉴定,转化产物经分离纯化后,运用核磁共振（NMR）、质谱（MS）、红外光谱（IR）分析进行结构鉴定。该菌株转化孕酮得到2个转化产物,转化产物分别为15β-羟基孕酮和6β,15β-二羟基孕酮。结果表明,桃拟茎点霉具有转化甾体的能力,对孕酮进行生物转化的主要产物是15β-羟基甾体衍生物。     

内生真菌拟茎点霉B3产漆酶分批发酵动力学

通过建立内生真菌拟茎点霉（Phomopsis liquidambari）B3产漆酶分批发酵动力学模型,发现B3菌漆酶生成与菌体生长呈现部分生长偶联型。应用MATLAB软件将实验数据与符合B3菌菌体生长的Logistic模型、漆酶生成的Luedeking-Piret模型和基质消耗的Luedeking-Piret-Like模型进行非线性拟合,求得最优参数估计值和发酵动力学模型。分析动力学模型的拟合曲线,发现模型的曲线与实验值能较好地拟合。验证实验表明模型计算值与实验值的偏差大部分都低于10%,说明该动力学模型能很好地描述B3菌的实际发酵情况。     

枫香拟茎点霉B3生物转化花生废弃物合成白藜芦醇

为探究接种枫香拟茎点霉B3对花生废弃物中白藜芦醇含量的影响,采用代谢物检测和关键酶基因的定量实验进行验证。结果表明,枫香拟茎点霉B3既可以通过分泌糖苷酶转化花生废弃物中的白藜芦醇苷为白藜芦醇,又可以通过从头合成途径利用花生废弃物中的对香豆酸生产白藜芦醇。在此基础上,采用响应面优化发酵条件对白藜芦醇转化效率的影响,得到在料液比为1∶30(g/mL)条件下,培养基初始pH 5.2、接种量9%、发酵温度28.6℃、发酵转速204 r/min时,菌株转化合成白藜芦醇效率最显著。在5 L发酵罐进行中试放大实验,经过实测经过36 h白藜芦醇含量提高了1.9倍。结果表明,接种枫香拟茎点霉B3可以显著提高花生废弃物中白藜芦醇含量,本研究对于花生中高附加值产物的开发和花生综合利用途径的拓宽具有重要的参考意义。     

烟草鸢尾丝囊霉菌的生物学特性及分子检测

针对近年来河南省许昌、洛阳、三门峡等部分烟区烟草漂浮苗鸢尾丝囊霉根腐病发生较重的问题,采用菌丝生长速率法初步分析了病原菌的生物学特性,并根据NCBI数据库中鸢尾丝囊霉菌代表菌株CBS 524.87的5个编码基因CDS序列,设计筛选鸢尾丝囊霉菌的特异性扩增引物并应用。生物学特性分析结果表明,在PDA培养基上,鸢尾丝囊霉菌菌丝生长的适宜温度为25～35℃,致死温度为50℃,处理10min;生长适宜pH为4.0～8.0,最适pH为6.0;连续光照条件有利于菌丝生长。筛选获得了特异性扩增鸢尾丝囊霉菌的引物对7个,对于基因组DNA扩增的灵敏度约为0.182 ng/μL;利用特异引物AiT3分别对接种育苗基质和烟苗进行分子检测,可特异性的检测出鸢尾丝囊霉菌,检测的灵敏度分别为每克基质含菌量为2.5×10^-2 g病原菌菌丝和0.5 ng/μL烟苗根系基因组DNA。本研究为鸢尾丝囊霉菌的快速分子检测提供了技术支撑,对苗期鸢尾丝囊霉根腐病病原的准确识别和预测预报提供了依据。     

烟草野火病菌特异性检测引物筛选及应用

烟草野火病是山东烟区主要的细菌性病害之一,为了能够对该病害进行精确、快速的分子鉴定,本研究从NCBI基因组数据库中下载了假单胞菌属25个不同菌种和127个丁香假单胞菌属不同致病变种的全基因组数据,利用Mauve 2.3.1进行全基因组比对,获得丁香假单胞菌烟草致病变种特异性区段。在此基础上利用Primer Premier 6.0进行引物设计,筛选出了一对丁香假单胞菌烟草致病变种的特异性检测引物40429。利用7株山东不同烟区的烟草野火病菌和4株分别来自于贵州、湖北、福建及云南的烟草野火病菌进行了该引物的适用性验证,结果表明该引物均可扩增得到大小为203 bp的单一目的条带。说明该引物对于烟草野火病菌的检测具有良好的适用性,可以用于野火病菌的分子鉴定。     

应用PCR快速检测食品中沙门氏杆菌方法的研究

本研究利用沙门氏杆菌属特异的inv A基因序列，设计一对特异性的引物，通过PCR反应来检测多种样品中的沙门氏杆菌；反应的特异性和敏感性以及反应的最适条件等试验的结果显示：此PCR方法检测沙门氏菌属的特异性为100％：样品中活菌的检测限为1．43CFU／10g：反应的最佳退火温度是65℃；最适模板浓度范围在10^4～10^8copies／ml。本方法与传统的沙门氏杆菌检测方法以及最近报道的相关方法比较，具有简单、快速、特异性好和敏感性高、经济等特点，并可满足大批样品沙门氏杆菌筛选检测的要求。     

千里香杜鹃提取物对赤拟谷盗、烟草甲和马铃薯茎线虫的防治作用

本文以千里香杜鹃枝、叶为实验材料,石油醚、甲醇、95%乙醇为提取剂,采用热浸法得到提取物,测试提取物对赤拟谷盗和烟草甲的触杀、熏蒸和拒食活性以及其对马铃薯茎线虫的毒杀活性,以此初步筛选出对赤拟谷盗、烟草甲和马铃薯茎线虫具有较好抗虫活性的提取物。结果表明：千里香杜鹃叶95%乙醇提取物对赤拟谷盗、烟草甲和马铃薯茎线虫具有明显杀虫活性(LD50分别为15.32、16.18 μg/头;LC50=0.78 mg/mL);对赤拟谷盗具有明显拒食活性,在质量浓度（2 mg/mL）测试条件下,拒食率为78.17%。各提取物对赤拟谷盗和烟草甲无明显熏蒸活性。综上,千里香杜鹃叶95%乙醇提取物对赤拟谷盗、烟草甲和马铃薯茎线虫具有一定防治作用。     

基于RAPD分子标记的烟草青枯病菌特异引物筛选及效果评价

为建立烟田土壤和烟株烟草青枯病菌的快速诊断方法,本研究采用RAPD方法筛选获得了6对可用于烟草青枯病菌PCR检测的特异性引物.经对6种细菌(烟草青枯病菌、多粘芽孢杆菌、枯草芽孢杆菌、节杆菌、荧光假单胞菌、野火病菌)和2个烟草品种(中烟100和云烟87)的检测验证,6对引物均具有良好的灵敏度、特异性和可靠性.对青枯菌菌液...     

转基因烟草检测方法研究进展

随着烟草国际贸易的发展,转基因烟草产品的检测成为烟草贸易关注的热点。目前对于转基因烟草产品的检测方法主要有两种,一种是以DNA为基础的PCR法,另一种是以蛋白质为基础的ELISA法。笔者主要论述了转基因烟草PCR检测法的研究进展,这对开展转基因烟草检测的研究工作有一定的参考价值。     

烟草五种土传病原菌的多重PCR快速检测

【目的】建立一种可同时检测烟草黑胫病菌（Phytophthora parasitica）、青枯病菌（Ralstonia solanacearum）、立枯病菌（Rhizoctonia solani）、根腐病菌（Fusarium oxysporum）和根黑腐病菌(Thielaviopsis basicola)5种烟草重要土传病原菌的五重PCR快速检测方法。【方法】筛选5种病原菌的特异性引物组合,通过不同的引物浓度和退火温度对多重PCR体系进行优化,检测体系的灵敏度,并对土壤和植株样品进行测试,验证其实用性。【结果】根据青枯病菌fliC基因、立枯病菌RPB2基因、根腐病菌COI基因以及根黑腐病菌TEF1基因设计特异性引物,并结合已报道的黑胫病菌parA1基因的特异性引物,成功建立5种烟草土传病害的多重PCR检测方法。反应体系(25μL):Sf1/Sr1、Ff1/Fr1、Pf1/Pr1每条引物分别0.3μL,Rf1/Rr1每条引物各1.8μL,TBf1/TBr1每条引物各1μL,2×PCR Mix 12.5μL,退火温度为58℃,灵敏度可达到100 pg/μL。【结论】本研究建立的多重PCR体...     

基于干燥动力学的烟草含水率快速检测方法

为提高烟草物料含水率测试效率,优化湿热处理单元工况调控响应,以Newton干燥动力学模型为基础,利用宏量热分析仪获取了10%~30%含水率区间内烟草物料的干燥速率与含水率之间线性相关规律,开发了一种烟草含水率的快速检测方法,并对该方法的准确度和重复性等进行了评价。结果表明:①5个标准样品检测结果的相对偏差小于2.89%,变异系数均小于3.71%,表明该方法准确度和重复性良好。②本方法试验参数为:干燥速率常数k=-0.004,干燥温度100 ℃,样品质量0.5 g,检测时间20 min。③该检测方法具有较好的普适性,对2019年福建南平4个等级、3个部位和5个含水率的样品检测绝对偏差均在0.46%以内。④该方法与烘箱法的相关系数R高达0.995 6。⑤与4种常用含水率检测方法比较,快速含水率检测法具有精度较高、检测时间较短,控制精准及检测精度可进一步提高的特点。      

Evaluation of a rapid detection method for Escherichia coli in foods using fluorogenic assay

The fluorogenic assay with the 4-methylumbelliferone glucuronide (MUG) incorporated in lauryl tryptose broth (LTB) was compared with the conventional FDA's Bacteriological Analytical Manual MPN Method for the detection and confirmation of E. coli in foods. One-hundred and one food samples comprising froglegs, oysters, mussels, shrimps, red meats, fish, dried prawns and dried beef were tested. Ninety one per cent of the LTB-MUG tubes were positive for E. coli using the fluorogenic assay and 81% using the MPN method. Analysis time was reduced from 5 days for the MPN method to only 2 days for the fluorogenic assay. In spite of problems posed by false positive and negative reactions, the fluorogenic assay remains a much more sensitive and rapid method.     

烟草赤星病病原长柄链格孢菌快速检测胶体金试纸条的研制

为实现大田期烟叶烟草赤星病的快速测定,以胶体金标记特异性识别长柄链格孢菌的单克隆抗体,利用侧流免疫层析技术构建了长柄链格孢菌快速检测试纸条.结果表明,该试纸条对目标真菌的检出限为20 ng/mL,且与烟草相关病原体无交叉反应,灵敏度高、特异性好.利用该试纸条对加标鲜烟叶进行检测,10 min内可完成检测,操作简便快速,...     

A rapid method for detection of histamine-producing bacteria

A method for detecting histamine-producing bacteria is described. The method is based on automated conductance measurements in a histidine-containing medium (HDB) incubated at 25°C, with a large and distinct increase in the conductance being indicative of the presence of histamine-producing bacteria. An initial low pH (5.5) is optimal for measuring the histidine decarboxylating activity of Morganella morganii, but clear results are obtained at higher pH values (6.0–7.0) as well. It is shown that the histidine decarboxylating activity of M. morganii is unaffected by the presence of non-histamine producing bacteria. Using this new method in the examination of mackerel spoiled at high temperatures, the results obtained indicated the presence of large amounts of histamine-producing organisms. These were subsequently isolated and identified as belonging to the family Enterobacteriaceae.     

A microwire sensor for rapid detection of Escherichia coli K-12 in fresh produce

A rapid immunofluorescence method for foodborne pathogens in food systems using microwire sensor coupled with high frequency alternating current was developed. The method was intended to enrich and quantify E. coli cells internalized in baby spinach leaves. The targeted bacterial cells in the sample solution were captured on microwires in a diameter of 25 μm, and were bound to fluorescein isothiocyanate (FITC) labeled polyclonal E. coli antibodies. Fluorescent images of the FITC antibodies were obtained using a fluorescence microscope equipped with a charge-coupled-device (CCD) camera, and the fluorescent intensity (FI) was quantified through image processing. The capture of E. coli K-12 in PBS buffer was optimized when the electric field was generated at the frequency of 3 MHz and 20 Vpp with bacterial concentration of 10⁷ CFU/mL. The detection limit of our sensing device was determined to be 10³ CFU/mL. Field emission scanning electron microscopy (FESEM) was used to validate and visualize E. coli cells captured on the tip surface. The sensitivity and specificity of the developed sensor has been successfully validated by testing E. coli internalized in baby spinach leaves. The immunofluorescence detection has been completed within 15 min. Moreover, it was found that the enrichment process of E. coli cells using the dielectrophoretic force was rarely affected by food particles, which proved the sensing selectivity. The developed sensor is expected to meet the steady demand for a simple, rapid, highly sensitive detection approach to quantify the targeted microbes in food systems.

Industrial Relevance

There has been an increase in the number of foodborne illnesses linked to the consumption of fresh and minimally processed fruits and vegetables. Some E.coli strains such as E.coli O 157:H7, can cause a variety of diseases, including diarrhea, urinary tract infections, respiratory diseases, meningitis and more. In general, consumers wash the fresh produces under cold running tap water to remove any lingering dirt on the surface of the produces before eating or preparing. However, how do the consumers know if there is any possible pathogen hiding inside of the fresh produce after rinsing? It was reported from many researchers that, the E.coli internalization, which may occur when fresh produces intake E. coli containing water or manure from the soil, would be a main cause of the foodborne illness outbreak. To ensure the safety of drinking water, E.coli concentration cannot be higher than 1 CFU/mL. How can we detect such a low level of E.coli in an easy yet efficient way? To our knowledge, none of the traditional detecting approaches such as cultural based method, polymerase chain reaction(PCR), surface plasmon resonance (SPR) biosensor, and Latex Agglutination, has performed perfectly. Hence, a rapid and accurate technique for detecting foodborne pathogens in fresh produce is urgently needed in order to secure the food safety.To overcome this issue, a simple detection method for foodborne pathogens in food systems using the microwire sensor coupled with high frequency alternative current was developed. The sensitivity and specificity of the developed sensor have been successfully validated by testing with E. coli internalized inside baby spinach leaves. It was found that spinach particles rarely affect the performance of our sensing device, which shows a promising prospect of its application in food industries.     

一株鞘氨醇单胞菌对复烤后烟叶多酚物质的降解作用

为开发促进烟叶品质改善的新型生物制剂,研究了鞘氨醇单胞菌Sphingomonas sp.XP对降解复烤后烟叶中多酚类的作用,优化了发酵条件,并对发酵后烟叶中主要多酚物质、常规化学成分和感官质量进行了评价。结果表明:①在菌液接种量15 wt%,相对湿度65%,30 ℃条件下发酵30 d,烟叶中多酚降解效果最佳;②菌株XP发酵处理对烟叶中绿原酸和芸香苷具有显著降解作用（P < 0.05）,而对于莨菪亭的降解没有显著作用（P > 0.05）;③经鞘氨醇单胞菌发酵处理后烟叶淀粉含量显著降低（P < 0.05）;总糖、还原糖含量显著增加（P < 0.05）;总氮、总植物碱和蛋白质含量没有显著变化（P > 0.05）;感官质量提高。      

银白色葡萄球菌实时荧光PCR快速检测方法的建立

本文旨在建立一种银白色葡萄球菌实时荧光PCR检测方法。根据对NCBI数据库中银白色葡萄球菌非核糖体多肽合成酶(NRPS)基因序列的比对分析,设计引物及探针,并基于所设计的引物及探针建立Taq-man 实时荧光PCR的检测方法。结果表明,本实验所建立的方法特异性较强,只能扩增出银白色葡萄球菌,而其他常见菌株的检测结果呈阴性;该方法的灵敏度为 10 pg/uL;在对实验室通过传统方法分离的金黄色葡萄球菌的检测中,发现有两个分离菌株呈阳性,其结果与普通PCR方法完全一致。     

快速检测技术在食源性沙门氏菌检测中的应用研究进展

沙门氏菌是一种广泛存在于自然界中的食源性致病菌,能导致人食物中毒,引发胃肠炎、败血症等疾病,严重时危及生命。建立快速、准确的沙门氏菌检测方法是预防和控制沙门氏菌疾病的关键,发展快速灵敏的检测方法对于保障食品质量与安全具有重要意义。本文通过对免疫学检测技术、分子生物学技术和生物传感器检测技术等在食源性沙门氏菌的检测方面的应用进行综合分析,阐明各种食源性沙门氏菌快速检测技术的原理、优缺点及研究进展,为优化食源性沙门氏菌快速检测方法提供参考。     

流式细胞术快速检测生乳中细菌总数

研究了应用流式细胞术检测生乳中细菌总数的方法。实验对生乳采用酶法消化蛋白质颗粒、高速离心脱脂的方式来消除大颗粒物质对流式检测的影响。并对菌体进行热处理,使其能被荧光性染料碘化丙锭染色。经过前处理的乳液进入流式细胞仪分析、计数。结果表明,流式细胞术与平皿菌落计数法呈正的直线相关(P<0.01),相关程度为显著相关(r=0.9360)。该方法极大的缩短了检测时间,检出限为104/mL。     

木薯叶粉中赭曲霉毒素A快速检测方法的建立

目的 开发快速检测方法检测木薯叶粉中的赭曲霉毒素A(Ochratoxins A, OTA)。方法 利用单克隆抗体和酶标抗原, 建立了竞争酶联免疫吸附测定法(enzyme-linked immunosorbent assay, ELISA)快速检测方法。用不同浓度的氯化钠甲醇水溶液、乙腈和磷酸盐缓冲液等作为样本提取液提取木薯叶粉中OTA, 用活性炭和稀释法减少样本基质干扰。结果 ELISA灵敏度为0.2 ng/mL, IC50为0.878 ng/mL。木薯叶粉用含氯化钠的70%甲醇水溶液提取, 离心后取上清5倍稀释, 样本回收率80%~121%, 检测限为5 μg/kg, 满足国标规定食品中OTA不超过5 μg/kg的要求, 检测时间为30 min, 一次可批量检测90个样本。结论 用本研究建立的ELISA方法检测木薯叶粉及其食品中的OTA含量结果准确、操作简单、成本低、时间短、重复性好, 适合基层批量筛查。