乳制品中金黄色葡萄球菌PCR快速检测方法的建立

为快速检测乳制品中的金黄色葡萄球菌,本研究将纳米氢氧化钛和纳米氢氧化锆固定在滤纸片上以去除PCR反应抑制因子,并以Nuc为靶基因,建立一种无需样品前增菌的PCR快速检测方法,同时确定该方法的特异性、灵敏度和实用性,并评估该方法所用试剂在冷冻保存条件下的稳定性与实用性。结果表明,该PCR快速检测方法在检测97株目标菌及83株非目标菌后未出现假阳性或假阴性结果;该方法无需增菌处理,可在4 h内完成检测,检测限为10¹ CFU·25 g^-1或10¹ CFU·25 mL^-1;该方法与传统检测方法在实际乳制品样品中金黄色葡萄球菌的检出率及活菌检测率均一致(符合率100%);此外,该检测方法所用试剂在-20℃冷冻条件下可稳定保存至少12个月。综上所述,本研究建立的PCR快速检测方法特异性强、灵敏度高,且实用性良好,能有效去除样品基质中的PCR反应抑制因子,为乳制品中金黄色葡萄球菌的快速检测提供了参考依据。     

金黄色葡萄球菌乳房炎奶牛血浆的比较蛋白质组研究

为了分析金黄色葡萄球菌(Staphylococcus aureus)自然感染引起的隐性乳房炎奶牛血浆蛋白的表达变化,经细菌培养分离鉴定了乳中金黄色葡萄球菌,选择其感染的奶牛.采用二维凝胶电泳技术分离了临床健康奶牛和乳房炎奶牛的血浆蛋白,考马斯亮蓝G-250染色后PDQuest 8.0软件检测差异表达蛋白点,高效液相色谱串联离子阱质谱鉴定.结果发现,金黄色葡萄球菌乳房炎奶牛血浆中有10个蛋白点的表达量发生改变,其中6个蛋白点经质谱鉴定为结合珠蛋白、转甲状腺素蛋白和α1酸性糖蛋白等4种蛋白.金黄色葡萄球菌感染可造成奶牛血浆结合珠蛋白、α1酸性糖蛋白和血清淀粉样蛋白A的表达量增加.ELISA法测定血浆结合珠蛋白的结果也发现,金黄色葡萄球菌感染奶牛血浆结合珠蛋白水平显著高于健康牛(P＜0.01).结果提示乳房炎奶牛血浆蛋白的变化可为揭示金黄色葡萄球菌感染乳腺炎症的应答提供依据.     

兔出血症病毒NJ85株衣壳蛋白基因在原核系统中的高效表达

从兔出血症病毒(Rabbit hemorrhagic disease virus，RHDV)中国早期流行株NJ85中成功地克隆出衣壳蛋白(VP60)基因。序列分析表明该基因长度为1740nt，编码579aa，与已报道的另2个RHDV中国毒株WX84和TP的VP60基因的同源性分别为92．7％和97．2％。构建了NJ85株VP60基因原核表达质粒pET—VP60，用SDS—PAGE分析，重组VP60蛋白的表达量占菌体总蛋白的40％，分子量约62kD，在菌体内以包涵体形式存在。免疫印迹实验表明，重组VP60蛋白与RHDV抗体反应形成1条带，证明它具有抗原性。     

苦瓜Mp-21.6蛋白的分离及生化特性

为研究苦瓜蛋白的有效分离技术与抑菌活性,本实验采用(NH4)2SO4分级分离油苦瓜(Momordic a charantiaL.)蛋白质,将SephadexG-100凝胶柱在DEAE-52阴离子交换柱色谱工作站上串联,分步聚集纯化出苦瓜蛋白Mp-21.6。SDS-PAGE凝胶电泳显示单一蛋白条带;经生化分析鉴定,Mp-21.6(Momordica protein of 21.6kD)蛋白的分子量为21.6kD,等电点pI=9.6;双向还原SDS-PAGE确定苦瓜蛋白Mp-21.6仅存在链内二硫健;Aacomplement软件的基于氨基酸组成比较发现,Mp-21.6类似于RNMC_MOMCH[Ribonuclease MC (RNase MC,EC=3.1.27.1)]。抑菌试验显示Mp-21.6对大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、沙门氏菌(Salmonellab sp)、枯草芽胞杆菌(Bacillus sabtilis)、啤酒酵母(Saccharomyces cerevisiae)和黄曲霉(Aspergillus flavus)产生明显...     

酸马奶酒中微生物的分离鉴定及抗菌特性的研究

从内蒙古锡盟不同地区采集15份酸马奶酒样品,经分离纯化、归属种鉴定,并单菌发酵液经调pH值、蒸发酒精、浓缩后,对李斯特杆菌、金黄色葡萄球菌、大肠杆菌进行抑菌研究。结果表明,酸马奶酒是由多种乳酸菌和酵母菌共同发酵的乳饮料。其中乳酸菌中有球菌5个属,杆菌6个属,酵母菌6个属。乳酸菌中有9株乳球菌和12株乳杆菌对李斯特杆菌有抑制作用,对大肠杆菌和金黄色葡萄球菌无抑制作用。酵母菌中有4株对大肠杆菌有抑菌作用,其中有2株同时对金黄色葡萄球菌有抑制作用,对李斯特杆菌无抑制作用     

金黄色葡萄球菌α溶血素的原核表达及其生物活性分析

摘要：α溶血素（α-Hemolysin, α-HL）是金黄色葡萄球菌的主要毒力因子之一，利用PCR技术从金黄色葡萄球菌临床分离株扩增出编码α溶血素的基因(α-HL)，经克隆筛选和测序鉴定后，构建成该基因的原核表达质粒pET32a+-α-HL。经诱导表达后，进行SDS-PAGE分析，结果表明:α-HL在大肠杆菌中已融合表达,融合蛋白的分子量为53kD。溶血实验证明该融合蛋白具有较强的溶血作用,其溶血效价达2.26×104 HU/mg,表明表达产物具有生物活性,这为进一步研究其致病与免疫机理、疫苗设计提供了条件。     

辐照对冷鲜熟制水饺微生物安全质量的影响

本试验主要研究了辐照对冷鲜熟制水饺中可能存在的致病微生物肠炎沙门氏菌、金黄色葡萄球菌和李斯特菌存活状况的影响。研究发现,肠炎沙门氏菌、金黄色葡萄球菌、李斯特菌的D10值分别为0.31、0.44和0.45kGy。随着辐照剂量的增大和贮存期的延长,不同致病菌数量变化趋势存在差异。4kGy辐照可保障冷鲜熟制水饺中3种致病菌达到不得检出的要求。     

百菌清降解菌BJQ2的分离、鉴定及影响因素研究

采用未有百菌清用药史的土壤定向培养49d,分离出50株百菌清耐受菌株,经初筛、复筛后得到1株降解能力最强的细菌BJQ2,经形态学、生理生化试验、16SrDNA序列同源性比对及系统发育树的分析,初步确定为阴沟肠杆菌（EnterobactercloacaeB9）,BJQ2可以以百菌清为唯一碳源于基础培养基中生长。BJQ2降解百菌清的最适宜条件为：培养温度30~35℃,初始pH值为7.0~8.0,百菌清的初始浓度为20mg·L-1,选用菌体母液（OD600=0.35）,以10%的接种量接种,6d后提取百菌清,利用气相色谱进行检测分析,计算后发现百菌清的降解率可以达到79.2%。     

4320菌体蛋白饲料中双菌作用机制的研究

该文报导新发现的利用粗淀粉料及渣粕类原料不灭菌固体发酵生产4320菌体蛋白饲料时所用的两个菌株—优良黑曲霉选拔株No.303和白地霉菌As.2.361间的关系是一种偏利生关系,在利用这种关系进行混菌固体发酵生产4320菌体蛋白饲料时,发现有类似杂交现象的“菌丛”,通过试验,证明“菌丛”是这两个菌间偏利生关系的特殊表现。通过对偏利生作用因子的研究表明,No.303的菌株分泌的柠檬酸、酶等都是直接或间接或是综合作用的偏利生因子。特别是No.303分泌的柠檬酸及由蛋白酶分解的谷氨酸等能明显刺激As.2.361生长。通过正交试验得知,它们是两个最大的作用因子     

抗鸡贫血病毒结构蛋白单克隆抗体制备及其对应抗原表位分析

以纯化的原核表达的鸡贫血病毒结构蛋白为抗原，免疫6-8w的雌性Balb/c鼠, 经过三次免疫后，取其脾细胞与SP2/0骨髓瘤细胞进行融合，以纯化的蛋白为抗原进行ELISA检测，将阳性细胞孔经过三次有限稀释，共获得6株能稳定分泌特异性抗体的阳性细胞株。间接免疫荧光试验证实6株单抗可以与鸡贫血病毒感染的MDCC-MSB1细胞发生特异性反应，Western blot结果显示单抗与杆状病毒表达的VP1重组蛋白可以发生特异性反应。利用单克隆抗体亚型鉴定试剂盒进行亚型鉴定，结果表明6株单抗均为IgG1亚型，且所有单抗的轻链均为κ链。用单克隆抗体对分段表达的VP1蛋白进行免疫印迹分析，初步鉴定了筛选出的单抗所对应的抗原表位，其中有四株单抗识别的表位位于VP1 218-274位氨基酸之间，另外两株单抗识别的表位分别位于275-301位、324-369位氨基酸之间。     

Screening of rhizobacteria isolated from maize (Zea mays L.) in Rio Grande do Sul State (South Brazil) and analysis of their potential to improve plant growth

Plant growth-promoting rhizobacteria (PGPR) are considered to have a beneficial effect on host plants and may facilitate plant growth by different mechanisms. In this work, the influence of different soil types on the bacterial diversity and the stimulatory effects of selected PGPR on two cultivars of maize were investigated. A set of 292 strains was isolated from the roots and rhizosphere soil of maize cultivated in five different areas of the Rio Grande do Sul State in Brazil. 16S rDNA-PCR-RFLP and 16S rDNA partial sequencing were used for identification, and the Shannon–Weaver index was used to evaluate bacterial diversity. We evaluated the ability of each isolate to produce indole acetic acid (IAA), siderophores and solubilize phosphates. On the basis of multiple PGP traits, six isolates were selected to test their potential as plant growth-promoting rhizobacteria on maize plants. In both the roots and the rhizospheric soil of maize, the dominant bacterial genera identified were Klebsiella and Burkholderia. IAA producers were distributed widely among isolates, regardless of the sampling site. Approximately 42% of the isolates exhibited at least two attributes, and 24% showed all three PGP traits. Three strains, identified as Achromobacter, Burkholderia, and Arthrobacter, were effective as PGPR in both of the cultivars evaluated.     

Bacterial numbers on penicylinders used in disinfectant testing: use of 24 hour adjusted broth cultures

The current AOAC use-dilution methods of disinfectant efficacy testing require the use of 48-54 h unadjusted broth cultures of Salmonella choleraesuis, Staphylococcus aureus, and Pseudomonas aeruginosa for the inoculation of stainless steel penicylinders. The use of unadjusted broth cultures contributes to noncomparable numbers of organisms on penicylinders among the test strains due to relative efficacy of bacterial attachment to penicylinders and to bacterial numbers in broth. To achieve comparable numbers of cells on the penicylinders among the 3 test strains, the cell densities of S. aureus and P. aeruginosa in broth culture were visually adjusted. Growth studies were conducted using S. choleraesuis and P. aeruginosa to determine the numbers of cells in broth at timed intervals and the corresponding numbers of cells attaching to the penicylinders. Results showed that the use of the 24 h broth cultures for all 3 test strains, with adjustment of S. aureus and P. aeruginosa broths, contributes to more comparable numbers of organisms attached to the penicylinders used in disinfectant testing.     

一株信鸽新城疫强毒的分离鉴定和分子特性分析

从发病鸽的体内分离到一株新城疫病毒（Newcastle Disease Virus，NDV）PB9601，经病毒蚀斑克隆纯化后动物回归试验可产生典型的NDV病变，其MDT、ICPI、IVPI分别为50.5、1.65和2.36，表明为NDV强毒。对其F和HN蛋白分别进行基因克隆测序，发现F蛋白多肽裂解位点的序列为111 KRQKRF 117，符合鸽源NDV强毒氨基酸基序。F基因分型及同源性比较显示：PB9601与YN-03、TW-98等鸽源毒株属于VI型，其F基因与基因Ⅱ型疫苗株LaSota氨基酸同源性为88.6%，与基因Ⅰ型疫苗株V4的氨基酸同源性为91.0%，与基因Ⅸ型传统强毒F48E9等的氨基酸同源性为90.6%；与国内鸽分离株GX-05、YN-03、TW-98的氨基酸同源性分别为92.1%、95.5、98.6%、与国外鸽分离株Capital3-97、Tigre6-99、1166-00、IT-227-82、2736-00的同源性分别为93.3%、91.9%、94.6%、95.1%、93.6%。HN基因氨基酸同源比较显示：PB9601与LaSota、F48E9、V4等的氨基酸同源性分别为87.2%、89.2%、87.6%；与国外鸽分离株1166-00、IT-227-82、2736-00的同源性为93.7%、95.5%、92.3%。由此看出PB9601与国内外分离株、LaSota、F48E9、V4等相比，具有明显的地域性和种属性。     

根际ACC脱氨酶活性细菌的分离及其促生作用研究

从不同来源的植物根际土壤中分离出能以1-氨基环丙烷-1-羧酸(ACC)为唯一氮源生长的细菌4株,经测定均具有较高的ACC脱氨酶活性,并进一步研究了菌株对植物的促生长作用。首先进行了种子根长试验,利用菌株菌悬液处理玉米和番茄种子,结果表明,经过细菌1101、2101、4201和GXGD002处理,种子的生根长度较对照组均有明显增长,玉米种子根长相对伸长率依次为78.73%、61.72%、47.37%、47.08%;番茄种子根长相对伸长率依次为61.19%、55.84%、54.34%、51.19%。其次进行了番茄穴盘栽培试验,分别在播种后40 d和55 d收苗,测定植株的株高、茎粗、根长等生长指标,结果表明,菌株4201和GXGD002具有显著促进生长作用,其处理组植株在株高、根长、根系活力等方面较对照组均有明显增强。     

Diversity and plant growth promoting evaluation abilities of bacteria isolated from sugarcane cultivated in the South of Brazil

Plant growth promoting (PGP) bacteria associated with sugarcane are a promising alternative for the expansion of this crop in Southern Brazil. In this study bacterial strains from different sugarcane fields were isolated to estimate their diversity, to evaluate some of their PGP activities and to use them as inoculant strains in field experiment. Samples of rhizospheric soil, roots, and stems of sugarcane were collected in six Rio Grande do Sul localities. The isolation of bacteria was made in three different N-free media. DNA from each isolate was subjected to nifH or 16S rDNA PCR-RFLP, and to the 16S rDNA partial sequencing. Five hundred and sixteen strains were isolated and several PGP characteristics were analyzed. Shannon index was used to evaluate the bacterial diversity. Indexes varying from 0.94 to 2.46 were obtained. Soil pH and clay were the characteristics most closely related to bacterial diversity. Achromobacter, Agrobacterium, Burkholderia, Gluconacetobacter, and Stenotrophomonas were the most abundant genera. Concerning the PGP activities, indolic compounds production was detected in 368 isolates; 138 isolates were able to solubilize phosphate; and 390 were siderophores producers. The inoculation of sugarcane with Gluconacetobacter diazotrophicus VI27 strain showed a significant increase in the number of sets germinated, in the amount of soluble solids, and in the yield of sugarcane juice compared with the control. As a conclusion, a diverse population of PGP bacteria was found in the sugarcane samples. These bacteria, especially G. diazotrophicus strain VI27, could be used as biofertilizers of sugarcane as well as other cereal crops under controlled conditions to avoid or reduce the use of standard N fertilizers.     

泰州市奶牛乳房炎调查及其病原菌的分离

奶牛乳房炎是奶牛场的常发病和难以防治的疾病,为进一步了解引起奶牛乳房炎的病原微生物,本研究通过对泰州市3个奶牛场572头泌乳牛的乳房炎发病率进行了调查并对乳房炎主要病原菌进行分离和鉴定。结果表明,奶牛临床型乳房炎的发病率为7.69%,隐性型乳房炎发病率为56.64%,乳区阳性率为33.8%,其中临床型：隐性=1：7.36。从65份确定患有乳房炎的奶样中共分离获得8种细菌、156株分离株,且引起乳房炎的主要病原菌是金黄色葡萄球菌和表皮葡萄球菌,其次是停乳链球菌、大肠杆菌、乳房链球菌和无乳链球菌,其检出率分别为42.31%、23.08%、8.97%、6.41%、5.13%和1.28%。该项调查结果初步明确了泰州市乳房炎发病情况,同时为进一步综合防治奶牛乳房炎和研制乳房炎治疗药物提供了科学依据。     

苹果树根际高效解钾菌的筛选及鉴定

为探明苹果树根际解钾菌的种群特征和解钾性能,从苹果树根际土壤中分离获得解钾能力较强的高效解钾菌。本研究以钾长石为唯一钾源,分离获得118株具有解钾活性的菌株,重复片段PCR基因指纹分析(Repetitive-element PCR,rep-PCR)聚为29个类群。利用火焰分光光度法测定菌株解钾能力,获得5株高效解钾菌,平均解钾活性达到41.47mg·L-1,其中K105的解钾能力最强,有效态钾增长23.09%。采用H2O2消煮后测得的K+浓度升高,有效态钾增长最高达31.22%。采用形态特征观察、生理生化特性检测和基于16S r RNA基因序列的系统发育分析对高效解钾菌株进行鉴定。结果表明:K1为不动杆菌属(Acinetobacter sp.)、K98、K105和K115为假单胞菌属(Pseudomonas sp.)、K168为芽孢杆菌属(Bacillus sp.)。研究结果可为开辟新型高效解钾菌,为连作土壤的改良和苹果专用微生物菌肥的开发提供依据。     

Isolation and characterization of phosphate solubilizing bacteria from the rhizosphere of crop plants of Korea

Whole-cell fatty acids methyl ester (FAME) profile and 16S rDNA sequence analysis were employed to isolate and identify the bacterial groups that actively solubilized phosphates in vitro from rhizosphere soil of various crops of Korea. Out of several hundred colonies that grew on Pikovskaya's medium 13 best isolates were selected based on the solubilization of insoluble phosphates in liquid culture and further characterized and identified. They were clustered under the genera Enterobacter, Pantoea and Klebsiella and the sequences of three representative strains were deposited in the GenBank nucleotide sequence data library under the accession numbers AY335552, AY335553, AY335554.     

Isolation and characterization of potassium solubilizing bacteria in some Iranian soils

The ability of a few soil bacteria to transform unavailable forms of potassium (K) to an available form is an important feature in plant growth-promoting bacteria for increasing plant yields of high-K-demand crops. In this research, isolation, screening, and characterization of six isolates of K solubilizing bacteria (KSB) from some Iranian soils were carried out. The ability of all isolates were tested in three treatments including acid-leached soil, biotite, and muscovite by analyzing the soluble K content after 5 days of incubation at 28 ± 2°C. Identification and phylogenetic analyses were also carried out by morphological, biochemical, and 16S rDNA analyses. Among the six efficient isolates, five isolates belonged to Bacillus megaterium (JK3, JK4, JK5, JK6, and JK7), while isolate JK2 belonged to Arthrobacter sp. The soluble K contents in all isolated-treatments were significantly (p < 0.01) higher than the contents in nonbacteria treatment. Herein, isolate JK2 had lower potential for K solubilization (910 mg kg^?1) compared with other isolates in acid-leached soils. The six bacterial strains showed higher solubilized K in biotite treatment than other two treatments. Overall, it can be concluded that the isolates belong to B. megaterium are the most efficient KSB under in vitro condition.     

Isolation and identification of locally isolated bacterial strains effective against whitefly Bemisia tabaci

Potent bacterial strains effective against the whitefly, Bemisia tabaci, nymphs (second instar), were isolated from tomato cultivated fields at Fayoum governorate, Giza, Egypt. Of 72 isolates, 12 with the most morphologically distinct-looking bacterial colonies were selected and named A1, A2, A3, A6, A7, A9, A12, A13, A107, B37, B45 and B100. All isolates were preliminarily identified as members of the genus Bacillus based on morphological, physiological and biochemical characteristics. When tested for their pathogenicity against Bemisia tabaci, the 12 isolates revealed varying efficiencies with isolates A1 and A9 being superior, exhibiting maximum mortality of 92.2 and 90.8% on day 10, respectively. Isolate A7 recorded the lowest percentage at 18.3%. Further genetic characterization of the 12 isolates was performed using inter simple sequence repeat (ISSR), randomly amplified polymorphic DNA (RAPD) and 16S rDNA gene sequencing analysis. RAPD and ISSR results confirmed each other. The combined ISSR and RAPD phylogenetic tree showed two major clusters. With 16S rRNA gene analysis, isolate A1 and A12 sequences recorded 100% identity with Bacillus thuringiensis, while isolates A7 and B100 showed 95.7% and 95.6% identity with Bacillus cereus and Bacillus sphaericus, respectively.