牛奶中磺胺类抗生素的快速检测分析

建立牛奶中磺胺类抗生素残留检测的放射免疫方法。通过对磺胺嘧啶、磺胺甲基嘧啶、磺胺二甲基嘧啶、磺胺甲氧哒嗪、磺胺甲基异恶唑、磺胺间甲氧嘧啶、磺胺间二甲氧嘧啶和磺胺喹恶啉8种磺胺类药物在空白牛奶样品中的加标试验,确定该方法的筛选水平。结果发现,采用放射免疫法检测牛奶中的磺胺类抗生素,筛选水平可达到10μg/L,变异系数在1.79%￣5.56%之间;对8种磺胺类药物的混合标准溶液进行检测,100%可以被检测出,检测限达5μg/L。放射免疫法检测牛奶样品中的磺胺类抗生素,方法简便、快速,灵敏度和精确度能够达到检测要求。     

赖氨酸磺胺嘧啶的合成及其在猪体内的药动学研究

将磺胺嘧啶钠与赖氨酸在稀酸环境中反应生成水溶性较好的赖氨酸磺胺嘧啶，并作成适宜注射使用的制剂；用赖氨酸啶和磺胺嘧啶钠做药代动力学比较试验，结果表明：在一定条件下，赖氨酸与磺胺嘧啶易结合，赖氨酸磺胺嘧啶的动力学规律较之磺胺嘧啶有较大变化，在猪体内具有一定的长效作用。     

恩诺沙星注射液中非法添加磺胺嘧啶HPLC-PDA检查法的建立

对恩诺沙星注射液中非法添加磺胺嘧啶的检测方法进行了研究.通过比较受试样品中未知峰与对照品色谱图保留时间的一致性和光谱指数图的匹配,建立了HPLC-PDA测定法,并进行相应的方法学研究.结果显示,磺胺嘧啶回收率为99.5％,RSD=0.4％;线性方程为y=873577x＋98154,R2 =1;检测限为5μg/mL,表明该检测方法准确可靠,可用于恩诺沙星注射液中非法添加磺胺嘧啶的检测.     

HPLC法同时测定禽肉中氯羟吡啶和多种磺胺类药物残留

建立了用于禽肉中氯羟吡啶和磺胺嘧啶、磺胺甲基嘧啶、磺胺二甲基嘧啶、磺胺甲氧嘧啶、磺胺-6-甲氧嘧啶、磺胺甲基异噁唑、磺胺二甲氧嘧啶、磺胺喹噁啉等残留测定的高效液相色谱法。样品以乙腈-氯仿提取,正己烷脱脂净化,以Symmetry C18 5μm(4.6 mm×150 mm)色谱柱进行分离,二极管阵列检测器检测,梯度洗脱测定氯羟吡啶和8种磺胺。回收率在71.7%~99.7%,相对标准偏差为1.5%~3.8%,最低检测限除磺胺二甲氧嘧啶、磺胺喹噁啉为0.02 mg/kg外,其余都可达到0.01 mg/kg。该方法已成功应用于禽类样品多兽药残留的检测。     

产蛋鸡慎用的几类药

1 磺胺类药物 常见的有磺胺嘧啶、磺胺噻唑、磺胺氯吡嗪、增效磺胺嘧啶等药物.产蛋鸡如果使用了上述药物,通过与碳酸酐酶结合,使其降低活性,从而使碳酸盐的形成和分泌减少,使鸡产软壳蛋和薄壳蛋.     

ELISA测定乳中磺胺甲基嘧啶残留

以牛血清白蛋白（ＢＳＡ）为载体,合成２种不同的物质的量的比值（约１∶１３和１∶４２）的磺胺甲基嘧啶抗原,并比较了两者的免疫原性;以卵白蛋白（ＯＡ）为载体,合成１种包被抗原;用辣根过氧化物酶标记羊抗兔ＩｇＧ,工作浓度１∶１０００;建立的乳中磺胺甲基嘧啶残留ＥＬＩＳＡ测定法,抗原亲和常数分别为１．２８×１０７（１∶４２）和３．２１×１０７（１∶１３）,磺胺甲基嘧啶检测质量浓度为５～２２０μｇ／Ｌ（１．８７×１０－８～８．２×１０－７ｍｏｌ／Ｌ）,检测限为２．４μｇ／Ｌ,回收率为８８％～１１８％,与磺胺二甲基嘧啶和磺胺噻唑交叉率分别为６．０％和０．８４％,与磺胺二甲氧嘧啶和磺胺脒无交叉反应     

利用表面等离子体共振生物传感器快速检测牛乳中的三聚氰胺

牛乳中残留的三聚氰胺对人类健康具有重要危害.利用表面等离子体共振(surface plasmon resonance,SPR)技术,建立一种快速定量检测牛乳中三聚氰胺的方法.以纳米金为媒介将三聚氰胺(melamine,MEL)连接到SPR传感芯片表面,优化最佳抗体用量和再生条件,测试芯片稳定性和方法的准确性.在无污染的牛乳中添加系列质量浓度的MEL,采用竞争抑制法建立抑制标准曲线,并对实际样品进行检测.结果表明:芯片重现性好,重复检测60次相对标准偏差为5.05％,方法检测限为3.24 ng/mL,远低于国际食品法典委员会和国家有关部门规定的残留量.该方法在30 min内完成样品的前处理和检测,适合牛乳生产现场快速定量检测.     

TTC法检测牛奶中的抗菌药物

针对用TTC法检测牛奶抗菌药物残留过程中因为在被检乳样中加入指示菌的数量不稳定而影响检测的问题,本研究增设了菌液稀释度试验以确定加入的菌液量。同时,采用TTC法对奶牛场常用的几种抗菌药物检测其在乳中的最低检测浓度。结果是青霉素3μg/kg,链霉素660μg/kg,庆大霉素620μg/kg,卡那霉素6200μg/kg,红霉素30μg/kg,环丙沙星500μg/kg,土霉素100μg/kg,金霉素100μg/kg,磺胺嘧啶100μg/kg。本研究用TTC法对青霉素、红霉素、土霉素、金霉素和磺胺嘧啶的检测灵敏度均达到农业部规定的牛乳中兽药残留最高限量要求。TTC法具有简便、价廉的优点,并且所检测的抗菌素范围较广,符合我国奶牛场中使用抗菌药物种类较多的现状。     

动物磺胺类药物中毒的病因、诊断与预防

磺胺素药物包括氨苯磺胺、磺胺噻唑、磺胺嘧啶、磺胺甲基嘧啶、磺胺二甲嘧啶、磺胺喹恶啉、磺胺异恶唑、磺胺甲基异恶唑、磺胺-6-甲氧嘧啶和磺胺二甲氧嘧啶等多种最常见的药物。其中动物肠道易吸收的磺胺类药物较易引起急性中毒。     

固相萃取-高效液相色谱法测定饲料中5种磺胺类药物的研究

建立了固相萃取-高效液相色谱(SPE-HPLC)测定饲料中磺胺嘧啶、磺胺间甲氧嘧啶、磺胺二甲基嘧啶、磺胺甲恶唑、磺胺喹恶啉5种磺胺类药物的方法。考察了碱性氧化铝小柱的最大饱和容量及洗脱条件,优化了色谱条件:流动相为乙腈和水(257∶5,v/v),流速为1.0 mL/min,检测波长为270 nm。在优化条件下,方法的最低定量限分别为磺胺嘧啶、磺胺间甲氧嘧啶5 mg/kg;磺胺二甲基嘧啶、磺胺甲恶唑、磺胺喹恶啉2 mg/kg。方法的线性范围在0.2～200μg/mL之间,线性相关系数高于0.9996。将该方法用于鸡配合饲料、猪配合饲料和浓缩饲料中磺胺类药物残留的测定,不同添加浓度的回收率均高于80%,相对标准偏差≤10%,能够满足实际样品的分析要求。     

复合酶制剂对瘤胃发酵及泌乳早期奶牛生产性能的影响

本试验旨在研究添加不同水平复合酶制剂对瘤胃发酵及奶牛生产性能的影响。试验一以奶牛全混合日粮作为底物进行体外瘤胃发酵试验,分为4组,即对照组不添加酶制剂,试验1、2和3组的酶制剂添加量分别为日粮浓度的0.10%、0.15%和0.20%,每组设9个重复。每个重复准确称取0.500 g底物,在体外发酵产气自动记录装置上发酵48 h,测定其发酵参数和营养物质降解率。结果表明:复合酶制剂显著提高发酵液中总挥发性脂肪酸和乙酸浓度(P<0.05);试验组中粗蛋白(P<0.05)和中性洗涤纤维(P<0.01)降解率显著高于对照组。试验二选择体重、胎次、泌乳天数和产奶量相近的泌乳早期荷斯坦奶牛36头,采用随机区组设计分为4组,即对照组和试验1、2和3组,对照组不添加酶制剂,试验1、2和3组分别添加0.10%、0.15%和0.20%的酶制剂,每组9个重复,试验期8周,测定产奶量和乳成分含量,计算3.5%乳脂校正乳。结果表明:复合酶制剂显著提高3.5%乳脂校正乳产量(P<0.05),0.10%、0.15%和0.20%组比对照组分别提高3.88、4.27和2.26 kg·d^-1。0.15%组的乳脂率显著高于对照组(P<0.05),比对照组高12.7%。结论添加复合酶制剂有利于瘤胃发酵和提高生产性能,且添加量为0.15%时效果较好。     

红花粕和水飞蓟粕对生长獭兔的营养价值评定

本试验旨在通过饲养试验和消化试验来评定红花粕和水飞蓟粕在生长獭兔上的营养价值。选取18只60日龄左右、平均体重(1.73±0.21)kg、健康状况良好的白色獭兔,随机分为3个组,每组6个重复,每个重复1只兔。各组分别饲喂基础饲粮、红花粕饲粮(85%基础饲粮+15%红花粕)和水飞蓟粕饲粮(85%基础饲粮+15%水飞蓟粕)。预试期和正试期各5 d。采用化学分析法测定红花粕和水飞蓟粕的总能(GE)及各营养物质含量,采用全收粪法测定生长獭兔对各营养物质的表观消化率。结果表明:红花粕和水飞蓟粕中的GE、干物质(DM)、粗蛋白质(CP)、粗纤维(CF)、中性洗涤纤维(NDF)、酸性洗涤纤维(ADF)、酸性洗涤木质素(ADL)、粗脂肪(EE)、粗灰分(Ash)、钙(Ca)、磷(P)以及无氮浸出物(NFE)的含量分别为:18.81 MJ/kg、93.76%、23.94%、14.95%、19.92%、11.99%、2.19%、1.64%、4.93%、0.37%、0.57%、49.32%与17.12 MJ/kg、91.94%、23.62%、16.21%、38.57%、22.73%、4.04%、2.07%、5.31%、0.29%、0.68%、42.09%。生长獭兔对红花粕和水飞蓟粕中GE、DM、CP、CF、NDF、ADF、EE、Ash、Ca、P和NFE的表观消化率分别为62.60%、61.72%、62.39%、15.68%、26.30%、14.75%、80.69%、38.35%、59.35%、31.98%、79.61%与63.13%、61.94%、68.01%、15.74%、27.64%、14.98%、79.90%、38.20%、60.44%、32.99%、79.81%。由此可见,生长獭兔对红花粕和水飞蓟粕中的不同营养成分的表观消化率存在一定差异,结合2种原料各营养物质含量总体分析,2种原料均可作为獭兔的蛋白质饲料资源应用,并且对生长獭兔的营养价值相近。     

绿豆蛋白乳饮料的制备及其稳定性

通过单因素试验确定复合稳定剂中黄原胶、羧甲基纤维素(carboxy methyl cellulose,CMC)、海藻酸丙二醇酯(propylene glycol alginate,PGA)的复配比例为1∶1.5∶6.25。在乳饮料生产中添加绿豆蛋白,以感官评分和产品的乳化稳定性为考核指标,通过正交试验确定产品的最佳配方,同时考察其热稳定性的变化。结果表明:最佳配方为乳粉8.0％、绿豆蛋白1.0％、绵白糖10.0％、复合稳定剂0.4％及柠檬酸0.3％(以上均为质量分数),按此配方生产的绿豆蛋白乳饮料乳香浓郁、口感细腻、甜度适中,乳化稳定性为98.60％。饮料中乳蛋白含量为1.95％,符合GB/T21732-2008《含乳饮料》中的标准要求。     

Limulus test--a rapid and simple method for the detection of endotoxins produced by gram-negative bacteria in mastitis milk

According to the present study the limulus amebocyte lysate test (LAL) seems to be a convenient test to detect endotoxin in milk from udder quarters with and without inflammation. The correlation between endotoxin concentration and the results from the bacteriological investigation of 79 milk samples was good (Table I). Determination of endotoxin in 20 milk samples from cases of acute clinical mastitis with high cell count and a negative bacteriological culture showed that all but one had an endotoxin concentration of greater than 1.0 ng/ml milk (Table II). By using a micromethod of the LAL it is possible to detect cases of mastitis caused by gram-negative bacteria about one hour after the sample has reached the laboratory. In a preliminary field study milk from 13 cases of acute clinical mastitis were tested by a modified limulus amebocyte lysate (LAL) test ("cowshed test"). A 100% correlation to bacteriological findings was observed (Table IV). By using the LAL test to detect mastitis cases caused by gram-negative bacteria great economic advantages and less risk for resistance problems can be achieved by using proper antibiotics. This is the fact in Sweden where the frequency of acute clinical mastitis caused by streptococci (100% of strains sensitive for penicillin) and Staphylococcus aureus (about 90% of strains sensitive for penicillin) is high (70-80%) and about 20% are caused by gram-negative bacteria, mostly E. coli.     

Enzyme immunoassay using mouse monoclonal anti-bovine antibodies for the detection of Brucella abortus antibodies in cow milk

Individual milk samples and artificially constructed tank milk samples from cows with naturally occurring brucellosis were examined by the enzyme-linked immunosorbent assay (ELISA) using sonicated B. abortus S-99 antigen, and mouse monoclonal anti-bovine IgM, IgA, and IgG1 conjugates. ELISA results were compared with the results of the milk ring test using either 1 ml milk (MRT-1) or 8 ml milk (MRT-8). The ELISA using mouse monoclonal anti-bovine IgG1 conjugate was sensitive and specific. In testing individual milk samples and constructed tank milk samples containing milk with low titers in the MRT-1 the ELISA was superior to the MRT-1, and MRT-8. In testing other milk samples, the ELISA was as sensitive or slightly less sensitive than the MRT-8. From a total of 5,910 milk samples collected from cows free from brucellosis, only 24 (0.4%) samples tested positive in the ELISA. All 500 tank milk samples collected from farms negative for brucellosis tested negative in the ELISA. We concluded that the ELISA is a good substitute for the MRT-1 to detect antibodies against Brucella in milk from individual cows. When tank milk is tested for antibodies against Brucella, however, both the MRT-8 and the ELISA should be used.     

高效液相色谱法测定发酵乳饮料中果糖、葡萄糖、蔗糖、麦芽糖及乳糖含量

建立基于高效液相色谱(high performance liquid chromatography,HPLC)法测定发酵乳饮料中果糖、葡萄糖、蔗糖、麦芽糖及乳糖含量的方法。用水作为提取剂,乙腈-水混合溶液作为流动相,采用糖柱进行分离,示差检测器检测。当果糖、葡萄糖、蔗糖、麦芽糖及乳糖的添加量在0.2％～10.0％时,加标回收率为92.6％～102.0％,相对标准偏差(n=6)小于3.00％,定量检出限为0.2％。该方法具有快速、准确、稳定性好、灵敏度高等优点,可用于发酵乳饮料中果糖、葡萄糖、蔗糖、麦芽糖及乳糖含量的检测。     

Serodiagnostic comparison between two methods, ELISA and surface plasmon resonance for the detection of antibodies of classical swine fever

A protein chip based on surface plasmon resonance (SPR) was developed to measure the antibody (Ab) titers of classical swine fever virus (CSFV) using the recombinant gp55 protein as an antigen. The diagnostic potential of this SPR assay for detecting the Ab titers to CSFV gp55 was compared that of the enzyme-linked immunosorbent assay (ELISA) using 170 serum samples from 14 pig farms. The SPR assay was highly specific and sensitive, and there were no cross-reactions detected. There was a strong positive correlation between the SPR and ELISA titers (n=170, r=0.869, p<0.01). Therefore, the SPR label-free method is a valuable tool in the serodiagnosis of CSFV infection and determining Ab titers after vaccination.     

Real-time polymerase chain reaction assays for the detection of members of the Mycoplasma mycoides cluster

AIM: To develop real-time PCR assays for the detection and differentiation of members of the Mycoplasma mycoides cluster. METHODS: Five real-time PCR assays were designed to allow differentiation of members of the M. mycoides cluster: an assay for detection of the M. mycoides subspecies, viz M. mycoides subsp mycoides large colony (MmmLC), M. mycoides subsp capri (Mmc), and M. mycoides subsp mycoides small colony (MmmSC); one for the detection of the M. capricolum subspecies, viz M. capricolum subsp capricolum (Mcc), M. capricolum subsp capripneumoniae (Mccp), and Mycoplasma sp bovine group 7 (BG7); and three for the specific detection of MmmSC, Mccp, and BG7. A panel of 74 Mycoplasma isolates from various geographical origins and a panel of 21 other bacterial isolates were used to evaluate the sensitivity and specificity of the assays. RESULTS: The assays displayed 100% analytical sensitivity in detecting all target Mycoplasma isolates. The analytical detection limit for the assays to detect the M. mycoides subspecies, M. capricolum subspecies, and MmmSC was determined to be 100 fg of genomic DNA, while the Mccp and BG7 assays had a detection limit of 100 fg and 10 fg of genomic DNA, respectively. The M. mycoides subspecies assay had a detection limit of 10(3) (SD 10(2)) cfu/ml milk, 10(4) (SD 10(4)) cfu per swab, and 10(3) (SD 10(3)) cfu/g lung in inoculated samples. The assays displayed 100% specificity when applied to non-target bacterial isolates and to 110 culture-negative milk samples. CONCLUSIONS: The assays were highly sensitive and specific, and provide accurate detection and differentiation of the members of the M. mycoides cluster.     

快速检测发酵乳中葡萄糖杆菌的赖解旋酶恒温基因扩增方法的建立

建立赖解旋酶恒温基因扩增（helicase-dependent isothermal DNA amplification，HDA）快速检测发酵乳中葡萄糖杆菌的方法。选择葡萄糖杆菌ITS基因序列（基因号为KF896260.1）为目的基因，设计特异性引物，并优化反应体系中UvrD解旋酶和T4 gp32的添加量，建立最优反应体系。采用建立的HDA体系对多种菌株进行扩增和电泳检测，验证方法特异性，通过HDA法直接检测发酵乳中的葡萄糖杆菌，并确定其检出限。结果表明：采用HDA法检测发酵乳中葡萄糖杆菌，反应体系中UvrD解旋酶和T4 gp32的适宜添加量分别为0.10 μg和5.0 μg，扩增产物与设计序列长度（309 bp）一致，特异性良好，检出限为4.3×101 CFU/g。该方法用于检测发酵乳中葡萄糖杆菌的灵敏度高、耗时短。