乳品中β-内酰胺类抗生素快速检测试纸条研制

通过β-内酰胺类抗生素-载体蛋白偶联物的合成,单克隆抗体的制备与纯化,胶体金标记物的制备,β-内酰胺类抗生素单克隆抗体-胶体金标记物的制备并冻干到微孔试剂,样品吸收垫和反应膜的制备等研究过程,研制出乳制品中β-内酰胺类抗生素的快速检测试纸条。检测限分别为:青霉素G2μg/L、氨苄青霉素4μg/L、阿莫西林5μg/L、苯唑青霉素/邻青霉素/双青霉素均为6μg/L、头孢洛宁10μg/L、萘夫西林20μg/L、头孢喹肟20μg/L、头孢曲松25μg/L、头孢哌酮50μg/L、头孢噻呋90μg/L;本试纸条特异性好、假阳性率不高于3%、假阴性率为0,检测时间不超过15min,检测限量达到了我国和欧盟的要求,适用于乳品流通环节乳品中β-内酰胺类抗生素残留的检测。     

嗜热脂肪芽孢杆菌试管法检测牛乳中β-内酰胺类抗生素残留

采用嗜热脂肪芽孢杆菌试管法对牛乳中6种最为常见的β-内酰胺类抗生素残留进行检测,指出嗜热脂肪芽孢杆菌试管法为半定量检测法,对牛乳中β-内酰胺类抗生素残留具有较好的灵敏性.牛乳中青霉素G、氨苄青霉素、羟氨苄青霉素的检测限为6μg/kg,头孢氨苄的检测限为150μg/kg,苯唑青霉素的检测限为15μg/kg、头孢匹林的检测限为30μg/kg.在检测时间、操作难易程度上较BSDA法具有一定的优越性,可应用于牛乳中β-内酰胺类抗生素残留的检测.     

胶体金试纸条快速检测动物源性食品中头孢氨苄残留

头孢菌素类抗生素属于第二种类型的β-内酰胺类抗生素,其在动物源性食品中的残留问题引起普遍关注。为保障食品安全,实现对食品中头孢菌素类抗生素残留的快速检测,该研究利用金纳米颗粒标记头孢氨苄(cefalexin,CEX)兔多克隆抗体作为可视化信号探针,基于硝酸纤维素膜研制一种能够检测头孢菌素类抗生素的免疫层析检测试纸。试纸条以CEX包被原为检测线,以羊抗兔二抗为质控线,对试验条件进行优化后,所构建的试纸条在缓冲液体系中对CEX、头孢拉定(cefradine,RAD)和头孢羟氨苄(cefadroxil,CFR)的检测限分别可达到20、25、25μg/L。为验证方法的实用性,将一定浓度的CEX添加至虾肉、牛奶、牛肉、猪肉、生猪肝和熟猪肝6种常见的动物源性食品中进行添加回收测定。结果显示:经过简单的样品处理,所构建的试纸条在10 min内即可实现对目标物的快速可视化检测。该方法操作简便、快速,适用于大批量动物性食品中多种头孢菌素类残留的高通量筛查。     

液相色谱-串联质谱法测定禽蛋中β-内酰胺类药物残留

目的 建立了禽蛋中普鲁卡因青霉素、青霉素V、青霉素G、氨苄西林、氯唑西林、阿莫西林、头孢氨苄、头孢噻呋、头孢喹肟药物残留检测的液相色谱-串联质谱方法。方法 禽蛋用80%乙腈水溶液提取,PRiME HLB固相萃取小柱净化,用液相色谱-串联质谱测定,青霉素G-D7内标法定量。结果 在0.5-20μg/L的基质匹配标准溶液内9种β－内酰胺类药物均呈良好线性关系,相关系数均大于0.994。样品中9种β－内酰类药物的定量限为1μg/kg,在1μg/kg～10μg/kg的添加浓度范围内平均回收率为73%～96%,相对标准偏差(RSD)小于10%。本方法方便快捷准确,适于禽蛋中的9种β－内酰胺类药物残留检测。     

牛奶中泰乐菌素和替米考星的胶体金免疫层析法测定

建立一种快速、简便、同时针对牛奶中泰乐菌素和替米考星残留的检测方法.应用竞争抑制免疫层析原理,研制出泰乐菌素和替米考星胶体金试纸条,并对其性能进行了鉴定.结果表明,该试纸条对牛奶中泰乐菌素和替米考星的检测限分别为25 μg/L和50 μg/L.假阳性率低于5％,假阴性率为0,检测时间10 min,与其他大环内酯类抗生素无交叉反应.该方法操作简便、灵敏度高、特异性强,适用于乳品流通环节中泰乐菌素和替米考星残留的快速检测.     

应用酶联免疫法快速检测乳品中β-内酰胺类抗生素残留

建立乳品中β-内酰胺类抗生素残留酶联免疫快速检测方法。应用氨苄青霉素抗体,通过人工制备了氨苄青霉素(Amp)和辣根过氧化物酶(HRP)的结合物Amp-HRP,建立直接ELisa竞争法检测β-内酰胺类抗生素,确定了各种β-内酰胺类抗生素的检出限,并对检测条件进行了优化。检测了已知阴性和阳性的生鲜牛乳样品,结果表明对于阴性和阳性牛奶样品的检测未出现假阳性或者假阴性结果。该方法检测结果准确可靠,可广泛应用于检测乳品中β-内酰胺类抗生素残留的检测。     

LC-MS/MS测定动物源食品中10种β-内酰胺类抗生素残留

采用高效液相色谱-串联质谱法(LC-MS/MS)测定动物源食品中青霉素G、青霉素V、阿莫西林、氨苄西林、苯唑青霉素、萘夫西林、双氯西林、哌拉西林、头孢氨苄、头孢唑林等10种β-内酰胺类抗生素残留量,以青霉素G-D7为内标。样品用磷酸盐缓冲溶液提取(鸡蛋和牛奶样品提取后用乙酸锌和亚铁氰化钾沉淀蛋白),然后提取液用正己烷除脂,经PEP-2固相萃取柱净化,采用高效液相色谱-串联质谱测定,内标法定量。在相应浓度范围内,10种β-内酰胺类抗生素线性系数R2均大于0.99,检测限为0.25~2.0μg/L。在低、中、高3个浓度水平下,10种β-内酰胺类抗生素在不同基质中的回收率均在80%~120%之间,相对标准偏差小于10%。该方法可以满足不同基质的动物源食品中上述10种β-内酰胺类抗生素的测定需求。     

胶体金免疫层析试纸条在猪尿β-兴奋剂多残留检测中的应用

本研究采用柠檬酸三钠还原法制备胶体金溶液,并对β-兴奋剂单克隆抗体进行标记。通过棋盘滴定法和单因素实验确定最佳的实验参数,研制出能同时检测多种β-兴奋剂的胶体金免疫层析试纸条。结果表明,本方法对克伦特罗的灵敏度最高,检测线性范围(IC20~IC80)为0.31~4.52μg/L,IC50为1.18μg/L,检出限(LOD,IC10)为0.14μg/L,猪尿样品无需处理,直接检测,单个样品检测时间只需8 min;与沙丁胺醇、马布特罗、溴布特罗、氯丙那林、西马特罗等β-兴奋剂类药物都有交叉,能实现β-兴奋剂的多残留检测。对阴性猪尿的加标回收实验中,试纸条批内实验回收率在83.6%~102.2%之间,试纸条批间实验回收率在84.2%~101.5%之间,且相对标准偏差均在10%以内,与ELISA、HPLC/MS法的对比实验表明,该试纸条能应用于猪尿中β-兴奋剂多残留的定量检测。     

青霉素结合蛋白的重组表达及其在β-内酰胺类抗生素检测中的应用

目的 获得重组表达纯化的青霉素结合蛋白(penicillin binding proteins, PBPs),研究其在β-内酰胺类抗生素检测中的应用。方法 以来源于肺炎链球菌Streptococcus pneumoniae R6的PBP2x蛋白为对照,以PBP1a蛋白作为目标受体,研究其与β-内酰胺类抗生素的亲和作用。结果 经纯化后的重组蛋白PBP2x和PBP1a检测头孢噻呋、青霉素G、氨苄青霉素、喷沙西林、阿莫西林的半抑制浓度(inhibitory concentration, IC50)都在7.02 ng/mL以下,明确了PBP1a和PBP2x具有β-内酰胺类抗生素结合能力。结论 该研究为开发新的基于PBPs蛋白检测β-内酰胺类抗生素的免疫学方法奠定了基础。     

液相色谱-串联质谱法测定水产品中β-内酰胺类抗生素的残留

建立了同时测定水产品中,6种β-内酰胺类抗生素药物残留的高效液相色谱-串联质谱(HPLC-MS/MS)检测方法.样品经乙腈-水(15∶2,v∶v)混合液提取,HLB固相萃取柱净化.以乙腈-0.01mol/L醋酸铵溶液(pH4.5)作为流动相,C18色谱柱进行分离,电喷雾正离子多反应监测模式进行检测.方法检测限为0.2～2.0μg/kg,加标回收率为80.8％～92.9％,相对标准偏差为2.6％～9.1％(n=9).该方法准确、灵敏、简便,适用于水产品中6种β-内酰胺类抗生素残留的测定.     

基于信号增敏型试纸条三聚氰胺超灵敏检测方法

通过胶体金免疫层析技术建立一种能便捷、快速、特异检测食品中三聚氰胺的超灵敏快速检测技术。在传 统的基于竞争法层析试纸条的基础上增加一个增强垫组装成增敏型试纸条,在识别垫和增强垫上分别使用25 nm和 15 nm胶体金,使其在传统型试纸条的基础上对三聚氰胺的检测灵敏度大大提高。结果表明：增敏后的试纸条可在 10 min内实现食品中三聚氰胺的快速检测;实验条件下检测灵敏度为0.5 μg/L,是传统检测灵敏度10 μg/L的20 倍; 实际样品中检测范围为0.1～5 μg/L,检测灵敏度为1 μg/L,相比于传统检测灵敏度提高了5 倍。     

基于胶体金免疫层析技术的赤霉素快筛试纸条的研制

研制快速检测豆芽中赤霉素的快筛试纸条。采用免疫层析技术,以胶体金为标记物制备赤霉素单克隆抗体-胶体金偶联物,以竞争抑制模式制备赤霉素胶体金免疫层析试纸条。该试纸条对豆芽中赤霉素的检测限为100 μg/kg,灵敏度为98%,特异性为97%,假阴性率为2%,假阳性率为3%。该方法操作简便,具有较高的灵敏度和特异性,可广泛应用于豆芽中赤霉素残留的现场筛查和检测。     

超高效液相色谱-串联质谱法同时测定动物源性食品中9种多肽类抗生素残留

建立了动物组织中9种多肽类抗生素残留的固相萃取-超高效液相色谱串联质谱（UPLC-MS/MS）检测方法。样品经甲醇-0.1 mol/L盐酸（7:3,v/v）混合溶剂提取,酸性氧化铝除杂、正己烷去除油脂,亲水-亲脂平衡（hydrophilic-lipophilic balance,HLB）固相萃取柱净化,以0.2%的甲酸水溶液和乙腈为流动相,经过C₈色谱柱（100A, 150 mm×2 mm, 3 μm）梯度洗脱分离,使用电喷雾正离子化和多反应监测模式检测。结果表明,万古霉素和去甲万古霉素、恩拉霉素A、恩拉霉素B、太古霉素在2～200 μg/L范围内,粘杆菌素A、粘杆菌素B、杆菌肽A在5～500 μg/L范围内,维吉尼霉素M1在0.1～20 μg/L范围内,各化合物定量离子的峰面积和样品质量浓度之间呈现良好的线性关系（R2>0.99）,方法的定量限（S/N=10）为1～20 μg/kg,检出限为0.3~6 μg/kg;以鸡肉、猪肉、猪肝、猪肾为基质,在加标水平为1～200 μg/kg时,各个化合物的平均加标回收率为74.2%～96.3%,相对标准偏差为4.3%～14.6%。该方法简便、灵敏、准确,可用于动物组织中多肽类抗生素残留的同时测定。     

鸡蛋膜荧光传感器光谱信息表征及三聚氰胺检测方法的构建

为了实现奶制品中三聚氰胺的快速准确检测,基于溶液中三聚氰胺能使荧光素的荧光强度在一定浓度范围内线性增强的特点,利用天然食品分析物鸡蛋膜作为生物膜基质,建立一种固定荧光素到鸡蛋膜上的生物传感器并用于三聚氰胺检测的方法,优化传感器体系中的荧光素浓度及戊二醛质量分数。该鸡蛋膜传感器的线性工作范围为1～500μg/L,检出限为0.47μg/L。相比溶液中三聚氰胺的检测方法鸡蛋膜固相传感器检测方法特异性强,稳定性好,具有更高的选择性和低检出限。该种鸡蛋膜荧光传感器能成功应用于市售奶制品中三聚氰胺的检测。     

Effect of heat treatments on stability of β-lactams in milk

The presence of residues of antimicrobial substances in milk may have serious toxicological and technical consequences. To date, few studies have been done to evaluate the effect of heat treatments on β-lactam residues in milk. However, the few studies that have been conducted estimate losses of antimicrobial activity under different combinations of temperature and time using microbiological methods. The aims of this study were to calculate the kinetic parameters for the degradation of β-lactam antibiotics in milk and to develop prediction models to estimate the concentration losses of these compounds in conventional dairy heat treatments. To do so, we employed a quantitative HPLC method to calculate losses in concentrations of 10 β-lactam antibiotics in milk with different combinations of temperature and time. Increasing the temperature from 60°C to 100°C decreased the half-life of amoxicillin (372 to 50 min), ampicillin (741 to 26 min), cloxacillin (367 to 46 min), and penicillin G (382 to 43 min). These increases in temperature caused further degradation in cephalosporins, which was accompanied by a decrease in half-life times to reach very low values; for instance, 4, 5, and 6 min for cefoperazone, cephurexime, and cephapirin, respectively. Kinetic equations were applied to different heat treatments used in dairy processing. Heat treatments at high temperatures and long times (e.g., 120°C for 20 min) led to a further degradation of β-lactam antibiotics with percentages close to 100% for cefoperazone and cefuroxime. In contrast, when milk was subjected to heat treatments at lower temperatures and times (e.g., 72°C for 15 s), the degradation of β-lactam in milk did not exceed 1% for the 10 antibiotics tested.     

In-house validation of PremiTest, a microbiological screening test with solvent extraction, for the detection of antimicrobial residues in poultry muscles

PremiTest, a microbial inhibition test for the screening of antimicrobial residues, was validated according to the criteria established by Decision 2002/657/EC. Sensitivity, detection capability (CCβ), specificity, selectivity, robustness and applicability were evaluated. The methodology involves the technique of solvent extraction, which increases the detection capability of the test for a wider range of antibiotics. The following CCβ values in poultry muscle were found: penicillin G ≤ 12.5 μg kg(-1), total sulfonamides ≤ 75 μg kg(-1), erythromycin 75 μg kg(-1) and lincomycin 50 μg kg(-1). The detection capability of chlortetracycline was equal to its maximum residue limit (100 μg kg(-1)) and the method did not detect gentamicin (1000 μg kg(-1)), for which no MRL is established in poultry muscle. Specificity evaluated in relation to different analytes and matrices did not detect any interferences in the tests results; whilst the robustness showed that the pH neutralisation point of the extract affects the analytical results and the kits' performance. Only the screening of tetracyclines requires the analysis of extracts without pH neutralisation. The results of the validation process showed that this method is acceptable for screening β-lactam, sulfonamide and macrolide antimicrobial groups in the National Residues and Contaminants Control Programme (PNCRC), and that for this it is fit for purpose.     

荧光免疫层析法快速测定牛奶中头孢氨苄残留量

采用荧光免疫层析法结合现场检测仪建立牛奶中头孢氨苄残留的快速定量检测方法。方法 利用反向微乳技术合成稀土铕荧光纳米颗粒,与头孢氨苄单克隆抗体结合制备荧光检测探针,以头孢氨苄卵清蛋白全抗原和羊抗小鼠抗体分别作为检测线和质控线制备免疫层析试纸条,结合现场检测仪建立牛奶中头孢氨苄残留的快速定量检测方法。结果 试验结果表明,该方法对头孢氨苄的检测限为0.16 ng/ml,半数抑制浓度(IC₅₀)为0.6 ng/ml,线性范围为0.16～5 ng/ml,标准添加回收率为100%～115%之间,与头孢菌素类及青霉素等其他12种抗生素的交叉反应率均＜0.01%,与ELISA方法比较,测定结果相关性良好。结论 本试验所建立的荧光免疫层析法快速检测牛奶中头孢氨苄残留,具有简便、快速、灵敏、直观的优点,可用于抗生素残留的筛查,极具推广和应用价值。     

Rapid on‐site determination of melamine in raw milk by an immunochromatographic strip

A rapid and simple method was established based on gold nanoparticle‐labelled monoclonal antibody probes for the detection of melamine pollution in raw milk. The conditions for conjugation between the antibody and gold nanoparticles were optimised (pH 8.0, antibody concentration 5 μg mL^?1). It gives a single proportional to melamine concentration with a performance time of only 3 min. A practical calibration curve was established with a reader system with limit of detection calculated as 4.47 and 8.34 μg L^?1 with naked eyes. Three structural analogues, atrazine, desethyl‐desisopropyl‐atrazine and cyromazine, were used to test the specificity of the immunochromatographic strip, and small colour changes on the strip test line were found even at the 500 ng mL^?1 spiked level. Fifty raw milk samples were detected with this strip method, and the resulting data coincided well with results from gas chromatography tandem mass spectrometry. The above‐mentioned results showed that this test strip can be used for melamine screening in the daily monitoring of milk.     

Development of an Immunochromatographic Test Strip for Rapid Simultaneous Detection of Enrofloxacin and Ofloxacin in Tissue of Chicken Muscle and Pork

ENR and OFL are the most consumed quinolones on livestock in China. In this work, we developed a rapid immunochromatographic lateral flow test strip for simultaneous detection of the residues of enrofloxacin and ofloxacin in chicken muscle and pork. We screened an anti-ENR and OFL monoclonal antibody. The IC₅₀ of anti-ENR and anti-OFL were 6.67 and 7.13 ng/ml, respectively. The present immunochromatographic lateral flow test strip is a one-step assay and required much less professional personnel and experimental instruments. In the present study, the decision limit (CCα) of the test strip was calculated to be 0.089 ng/mL and detection capability (CCβ) was 0.217 ng/mL with the scanner. The limit of detection was estimated to be 10 ng/mL. According to parallel HPLC analysis with 47 blind samples, coincidence rate is 100 % when the contents of ENR and OFL were more than 10 ng/mL. Results indicated that the strip test we developed had good reliability, and the strip test gave neither false positive nor false negative results. It will provide results within 20 min without special equipment. Therefore, the test strip is very useful as a screening method for semi-quantitative or qualitative detection of enrofloxacin and ofloxacin in chicken muscle and pork.