HPLC法检测发酵玉米粉中的黄曲霉霉素B1、B2、G1、G2

发酵玉米粉用甲醇水(8+2,v+v)提取,经免疫亲合柱(IAC)分离纯化、在线光化学衍生器柱后衍生化后,反相HPLC荧光检测器测定AFT含量.4种毒素在0.5ng/g和2ng/g两个水平下的样品加标回收率平均值分别为B188.0%和93.6%、B285.2%和85.3%、G192.4%和91.5%、G289.6%和85.6%;5次测定的精密度SD值分别为B12.5和6.3、B23.5和6.0、G12.8和9.0、G22.3和6.9;4种毒素的方法检出限均达到0.05ng/g;样品中4种毒素测定值分别为B1(102±5.4);B2(47±9.2);G1(63±7.4);G2(30±4.9).     

HPLC法检测发酵玉米粉中的黄曲霉毒素B_1、B_2、G_1、G_2

发酵玉米粉用甲醇∶水(8+2,v+v)提取,经免疫亲合柱(IAC)分离纯化、在线光化学衍生器柱后衍生化后,反相HPLC荧光检测器测定AFT含量。4种毒素在0.5ng/g和2ng/g两个水平下的样品加标回收率平均值分别为B188.0%和93.6%、B285.2%和85.3%、G192.4%和91.5%、G289.6%和85.6%;5次测定的精密度SD值分别为:B12.5和6.3、B23.5和6.0、G12.8和9.0、G22.3和6.9;4种毒素的方法检出限均达到0.05ng/g;样品中4种毒素测定值分别为B1(102±5.4);B2(47±9.2);G1(63±7.4);G2(30±4.9)。     

用MycoSepTM净化柱和高效液相色谱法对谷物中黄曲霉毒素B1、G1、B2、G2的测定

用一种快速方便的MycoSep^TM净化柱和高效液相色谱法对谷物中黄曲霉毒素B1、G1、B2、G2进行测定。样品经乙腈-水（体积比84：16）提取，提取液通过MycoSep^TM净化柱净化、浓缩，三氟乙酸（TFA）柱前衍生，C18色谱柱分离，荧光检测器检测，外标法定量。对添加两个浓度黄曲霉毒素的玉米样品进行加标回收实验，4种毒素的回收率均在90．44％-101．23％之间，B1、G1、B2、G2的最低检测限分别为2．0、1．0、0．5、0．5ng／kg。实验结果表明，此法不仅定量准确，精密度高而且操作极为方便。     

免疫亲和-光化学柱后衍生高效液相色谱荧光法测定花生粕中黄曲霉毒素的研究

研究建立了光化学柱后衍生-高效液相色谱(HPLC)-荧光检测器测定花生粕中黄曲霉毒素B1、B2、G1和G2的方法。甲醇/水提取样品中黄曲霉毒素B1、B2、G1和G2,Afla-P黄曲霉毒素免疫亲和柱净化,经高效液相色谱分离后,由光化学柱后衍生,通过荧光检测器测定。结果表明:在优化条件下,黄曲霉毒素B1、B2、G1和G2的检出限分别为1.0、0.3、1.0μg/kg和0.3μg/kg,回收率为67.0%～117%,相对标准偏差(RSD)低于18.2%。该方法简便快速、灵敏度高、重现性好,满足饲料用花生粕中黄曲霉毒素检测的需要。     

免疫亲和柱净化-高效液相色谱法测定饲料中的黄曲霉毒素

建立免疫亲和柱净化-高效液相色谱法测定饲料中的黄曲霉毒素(AF)B1、B2、G1和G2。该方法样品用甲醇-水提取,稀释后经免疫亲和色谱柱纯化,应用高效液相色谱法检测。以Cloversil C18柱为分离色谱柱,甲醇-水(45∶55,体积/体积)溶液为流动相梯度洗脱,流速0.8 m L/min,柱温30℃,进样量20μL,荧光检测器检测,激发波长360 nm,发射波长440 nm。试验结果表明:空白样品分别按照1、10、20和50μg/kg添加黄曲霉毒素,平均回收率在85.9%~113.2%,精密度10%,4种毒素在标准溶液为0、1、5、10和50 ng/m L的条件下呈良好的线性关系,相关系数分别为0.999 8、0.999 9、1.000 0和0.9998,根据3倍信噪比的峰响应值,确定黄曲霉毒素B1、B2、G1和G2的检测灵敏度分别为0.2、0.2、0.3和0.3μg/kg。该方法简单快速、灵敏度高和溶剂用量少且环保,适用于饲料中的黄曲霉毒素总量的测定。     

光化学衍生法测定玉米及玉米制品中的黄曲霉毒素B1

为研究玉米及玉米制品被黄曲霉毒素B1污染的情况,确保市场上玉米及玉米制品的食用安全,本文旨在建立免疫亲和柱净化-高效液相色谱-柱后光化学衍生-荧光法测定玉米和玉米制品中黄曲霉毒素B1含量的方法。将玉米和玉米制品试样粉碎后,试样粉末在甲醇-水溶液（体积比为55:45）条件下提取,经免疫亲和柱富集和净化后,以甲醇-水溶液（体积比45:55）为流动相,采用XDB-C18色谱柱（5 μm×4.6 mm×250 mm）,由光化学柱后衍生,荧光检测器检测（Ex：360 nm,Em：440 nm）。结果表明：黄曲霉毒素B1的最低检出限和定量检出限分别为0.009 μg/kg和0.03 μg/kg,标准曲线的线性范围为0.1 ~ 0.5 mg/L（R=0.9990）,2个不同水平的加标平均回收率为88.4% ～ 90.1%,RSD值为0.47% ～ 1.07%。该方法具有操作简便、灵敏度高、重现性佳等特点,适用于玉米和玉米制品中黄曲霉毒素B1的检测。 [关键词] 免疫亲和柱|高效液相色谱|柱后光化学衍生|黄曲霉毒素B1     

高效液相色谱法测定饲料中黄曲霉毒素含量的研究

旨在建立检测饲料中黄曲霉毒素B1、B2、G1、G2含量的高效液相色谱分析方法。样品用甲醇溶液提取,利用免疫亲和柱净化,以C18反相色谱柱分离,光化学衍生器衍生后应用荧光检测器检测。结果表明:4种黄曲霉毒素在0.5~20ng/mL范围内呈现良好的线性关系,回收率为71.6%~89.0%。采用该方法检测5种饲料样品中的黄曲霉毒素总量,检出率范围在74.0%~86.7%,检出值的范围为0.5~4.3μg/kg。建立的方法精密度高、重复性好,为监测饲料中黄曲霉毒素含量水平提供了有效的分析方法。     

饲料中T-2毒素的高效液相色谱-荧光法检测研究

以甲醇和水(体积分数8∶2,v/v)为提取溶剂,采用硅镁吸附剂层析柱结合T-2毒素免疫亲和柱两阶段净化处理、柱前衍生,建立了饲料中T-2毒素的高效液相色谱法结合荧光检测方法。衍生液选用2-萘甲酰氯(2-NC),4-甲基氨基吡啶(DMAP)作为反应促进剂。确立的色谱检测条件为:流动相是乙腈和水(体积分数75∶25,v/v),激发波长(Ex)381 nm,发射波长(Em)470 nm,流速0.6 mL/min,柱温30℃。方法的检测限1.8 ng/g,定量限为6×103 ng/kg,标准方差(RSD)低于4.25%,回收率72.24%～83.31%,T-2毒素在25～800 ng/mL范围内线性回归良好,线性方程为:y=2186.2x-31953,标准方差R2=0.9992。本方法简单快速、灵敏、准确、重复性好,能满足饲料中检测T-2毒素的需要。     

不同乳酸菌剂对发酵全混合日粮霉菌毒素含量的影响

本试验研究了不同乳酸菌剂对发酵全混合日粮(FTMR)中6种霉菌毒素含量的影响。将不同乳酸菌剂组(无添加组、添加BM组、添加CL组和添加YX组)的FTMR贮存发酵64d,采用多功能色谱柱净化-电化学衍生-高效液相色谱法测定FTMR中的黄曲霉毒素(AFT),免疫亲和柱-高效液相色谱法测定玉米赤霉烯酮(ZEN)和呕吐毒素(DON)含量,外标法定量。结果表明,4个处理组AFB1含量分别为23.37μg/kg、19.71μg/kg、21.81μg/kg和20.39μg/kg,ZEN含量分别为1159.95μg/kg、665.71μg/kg、964.12μg/kg和851.74μg/kg,DON含量分别为137.84μg/kg、114.46μg/kg、129.71μg/kg和129.39μg/kg,4组中均未检测出AFG1、AFB2和AFG2。结果表明,乳酸菌剂能够显著降低FTMR中霉菌毒素含量,其中BM乳酸菌剂效果最好。     

气相色谱-质谱检测饲料中B类单端孢霉烯族毒素

建立了气相色谱-质谱联用同时测定饲料中脱氧雪腐镰刀菌烯醇和雪腐镰刀菌烯醇的方法。饲料样品中毒素提取液经Mycosep227多功能净化柱净化后,利用硅烷化试剂TBT(三甲基硅烷咪唑:双三甲基硅基乙酰胺:三甲基氯硅烷=3:3:2,V/V/V,与B类单端孢霉烯族毒素进行衍生化反应。HP-5MS毛细管柱作为色谱柱分离毒素,质谱进行检测。外标法定量。脱氧雪腐镰刀菌烯醇和雪腐镰刀菌烯醇标准溶液提取离子峰面积与标品浓度分别在10～1300ng/ml、8～1300ng/ml内呈良好的线性关系,饲料样品中两种毒素不同加入量的回收率分别在81.7%～92.6%、78.2%～87.8%之间,最低检出限为9ng/g、7ng/g,批间与批内的相对标准偏差均小于10%。该方法检测灵敏度高且具有良好的重现性。     

Equine uridine diphospho-glucuronosyltransferase 1A1, 2A1, 2B4, 2B31: cDNA cloning,expression and initial characterization of morphine metabolism

ObjectiveUridine diphospho-glucuronosyltransferases (UGTs) are membrane-bound enzymes that catalyze the conjugation of glucuronic acid onto a diverse set of xenobiotics. Horses efficiently and extensively glucuronidate a number of xenobiotics, including opioids, making UGTs an important group of drug-metabolizing enzymes for the clearance of drugs. Recombinant enzymes have allowed researchers to characterize the metabolism of a variety of drugs. The primary objective was to clone, express and characterize equine UGTs using drugs characterized as UGT substrates in other species. A secondary objective was to characterize the in vitro metabolism of morphine in horses.Study designIn vitro drug metabolism study using liver microsomes and recombinant enzyme systems.AnimalsLiver microsomes and RNA from tissue collected from two Thoroughbred mares euthanized for other reasons.MethodsBased on homology to the human UGT2B7, four equine UGT variants were expressed: UGT1A1, UGT2A1, UGT2B31 and UGT2B4. cDNA sequences were cloned and resulting protein expressed in a baculovirus expression system. Functionality of the enzymes was assessed using 4-methylumbelliferone, testosterone, diclofenac and ketoprofen. Recombinant enzyme, control cells, equine liver microsomes and human UGT2B7 supersomes were then incubated with morphine. Concentrations of metabolites were measured using liquid chromatography–tandem mass spectrometry and enzyme kinetics determined.Results4-Methylumbelliferone was glucuronidated by all expressed equine UGTs. Testosterone glucuronide was not produced by any of the expressed enzymes, and diclofenac glucuronide and ketoprofen glucuronide were produced by UG2A1 and UGT1A1, respectively. UGT2B31 metabolized morphine to morphine-3-glucuronide and low concentrations of morphine-6-glucuronide.Conclusions and clinical relevanceThis is the first successful expression of functional recombinant equine UGTs. UGT2B31 contributes to the glucuronidation of morphine; however, it is probably not the main metabolizing enzyme. These results warrant further investigation of equine UGTs, including expression of additional enzymes and further characterization of UGT2B31 as a contributor to morphine metabolism.     

Peptide mapping of ovoglobulins G2A and G2B in the domestic fowl

1. The purification of the two principal genetic variants of ovoglobulin G2 from egg‐whites is described.

2. Both ovoglobulin G2A and G2B had molecular weights of 47 ±2 kDa. They differed in their mobilities in non‐denaturing gels and in their isoelectric points, which were 4.9 and 5.3 for G2A and G2B respectively.

3. Peptide mapping using either chymotrypsin or V8 digestion revealed additional proteinase sensitive sites in ovoglobulin G2A. The results are consistent with one of the differences between the two ovoglobulins being the replacement of an acidic amino acid residue in G2A by a neutral residue in G2B.     

Echinococcus ortleppi and E. granulosus G1, G2 and G3 genotypes in Italian bovines

To increase the knowledge on Echinococcus genotypes infesting cattle and water buffaloes (Bubalus bubalis) born and bred in Italy, the germinal layer of hydatid cysts was collected from the liver and the lungs of 80 animals slaughtered in 2007. Two mitochondrial genes (the cytochrome c oxidase subunit I and the NADH subunit I) were tested by PCR. Four genotypes were identified: G1 (sheep strain), G2 (Tasmanian sheep strain), G3 (buffalo strain), and G5 (cattle strain). Fertile cysts were detected only in the lungs of 4.5% of the total G1 lung cysts, of 9.4% of the total G3 lung cysts, and in the only G5 infected animal. This is the first report of Echinococcus ortleppi (genotype G5) in Italy.     

Expression of TGFBR1, TGFBR2, TGFBR3, ACVR1B and ACVR2B is altered in ovaries of cows with cystic ovarian disease

The objective of this study was to examine the expression of transforming growth factor beta receptor (TGFBR)1, TGFBR2, TGFBR3, activin receptor (ACVR)1B and ACVR2B in ovaries of cows with cystic ovarian disease (COD). The expression of the selected receptors was determined by immunohistochemistry in sections of ovaries from cows with ACTH‐induced and spontaneous COD. Expression of TGFBR1 and TGFBR3 was higher in granulosa cells of cysts from cows with spontaneous COD than in tertiary follicles from the control group. Additionally, TGFBR3 expression was higher in granulosa cells of cysts from cows with ACTH‐induced COD than in those from the control group and lower in theca cells of spontaneous and ACTH‐induced cysts than in tertiary control follicles. There were no changes in the expression of TGFBR2. ACVR1B expression was higher in granulosa cells of tertiary follicles of cows with spontaneous COD than in the control group, whereas ACVR2B expression was higher in cysts of the spontaneous COD group than in tertiary follicles from the control group. The alterations here detected, together with the altered expression of the ligands previously reported, indicate alterations in the response of the ligands in the target cells, modifying their actions at cellular level.     

Total protein and immunoglobulins G1, G2, and M in serum of calves persistently infected with bovine viral diarrhea virus

Total serum protein and immunoglobulins (Ig) G1, G2, and M concentrations were investigated in 11 calves persistently infected with bovine viral diarrhea virus. These calves were allowed to suckle from their dams until weaned. A gradual increase in total protein was observed from birth to 12 months of age. There was a wide variation in Ig concentrations in pre- and postcolostrum sera. The IgG1 increased from the time of delivery of the calves to the 5th month, decreased by the 10th month, and then stabilized through the 12th month. The IgG2 increased from birth to 10 months and remained stable through 12 months. The IgM increased from birth to the 11th month, and then decreased sharply by the 12th month.     

17种植物性饲料原料中伏马毒素(B1+B2)、赭曲霉毒素A、T-2毒素、黄曲霉毒素B1污染状况调查检测分析

为了解掌握伏马毒素（B₁+B₂）、赭曲霉毒素A、T-2毒素、黄曲霉毒素B₁在植物性饲料原料中的污染状况,指导饲料生产企业和养殖企业开展霉菌毒素防控,降低霉菌毒素对饲料产品质量及畜禽养殖危害,减少经济损失,2019年对17种62份植物性饲料原料进行采集,采用液相色谱—串联质谱法、免疫亲和柱净化—高效液相色谱法检测,依据《饲料卫生标准》（GB 13078—2017）判定分析。结果表明：伏马毒素（B₁+B₂）、赭曲霉毒素A、黄曲霉毒素B₁在17种植物性饲料原料中的污染状况差别明显,伏马毒素（B₁+B₂）、赭曲霉毒素A、黄曲霉毒素B₁平均检出率分别为37.09%、8.06%、29.03%,最大检测值分别为15.96 mg/kg、26.60 μg/kg、351.00 μg/kg。从检测结果得出,4种霉菌毒素在17种植物性饲料原料中的污染较重,整体污染率达48.40%,玉米皮、喷浆玉米皮、花生粕3种植物性饲料原料中黄曲霉毒素B₁超标,污染率与超标率不一定呈正比,表明霉菌毒素在植物性饲料原料中污染普遍,对饲料产品及养殖安全造成严重影响。针对该问题,提出控制植物性饲料原料质量建议,为今后控制饲料原料中霉菌毒素含量提供参考。     

Full-length genome analysis of G2, G9 and G11 porcine group A rotaviruses

Group A rotaviruses with G2 and G9 VP7 specificity are common in humans, while G11 strains have been detected only sporadically. G2, G9 and G11 rotaviruses also circulate in pigs and swine rotaviruses have been suspected of interspecies and zoonotic transmissions in numerous studies. However, the complete gene constellation of G2 and G9 porcine rotaviruses has not yet been determined. In order to start filling this gap, the genomic make up of two G2, one G9 and one G11 porcine rotavirus strains, detected in Canada in 2005–2007, was determined. With the exception of a G2P[34] strain, with E9 NSP4 type and mixed I5 + I14 VP6 type, the constellation of genomic segments was rather conserved and were closely related to prototype porcine strains in the four viruses characterized (I5-R1-C1-M1-A8-N1-T7-E1-H1). Most notably, all the viruses displayed a rare NSP3 genotype, T7, which has also been identified in rare human reassortant strains and in the reference strain RVA/Cow-tc/GBR/UK/1973/G6P[5]. This study provides crucial genetic data on these complex viruses and will help understand the origin and ecological niche of gene segments and the role played by pigs in their evolution.