免疫层析法快速检测金黄色葡萄球菌的研究

为建立一种快速检测金黄色葡萄球菌的新方法,本研究采用柠檬酸三钠还原法制备胶体金,用胶体金标记鼠抗金黄色葡萄球菌凝集因子A(C1fA)重组蛋白特异性抗体,从抗金黄色葡萄球茵IgG包被于硝酸纤维素膜,制成免疫层析快速检测试纸条.该方法可以在10min内完成对含金黄色葡萄球菌奶样的检测;敏感度为1×104 cfu/mL以上,与PCR方法进行对比检测,相差一个数量级;与大肠杆菌、链球菌、李氏杆菌、巴氏杆茵及蜡样芽孢杆菌均无交叉;稳定性和重复性较好.该试纸条检测金黄色葡萄球菌临床样品表明其具有特异性好,灵敏度较高的特点.     

Rapid and sensitive detection of African horse sickness virus by real-time PCR

A highly sensitive and specific TaqMan-MGB real-time RT-PCR assay has been developed and standardised for the detection of African horse sickness virus (AHSV). Primers and MGB probe specific for AHSV were selected within a highly conserved region of genome segment 7. The robustness and general application of the diagnostic method were verified by the detection of 12 AHSV isolates from all of the nine serotypes. The analytical sensitivity ranged from 0.001 to 0.15 TCID₅₀ per reaction, depending on the viral serotype. Real-time PCR performance was preliminarily assessed by analysing a panel of field equine samples. The same primer pair was used to standardise a conventional RT-PCR as an affordable, useful and simple alternative method in laboratories without access to real-time PCR instruments. The two techniques present novel tools to improve the molecular diagnosis of African horse sickness (AHS).     

Rapid detection of viral-specific antibodies by enzyme-linked immunosorbent assay (ELISA)

The development of three separate rapid ELISAs for detecting antibodies in host serum to three different viruses is described. These include: 1. A direct antigen assay using enzyme labelled anti-canine Ig for detecting antibodies to canine parvovirus, 2. A competitive ELISA using a feline infectious peritonitis virus-specific monoclonal antibody labelled with enzyme, and 3. A competitive ELISA using an equine infectious anemia virus-specific monoclonal antibody and enzyme labelled antigen, p. 26. The utility and benefits of each of the three approaches is emphasized.     

荧光定量PCR检测牛乳中金黄色葡萄球菌肠毒素A基因方法的建立

为建立检测牛乳中金黄色葡萄球菌(S.aureus)肠毒素A基因(SEA)定性定量的检测方法,本研究针对S.aureus SEA基因片段设计1对引物,将构建的重组质粒作为阳性对照,建立了S.aureus SEA DNA的SYBR Green I real-time PCR检测方法.结果显示,特异性产物Tm值为78.2℃～78.5℃,最低可检测到49.5 fg/μL(16.5拷贝)的阳性质粒.标准曲线的相关系数为0.99.与其他常见的产SEB的S.aureus、产SEC的S.aureus、无乳链球菌、大肠杆菌、嗜热链球菌、伤寒沙门氏菌、大肠杆菌DH5 α及JM109均无交叉反应.该检测方法具有较好的特异性和敏感性,为牛乳中S.aureus的快速检测提供了新的技术手段.     

ROSA法快速检测婴儿奶粉中四环素类和β-内酰胺类药物残留

为了建立同时快速检测婴儿奶粉中四环素类和β-内酰胺类药物残留的方法,试验采用RO-SA法测定了市购婴儿奶粉中的药物残留。结果表明:ROSA法的灵敏度和特异性均能达到检测要求,15 min可获得检测结果。说明ROSA检测方法可以用来检测婴儿奶粉中的药物残留。     

Antimicrobial susceptibility of coagulase-negative Staphylococcus species isolated from bovine milk

Coagulase-negative Staphylococcus (CNS) isolates (n=168) obtained from milk from heifers and dairy cows were screened for minimum inhibitory concentration (MIC) to antimicrobials used commonly for mastitis therapy. Of the 10 CNS species included in the study, the predominant species were Staphylococcus chromogenes (n=61), Staphylococcus epidermidis (n=37), Staphylococcus hyicus (n=37), and Staphylococcus simulans (n=16). The majority of CNS was susceptible to ampicillin, oxacillin, cephalothin, and ceftiofur. Erythromycin and pirlimycin were also very effective in vitro inhibitors of CNS. The only exception was observed with S. epidermidis. Of 37 S. epidermidis evaluated, 13 (35%) exhibited efflux-based resistance to erythromycin (> or =16 microg/ml) encoded by msrA and one isolate carried ermC encoding ribosomal methylase-based resistance to both erythromycin (> or =64 microg/ml) and pirlimycin (> or =64 microg/ml). A total of 17 S. epidermidis, 11 S. chromogenes, and one S. hyicus exhibited phenotypic resistance to ampicillin (> or =0.5 microg/ml). Constitutive beta-lactamase production was observed in all ampicillin resistant isolates except 4 S. epidermidis that exhibited inducible beta-lactamase production. Induced beta-lactamase production was also observed in 13 S. epidermidis that were phenotypically susceptible to the entire MIC panel. All isolates that produced beta-lactamase either constitutively or by induction carried blaZ. S. epidermidis (n=12, 32%) that were resistant to methicillin (oxacillin > or =0.5 microg/ml) carried low affinity penicillin-binding protein encoded by mecA. Most multi-drug resistant (MDR) S. epidermidis (> or =2 resistance genes) were resistant to ampicillin, erythromycin and methicillin. All except one MDR S. epidermidis had icaAB, which encodes for polysaccharide intercellular adhesion. Based on pulsed field gel electrophoresis, MDR S. epidermidis were closely related genotypically, and were isolated from different cows on the same farm suggesting clonal dissemination. Bovine S. epidermidis share antimicrobial resistance patterns and virulence determinants of strains observed in human infections. Studying CNS at the species level can provide valuable information about species-specific differences that can be vital data for effective mastitis therapy and management.     

牛奶中孕酮直接竞争ELISA检测方法的建立

采用直接竞争ELISA方法检测牛奶中孕酮含量.采用碳化二亚胺法,应用辣根过氧化物酶(HRP)标记11-α羟基孕酮琥珀酸酯(11α-OH-P4-HS),制备孕酮酶标抗原;酶标抗原与牛奶样品中的孕酮共同竞争结合固相包被的孕酮单克隆抗体,建立直接竞争ELISA反应体系.试验结果表明,最佳抗体包被浓度为1∶2 000,最佳酶标抗原工作浓度为1∶16 000,建立的标准曲线为y=-2.4598x+0.999(R2=0.996),牛奶中孕酮检测范围0.12～40 ng/mL,最低检测量为0.12 ng/mL,批内和批间变异系数分别为1.63%和2.49%.成功建立了可用于牛乳中孕酮快速检测的直接竞争ELISA方法.     

应用ELISA快速检测牛羊副结核病

在96孔微板中包被抗原，加入被检血清，经温育后，抗体特异性地与抗原相结合，形成抗原一抗体结合物。洗涤去除未结合的物质，与加入的酶结合物(HRPO)相结合。再洗去未结合的酶结合物，加入底物后显色．加入终止液终止反应。用装有630nm(或620nm、650nm)滤光片的酶标仪进行检测，通过计算S／P值，判定血清中副结核抗体阴、阳性。此方法具有快速、灵敏、准确的特点。     

奶牛乳汁体细胞数的快速检测及其临床意义

本文用新型便携式体细胞计数仪C-Reader对奶牛乳汁体细胞数(SCC)进行快速、精确地检测,同时对乳样中的病原菌进行分离鉴定,旨在指导奶牛乳房炎的诊断、预防及治疗。选用北京及河北地区10个奶牛场的111头奶牛共428份奶样,进行SCC计数。SCC20万/mL的奶样占63.6%,SCC介于20万/mL～50万/mL的奶样占13.1%,SCC50万/mL占23.3%。检测的111头奶牛中,60头奶牛患有隐性乳房炎,头发病率54.1%,乳区发病率23.3%。隐性乳房炎SCC变化范围50.6万/mL～984.3万/mL。对隐性乳房炎乳样进行致病菌分离培养,其阳性率高达82.0%。共鉴定出100株致病菌,其中金黄色葡萄球菌64株,表皮葡萄球菌11株,大肠杆菌8株,链球菌10株,其他菌7株。结果表明,C-Reader计数仪能够快速准确地检测北京及河北地区的奶牛隐性乳房炎,其病原菌以金黄色葡萄球菌为主。     

Rapid detection and identification of Clostridium chauvoei by PCR based on flagellin gene sequence

We developed a one-step polymerase chain reaction (PCR) system that specifically detects Clostridium chauvoei. Oligonucleotide primers were designed to amplify a 516-bp fragment of the structural flagellin gene. The specificity of the PCR was investigated by analyzing 59 strains of clostridia, and seven strain of other genera. A 516-bp fragment could be amplified from all the C. chauvoei strains tested, and no amplification was observed by using DNAs from the other strains tested, including Clostridium septicum. Similarly, this PCR-based method specifically detected C. chauvoei DNA sequences in samples of muscle and exudate of obtained from mice within 12h of inoculation. In tests using samples of muscle or liver, the limit of detection was about 200 organisms per reaction. These results suggest that the one-step PCR system may be useful for direct detection and identification of C. chauvoei in clinical specimens.     

Capture ELISA systems for the detection of bovine coronavirus-specific IgA and IgM antibodies in milk and serum

Isotype-capture ELISAs for BCV-specific IgA and IgM were developed and tested on milk and serum samples from Swedish cattle. The capture ELISAs showed higher sensitivity than indirect ELISAs for detection of BCV-specific IgA and IgM. In the capture ELISAs the agreement between detection in milk and serum samples was 94% for IgA and 86% for IgM. The correlation between log(10) titres in milk and serum was r=0.82 (P<0.001) for IgA and 0.84 (P<0.001) for IgM. Milk seemed a better target than serum for diagnosing specific IgA at low levels. There was no variation in the isotype-specific BCV antibody titres between healthy quarters of the same udder, but subclinical mastitis was associated with higher levels of IgA antibodies and weak false IgM positive reactions in undiluted milk. Bovine IgA and IgM antibodies in milk and serum showed high stability towards freezing and thawing and storage at room temperature.The antibody responses to BCV were followed in milk and serum from six dairy cows and in serum from four calves for a period of 1 year after an outbreak of winter dysentery (WD). In this outbreak some animals became reinfected with BCV. The IgA and IgM capture ELISAs differentiated between primarily BCV infected and reinfected animals. In the primarily infected cattle, IgM antibodies were first detected in milk and serum four to nine days after the first WD symptoms observed, and were subsequently detected for at least 2-3 weeks. IgM was also detected in the reinfected cows, but mostly at lower levels and for a shorter period of time than in the primarily infected animals. In milk, however, the IgM response of the reinfected cows was detected for a longer period of time than in serum. Six months after the outbreak, IgA was still detected in both serum and milk of all six cows and also in serum of one calf. The reinfected cows showed higher and more long-lasting peak levels of IgA in milk and serum than the primarily infected cows, indicating boosting of the IgA response.     

Efficacy of the API Staph-Ident system for identification of Staphylococcus species from milk

A total of 524 staphylococcal isolates from bovine milk were identified, using the API Staph-Ident system and conventional biochemical methods. The API Staph-Ident system correctly identified 192 of 201 (95.5%) Staphylococcus aureus isolates, but was correct on only 23 of 323 (7.1%) non-S aureus isolates.     

Prevalence of Coxiella burnetii infection in Dutch dairy herds based on testing bulk tank milk and individual samples by PCR and ELISA

A study using an ELISA and a real-time PCR assay based on the detection of the repetitive transposon-like gene of Coxiella burnetii revealed that infection with the bacterium was widespread among Dutch dairy herds, with antibodies detected in bulk tank milk (BTM) from 268 of 341 herds (78.6 per cent) and bacterial DNA detected in 193 of 341 herds (56.6 per cent). The BTM samples were taken in November and December 2007. Serological and molecular studies in young and adult cattle selected from 100 herds showed that antibodies were present in the blood of 470 of 2936 (16.0 per cent) lactating cows but only in 19 of 1831 (1.0 per cent) young animals. Bacterial DNA was detected in the milk of 254 of 2925 (8.7 per cent) lactating cows; bacterial DNA was not detected in any of the faecal samples obtained from youngstock. The blood and milk samples were taken from the cattle in the period January to April 2008.     

Adaptation of a commercial ELISA for the detection of antibodies against Neospora caninum in bovine milk

The aim of the present study was to determine whether a commercially available ELISA could be used to examine bovine milk for antibodies against Neospora caninum. In an initial titration experiment, a milk dilution of 1:2 was found optimal to obtain milk results that were linearly correlated to those obtained with corresponding sera. This dilution was then used to examine 791 milk samples from N. caninum infected herds in the commercial ELISA. Milk results of individual animals were compared with those obtained by the same ELISA for the corresponding serum samples. The linear correlation between milk and serum antibody results of individual animals was characterized by R2 = 0.702. Multiple linear regression indicated that the later the stage of lactation at which an animal was sampled, the higher the milk ELISA result was as compared to the serum ELISA result. The examination of the two-graph receiver operating characteristics revealed an optimal cut-off of 0.261 to obtain similar results in the examination of milk and serum. With this cut-off, the test had a sensitivity and specificity relative to the serum results of 90%. The milk-based commercial ELISA classified more aborting dams as positive than the serum-based ELISA with this cut-off. The milk ELISA may be a valuable tool to assess the herd status with regard to abortion caused by N. caninum.     

克伦特罗ELISA快速检测的几点操作体会

<正>克伦特罗属于β-兴奋剂类药物,可以提高脂肪型动物的瘦肉率和加速动物生长。目前由于多起克伦特罗中毒事件且出现了死亡案例,因此被禁止使用。克伦特罗ELISA快速检测试剂盒与经典色谱法相比,具有操作过程简单、样品处理快捷,可同时分析多个样品,分析时间短、成本低、灵敏度高且对环境污染小等优点。     

Evaluation of an iscom ELISA used for detection of antibodies to Neospora caninum in bulk milk

The intracellular parasite Neospora caninum is increasingly recognized as an important cause of abortion and stillbirth in cattle. Presence of specific antibodies indicates infection, and the immunostimulating complex (iscom) enzyme-linked immunoassay (ELISA) has previously been evaluated for use on individual milk and sera. In the present study, this test is investigated for use on bulk milk. In this study, 124 herds were used to analyse the relationship between within-herd prevalences based on individual sera and bulk milk optical densities. The individual test results were translated into a herd-level result, which enabled comparison of the bulk milk test result to the aggregate of individual serum results. The relative contribution of milk from cows with different milk yield and antibody status to the tank, i.e. its composition, was expected to influence the outcome of the bulk milk test. Therefore, sensitivity and specificity were calculated at different cut-off levels, not only using a standard cross-tabulation technique, but also a logistic regression model. By using the latter method, the sensitivity and specificity could be estimated adjusting for milk yield covariates. Specificity was estimated to be high ( approximately 98%) at the 0.20 cut-off, which can be used as a decision threshold to rule in infection. With more equal emphasis on sensitivity and specificity, a lower cut-off should be used. Although infection cannot be completely ruled out, herds with test results below 0.05 are highly likely to be non-infected. The within-herd prevalence of false negative herds is probably less than 10-15% at this level. From what is known about test performance at the individual level and the prevalence of infection, the estimate of the specificity of the bulk milk test should be quite accurate while the sensitivity is likely to be underestimated. We confirmed that the performance of the bulk milk test depends on the milk tank composition. In particular the milk yield of cows with high antibody levels affects the probability of a positive outcome of the bulk milk test.     

ELISA方法检测犬瘟热抗体初探

本实验以细胞培养犬瘟热病毒作为包被抗原,建立了检测犬瘟热病毒血清抗体的间接ELISA方法。该检测方法抗原最佳包被浓度为5μg/mL,血清最佳稀释度为1∶100。阻断实验、敏感性实验等结果表明该方法特异性强、重复性较好,可用于犬瘟热血清抗体的定量和定性检测。     

ELISA在检测饲料黄曲霉毒素总量中的应用

实验研究了酶联免疫吸附法(ELISA)测定饲料中黄曲霉毒素总量的方法。采用70%甲醇萃取饲料中黄曲霉毒素,应用固相直接竞争酶联免疫吸附原理,建立标准反应曲线Y=-21.1X+120.11,R2=0.998。该方法简单、快捷、准确,回收率在97.71%￣102.74%之间。