【作者】 张志刚；

【导师】 王哲；

【作者基本信息】 吉林大学 ， 临床兽医学， 2009， 博士

【摘要】 酮病是奶牛主要的群发性、高发性疾病之一,其病理学基础是能量负平衡。本研究的目的是研制一种拥有自主知识产权的乳汁β-羟丁酸(β-hydroxybutyric acid,BHBA)分析试纸条,用于奶牛亚临床酮病的诊断,填补国内空白。根据生物有机化学显色法原理,建立了用于检测乳汁BHBA的试纸条的反应原理,对试剂及其配比、反应条件等分别进行了筛选、优化和确定。成功建立了可用于奶牛乳汁BHBA定量检测的紫外分光光度计检测方法,检测性能较好,与荧光法检测具有良好的相关性(r=0.9939)。所建立方法需要仪器比较常规、检测成本相对较低,是一种可用于乳汁BHBA定量检测的方法。依据试纸条反应原理及试剂配比,配制了试纸条的反应液,采用薄型中速定量滤纸、聚乙烯塑料胶板为材料成功的组装了试纸条。制作了试纸条的标准比色卡,以200μmol/L作为试纸条阳性结果判读标准。确定试纸条与被检样品反应的结果判读时间为反应3 min后。稳定性试验表明,组装的试纸条可在2-8℃条件下保存18个月检测性能不降低,符合国家食品与药品监督管理局关于临床体外诊断试剂的相关标准。所研制试纸条的特异性、敏感性和重复性较好。已于2007年申报国家发明专利(申请号:200710056031.9),现正在起草乳汁BHBA分析试纸条的技术条件标准。以血液BHBA>1200μmol/L作为奶牛亚临床酮病的诊断标准,自制乳汁BHBA分析试纸条的检测敏感性和特异性与国外同类试纸条大致相当。自制试纸条和国外同类试纸条的价格比较结果表明自制乳汁BHBA分析试纸条的价格将远低于国外同类试纸条的售价。结合我国实际国情,自制乳汁BHBA分析试纸条具有广阔的应用前景和较大的经济价值。更多还原

【Abstract】 Dairy cows ketosis is a metabolic diseases that carbohydrates and fat metabolic disorder caused by a systemic dysfunction, It is one of major diseases which are harmful to dairy industry. The disease usually take place in high-yielding cow which day milk production are more than 30 Kg, the main pathology is characterized by low blood sugar, high blood ketone. Because of the introduction of high-yielding varieties, cultivation, high-energy feed, the development, use, large-scale breeding and improvement in the level and many other reasons, making the incidence of the disease has long been high. Ketosis in dairy cattle in our country the incidence of lactational cow account to15%-30%, 5.0% in American, 15.4% in Iran,14.69%in India, while has up to 43.1% in Japan. The disease cause huge economic losses to the dairy industry, is an important factor that constrain the development of dairy industry. It is reported that a clinical treatment of ketosis in dairy cattle only drugs and the decline in milk production losses, which amounts to 145 American dollars. Ketosis in dairy cattle caused not only the economic loss but also displaced abomasums, retained placenta, decreasing of reproductive performance, perinatal paralysis et al. Therefore, ketosis in dairy cattle have been classified as the most important dairy cattle diseases to study in many countries.Ketosis in dairy cattle according to their availability of obvious clinical symptoms can be classified into clinical and subclinical ketosis. Clinical ketosis in accordance with its obvious clinical symptoms (such as loss of appetite, constipation, fecal mucus overlying have mental gloomy or mental excitement, milk yield decreased, exhaled gas has ketone flavor) is not difficult to make a diagnosis, and is relative rare in clinical practice. Subclinical ketosis without obvious clinical symptoms, it is reported that each of the dairy cow with subclinical ketosis caused economic losses of about 80 American dollars. Ketones are the material produced by partial oxidation of liver nonesterified fatty acids (NEFA).Animals includes three ketones in vivo, acetone, acetoacetate andβ-hydroxybutyric acid (BHBA). Detection of ketones are diagnostic indicators of subclinical ketosis in cows. BHBA relatively stable, it is often used as a diagnostic indicator of ketosis, and acetoacetate can decompose into acetone, and acetone with volatility. Ketone detection including blood ketone detection, urine ketone testing and milk ketone body detection. Milk ketone body detection is easy to implement, cost far less than the blood ketone body detection; It is reported that the milk ketone body and blood ketone body content has strong correlation, milk ketone tests can be used to diagnose cows subclinical ketosis.At present, blood BHBA>1200μmmol/L is used as the diagnostic criteria for the dairy cows subclinical disease in the international arena. Reported diagnosis methods for subclinical ketosis include ketone powder, urine analysis strip, milk BHBA test strip and ketone tablet et al.As the milk samples are easier collected than urine and blood collection, it is a very convenient way for routine monitoring subclinical ketosis or diagnosis of subclinical ketosis in dairy cows. Study showed that detection sensitivity and specificity of the milk BHBA test strip were the best. Foreign milk BHBA test strip includes Ketolac strip, it is expensive, Ketolac strip can not be bought in domestic market so far also and is still at the blank stage. In this study, we first developed a milk BHBA detect strip strip for the diagnosis of subclinical ketosis in dairy cattle at the domestic, and carried out a preliminary clinical application research on the strip.Firstly, we set up the reaction principle of strip for detection of milk BHBA, the main reagent composition is dihydrouracil dehydrogenase (NAD), nitroblue tetrazolium (NBT), BHBA dehydr- odehydrogenase and diaphorase. Through comparison test between three commonly used buffer, sodium-hydrochloric acid buffer, Tris-hydrochloric acid buffer and borax-hydrochloric acid buffer, which the range of pH was 7.0-9.0, and determined to use the Tris-hydrochloride buffer as buffer of the test strip. The results of different concentration and pH experiments showed that optimum concentration and pH of Tris-hydrochloric acid buffer solution for preparation strip reaction solution were 0.01 mol/L and 8.5 respectively. Determining the sensitizer was Tween-20 based on different sensitizer test, and its optimum concentration was 0.1%. The study of effect of several metal ions on the reaction results showed that K+, Na+, NH4+, Mn2+, Zn2+, Ca2+, Ba2+ and Mg2+ inhibited differently degrees the reaction, the inhibition order was K+<Na+<NH4+<Mn2+<Zn2+ <Ba2+ <Mg2+<CK. Determining the optimum ratio of the main reagent of strip, NAD, NBT, BHBA dehydrogenase and diaphorase by orthogonal test.A simple UV detection method for milk BHBA quantitative detection was developed, which the linear range was in the range of 0.01-5 mmol/L, recovery was 99.35%-100.22%, better repeatability (CV<2 %), detection limit 0.01 mmol/L. And the assay has good correlation with fluorometric method (r= 0.9939). The equipment used in the developed method was conventional methods, and detection cost was relatively low. It was a kind of detection method could be used to assay milk BHBA.Strip reaction solution based on principle and reagent ratio was prepared. We assembled the strip with thin medium-speed quantitative filter paper, polyethylene plastic glue pad successfully. A standard color card for the strip was produced, which included 0.05 mmol/L, 0.10 mmol/L, 0.20 mmol/L, 0.50 mmol/L, 1 mmol/L, 0.20 mmol /L was used as the positive test standards results of strip. On the dipstick all the parameters studied. The study on the parameters of the strip were finished. Determining the interpretation time was 3 min after reaction. The results of different temperatures to preserve the strip test showed that the strip could be preserved for 18 months at 2-8℃, and reached to the relevant standards of the National Food and Drug Administration. The developed strip had good specificity, and did not react with L-lactic acid, L-succinate, fumarate, acetoacetate, acetone, L-proline and L-cysteine (These substances had similar structure or metabolic pathway with BHBA which were detected).Detecting the BHBA standard solution, BHBA positive milk samples, BHBA negative milk samples with different between-batches and intra-batches strip, 10 times duplicate detection for each sample. The coincidence of BHBA standard solution (200μmol/L) was 100%, BHBA positive milk was 100%, BHBA negative milk was 100%, indicating the repeatability of the test strip was good. Color sensitivity test showed that the self-test strip has a better sensitivity. Refering to foreign semi-quantitative diagnostic methods for subclinical ketosis in dairy cows, foreign strip, self-made strip, ketones powder, urine analysis strip were compared. The UV spectrophotometry was used for quantitative determination of milk BHBA. The test milk BHBA result of self-made and foreign congeneric strip showed that both the detection results of milk BHBA were high slightly.Blood BHBA>1200μmol/L as diagnostic criteria for subclinical ketosis, and analyzing the sensitivity and specificity of detection of self-test strip, congeneric foreign strip, ketones powder and urine analysis strip. The results showed that the sensitivity and specificity of milk test strip were higher than ketone powder and urine analysis strip; the sensitivity and specificity of self-made strip test reached to the level of foreign congeneric strip. The correlation of BHBA milk with blood BHBA was 63%, which maybe the main influencing factor for the sensitivity and specificity of self-made milk BHBA test strip and Ketolac strip, belonging to the limitations of detection methods.Self-made milk BHBA test strip is easy-to-use and inexpensive, detection performance of it reaches to the same level of foreign congeneric strip, can be applied to diagnose subclinical ketosis of dairy cows.更多还原