奶牛隐性乳房炎致病菌分离鉴定及LAMP检测方法的建立
发布时间：2018-10-24 22:27
【摘要】：奶牛乳房炎是奶牛养殖业中最常见疾病之一,该病严重阻碍奶牛养殖业向着安全健康的目标发展,给该行业和奶制品生产业造成严重的经济损失,甚至危害到人类健康。临床中隐性乳房炎的发病率比临床乳房炎的发病率更高,危害更大。病原微生物的感染是奶牛乳房炎的主要致病原因。为了了解咸阳市周边奶牛隐性乳房炎致病菌的分布情况,本试验选取了该地区2个规模化奶牛养殖场,对奶牛场隐性乳房炎病原菌进行分离鉴定和药敏试验,为该地区奶牛隐性乳房炎的预防和治疗提供参考依据。并针对其主要致病菌,建立环介导恒温扩增(loop-mediated isothermal amplification,LAMP)检测方法,为奶牛隐性乳房炎的快速准确诊断奠定基础。本试验主要结果如下:1.对陕西省咸阳市周边地区2个奶牛场的48份隐性乳房炎奶样进行了病原菌的分离培养、革兰氏染色与生化试验鉴定,共分离出9种92株病原菌,包括大肠杆菌、金黄色葡萄球菌、沙门氏菌、克雷伯氏菌、肠杆菌、真菌、变形杆菌、表皮葡萄球菌和志贺氏菌;2个奶牛场奶牛隐性乳房炎的主要病原菌均以金黄色葡萄球菌和大肠杆菌为主,占总检查细菌总数的60%以上,病原菌的混合感染率为83.33%。药敏试验结果表明,2个奶牛场中的革兰氏阴性杆菌均对头孢他啶敏感;2个奶牛场中分离的革兰氏阴性杆菌均对氨基糖胺类抗生素和大环内酯类抗生素产生了严重耐药性;2个牛场的革兰氏阳性球菌四环素和左氟沙星敏感,对青霉素G、苯唑西林和克林霉素严重耐药。2.针对大肠杆菌的特异性基因碱性磷酸酶(phoA)设计一套LAMP引物,优化反应条件后进行特异性及敏感性实验。LAMP反应体系在60~68℃水浴锅中50~60 min内即可完成反应,阳性结果呈翠绿色或绿色荧光。本方法对大肠杆菌的最低检测限为241 fg/μL,为PCR方法的100倍,且与奶牛乳房炎常见的致病菌无交叉反应。本研究成功建立了一种快速、特异、灵敏,操作和结果判定简便的大肠杆菌性乳房炎LAMP可视化检测方法。
[Abstract]:Cow mastitis is one of the most common diseases in dairy cattle breeding industry, which seriously hinders the development of dairy cattle breeding industry towards the goal of safety and health, and causes serious economic losses and even harm to human health. The incidence of occult mastitis is higher and more harmful than that of clinical mastitis. Infection of pathogenic microorganisms is the main cause of mastitis in dairy cows. In order to understand the distribution of recessive mastitis pathogens in dairy cows around Xianyang City, two large-scale dairy cattle farms in this area were selected to isolate and identify the pathogens of recessive mastitis in dairy farms and to test their drug sensitivity. To provide reference for the prevention and treatment of recessive mastitis in dairy cattle in this area. The method of ring mediated isothermal amplification (loop-mediated isothermal amplification,LAMP) was established for the rapid and accurate diagnosis of recessive mastitis in dairy cattle. The main results are as follows: 1. In this paper, 48 samples of recessive mastitis milk samples from 2 dairy farms in Xianyang City, Shaanxi Province were isolated and cultured. Gram staining and biochemical tests were used to identify 9 species and 92 strains of pathogenic bacteria, including Escherichia coli. Staphylococcus aureus, Salmonella, Klebsiella, Enterobacter, Fungi, Proteus, Staphylococcus epidermidis and Shigella. The mixed infection rate of pathogenic bacteria was 83.33. The results of drug sensitivity test showed that the Gram-negative bacilli in two dairy farms were sensitive to ceftazidime, and the Gram-negative bacilli isolated from two dairy farms had serious effects on aminoglycoamine antibiotics and macrolide antibiotics. Drug resistance; Gram-positive cocci tetracycline and levofloxacin were sensitive in two cattle farms. Severe resistance to penicillin G, oxacillin and clindamycin. 2. A set of LAMP primers was designed for the specific gene alkaline phosphatase (phoA) of Escherichia coli, and the specificity and sensitivity were tested after optimizing the reaction conditions. The LAMP reaction system could complete the reaction within 50 ~ 60 min in 60 ~ 68 鈩，
本文编号：2292746
[Abstract]:Cow mastitis is one of the most common diseases in dairy cattle breeding industry, which seriously hinders the development of dairy cattle breeding industry towards the goal of safety and health, and causes serious economic losses and even harm to human health. The incidence of occult mastitis is higher and more harmful than that of clinical mastitis. Infection of pathogenic microorganisms is the main cause of mastitis in dairy cows. In order to understand the distribution of recessive mastitis pathogens in dairy cows around Xianyang City, two large-scale dairy cattle farms in this area were selected to isolate and identify the pathogens of recessive mastitis in dairy farms and to test their drug sensitivity. To provide reference for the prevention and treatment of recessive mastitis in dairy cattle in this area. The method of ring mediated isothermal amplification (loop-mediated isothermal amplification,LAMP) was established for the rapid and accurate diagnosis of recessive mastitis in dairy cattle. The main results are as follows: 1. In this paper, 48 samples of recessive mastitis milk samples from 2 dairy farms in Xianyang City, Shaanxi Province were isolated and cultured. Gram staining and biochemical tests were used to identify 9 species and 92 strains of pathogenic bacteria, including Escherichia coli. Staphylococcus aureus, Salmonella, Klebsiella, Enterobacter, Fungi, Proteus, Staphylococcus epidermidis and Shigella. The mixed infection rate of pathogenic bacteria was 83.33. The results of drug sensitivity test showed that the Gram-negative bacilli in two dairy farms were sensitive to ceftazidime, and the Gram-negative bacilli isolated from two dairy farms had serious effects on aminoglycoamine antibiotics and macrolide antibiotics. Drug resistance; Gram-positive cocci tetracycline and levofloxacin were sensitive in two cattle farms. Severe resistance to penicillin G, oxacillin and clindamycin. 2. A set of LAMP primers was designed for the specific gene alkaline phosphatase (phoA) of Escherichia coli, and the specificity and sensitivity were tested after optimizing the reaction conditions. The LAMP reaction system could complete the reaction within 50 ~ 60 min in 60 ~ 68 鈩，
本文编号：2292746
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