奶牛隱性乳房炎致病菌分離鑒定及LAMP檢測方法的建立
發(fā)布時間：2018-10-24 22:27
【摘要】：奶牛乳房炎是奶牛養(yǎng)殖業(yè)中最常見疾病之一,該病嚴(yán)重阻礙奶牛養(yǎng)殖業(yè)向著安全健康的目標(biāo)發(fā)展,給該行業(yè)和奶制品生產(chǎn)業(yè)造成嚴(yán)重的經(jīng)濟(jì)損失,甚至危害到人類健康。臨床中隱性乳房炎的發(fā)病率比臨床乳房炎的發(fā)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。為了了解咸陽市周邊奶牛隱性乳房炎致病菌的分布情況,本試驗(yàn)選取了該地區(qū)2個規(guī)�；膛ｐB(yǎng)殖場,對奶牛場隱性乳房炎病原菌進(jìn)行分離鑒定和藥敏試驗(yàn),為該地區(qū)奶牛隱性乳房炎的預(yù)防和治療提供參考依據(jù)。并針對其主要致病菌,建立環(huán)介導(dǎo)恒溫?cái)U(kuò)增(loop-mediated isothermal amplification,LAMP)檢測方法,為奶牛隱性乳房炎的快速準(zhǔn)確診斷奠定基礎(chǔ)。本試驗(yàn)主要結(jié)果如下:1.對陜西省咸陽市周邊地區(qū)2個奶牛場的48份隱性乳房炎奶樣進(jìn)行了病原菌的分離培養(yǎng)、革蘭氏染色與生化試驗(yàn)鑒定,共分離出9種92株病原菌,包括大腸桿菌、金黃色葡萄球菌、沙門氏菌、克雷伯氏菌、腸桿菌、真菌、變形桿菌、表皮葡萄球菌和志賀氏菌;2個奶牛場奶牛隱性乳房炎的主要病原菌均以金黃色葡萄球菌和大腸桿菌為主,占總檢查細(xì)菌總數(shù)的60%以上,病原菌的混合感染率為83.33%。藥敏試驗(yàn)結(jié)果表明,2個奶牛場中的革蘭氏陰性桿菌均對頭孢他啶敏感;2個奶牛場中分離的革蘭氏陰性桿菌均對氨基糖胺類抗生素和大環(huán)內(nèi)酯類抗生素產(chǎn)生了嚴(yán)重耐藥性;2個牛場的革蘭氏陽性球菌四環(huán)素和左氟沙星敏感,對青霉素G、苯唑西林和克林霉素嚴(yán)重耐藥。2.針對大腸桿菌的特異性基因堿性磷酸酶(phoA)設(shè)計(jì)一套LAMP引物,優(yōu)化反應(yīng)條件后進(jìn)行特異性及敏感性實(shí)驗(yàn)。LAMP反應(yīng)體系在60~68℃水浴鍋中50~60 min內(nèi)即可完成反應(yīng),陽性結(jié)果呈翠綠色或綠色熒光。本方法對大腸桿菌的最低檢測限為241 fg/μL,為PCR方法的100倍,且與奶牛乳房炎常見的致病菌無交叉反應(yīng)。本研究成功建立了一種快速、特異、靈敏,操作和結(jié)果判定簡便的大腸桿菌性乳房炎LAMP可視化檢測方法。
[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，
本文編號：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，
本文編號：2292746
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