牛奶中支原体的检测及牛支原体LAMP诊断方法的建立

发布时间：2017-12-28 10:33

本文关键词：牛奶中支原体的检测及牛支原体LAMP诊断方法的建立　出处：《中国农业科学院》2016年硕士论文　论文类型：学位论文

更多相关文章： 支原体 奶样 PCR 牛支原 LAMP

【摘要】：支原体是一类没有细胞壁、具有多形性、能够通过细菌滤器、目前已知最小的、可以在非细胞培养基中生长的、特殊的原核微生物,能够引起人和动物的多种疾病,是一类重要的病原体。国外研究表明,支原体在奶牛乳房炎的发生方面具有重要的病原学地位,在美国和欧洲每年给奶牛业带来巨大的经济损失。但目前国内奶牛支原体乳房炎相关研究开展得很少,支原体在我国奶牛场奶样中的流行情况、流行的优势病原种类及其在奶牛乳房炎发生中的作用等都还不清楚。本研究拟采用支原体分子检测和病原分离鉴定等技术手段,对我国多个地区奶牛场的奶样进行检测和种类鉴定,初步探索支原体在奶牛场奶样中的流行情况及其在奶牛乳房炎发病、流行中的作用。由于已报道可引起牛乳房炎的支原体种类较多且有可能存在未知支原体病原,为了筛选一种可靠的支原体属特异性PCR方法作为初筛手段,本研究首先用实验室保存的17种55株支原体以及13种16株细菌病原体对5种已发表的支原体属特异性PCR方法进行了验证、筛选,结果显示方法4具有良好的特异性,能够完全区分支原体与非支原体细菌。以牛支原体为扩增对象,最低能够检出10 pg牛支原体基因组DNA以及浓度为100 ccu/mL的牛支原体培养物。利用该PCR方法和支原体分离方法检测人工感染牛支原体样品和临床采集样品73份,二者符合率为93.15%。结果表明方法4可作为实验室支原体常规快速筛查方法。应用筛选出的支原体属特异性PCR方法对我国北京、宁夏、内蒙等地区的10个奶牛场的1100份奶样进行了支原体初筛,结果隐性乳房炎样407份,支原体阳性率为11.06%(45/407);临床型乳房炎样品49份,支原体阳性率为10.20%(5/49);临床健康牛奶样644份,支原体阳性率为8.54%(55/644);对阳性样品进一步利用几种已报道的牛乳房炎支原体(牛支原体、牛生殖道支原体、加利福尼亚支原体、产碱支原体、牛鼻支原体)种特异性PCR,结果阳性样品中98.06%(101/103)为牛支原体、1.94%(2/103)为产碱支原体,没有检测到其它支原体。这些结果表明我国奶牛奶样中存在支原体流行,且优势支原体种类为牛支原体。为了能够快速、简便的检测奶样中的优势支原体——牛支原体,利用牛支原体uvrC基因为检测靶标,设计了一套LAMP引物,通过优化实验条件,建立了一种牛支原体特异性的LAMP检测方法。对11种(12株)不同来源的支原体和4种(4株)牛源细菌进行检测,结果除了牛支原体外,其他病原均为阴性,表明本方法具有良好的特异性。敏感性试验显示本方法最低能够检测1 pg的牛支原体基因组。本实验建立的牛支原体LAMP检测方法,只需要在61℃保温一个小时即可完成反应,反应完成后加入SYBR GreenⅠ显色液就能直接根据颜色变化用肉眼判断检测结果,是一种适合在基层推广应用的快速检测牛支原体的轻简化检测方法。
[Abstract]:Mycoplasma is a kind of special prokaryotic microorganism, which has no cell walls, polymorphic, and can be found through bacterial filters, and is currently the smallest known and can grow in non cell culture medium. It can cause a variety of diseases of human and animal, and is an important pathogen. Foreign studies have shown that Mycoplasma has important etiological status in the occurrence of Dairy Mastitis, which brings huge economic losses to dairy cattle industry in the United States and Europe every year. But at present, there are few researches on Mycoplasma mastitis in China. The prevalence of Mycoplasma in dairy farms in China, the prevalent dominant pathogens and their role in the occurrence of mastitis in dairy cows are not clear. The purpose of this study was to isolate and identify the pathogen and molecular detection of Mycoplasma and other technical means, in many areas of China dairy milk samples were detected and identified, preliminary exploration of the epidemic situation of Mycoplasma in dairy milk samples and the incidence of mastitis in dairy cow in effect. As has been reported by many kinds of Mycoplasma mastitis and potentially unknown pathogenic mycoplasma, a reliable means of screening for Mycoplasma specific PCR as a screening method, this research first used the laboratory preserved 17 55 mycoplasma strains of Mycoplasma and 13 species and 16 strains of bacterial pathogens of 5 genera have been published the specificity of PCR method was verified, screening, 4 results show that the method has good specificity and can distinguish between bacterial and non Mycoplasma mycoplasma. With Mycoplasma bovine as the amplification object, 10 PG of Mycoplasma bovine genome DNA and 100 ccu/mL of bovine mycoplasma were detected. The PCR method and Mycoplasma separation method were used to detect 73 samples of Mycoplasma bovine infection and clinical samples, and the rate of compliance was 93.15% in two cases. The results show that method 4 can be used as a routine rapid screening method for Mycoplasma in laboratory. The application of the screened Mycoplasma genus specific PCR method for 10 cows in Beijing, Ningxia, Inner Mongolia and other areas in the field of 1100 milk samples were Mycoplasma screening results of subclinical mastitis in 407 samples, the positive rate of Mycoplasma was 11.06% (45/407); clinical mastitis in 49 samples, the positive rate of Mycoplasma was 10.20% (5/49); clinical health milk 644 samples, the positive rate of Mycoplasma was 8.54% (55/644); the positive samples were further use of several reported bovine mastitis (bovine mycoplasma mycoplasma and Mycoplasma bovigenitalium, California mycoplasma, Alcaligenes mycoplasma and Mycoplasma bovirhinis) specific PCR, the results of 98.06% positive samples (101/103) bovine mycoplasma, 1.94% (2/103) for the production of alkali did not detect other Mycoplasma mycoplasma. These results indicate that mycoplasma is prevalent in dairy cows, and mycoplasma is the dominant mycoplasma. In order to rapid and simple detection in milk samples, using the advantage of Mycoplasma Mycoplasma bovis, Mycoplasma bovis uvrC gene as target detection, design a set of LAMP primers, by optimizing the experimental conditions, the establishment of LAMP method for detection of bovine mycoplasma specific. 11 strains (12 strains) of Mycoplasma and 4 strains (4 strains) of bovine origin bacteria were detected. The results showed that all of the other pathogens were negative except bovine body, indicating that this method has good specificity. The sensitivity test showed that this method could detect the genome of Mycoplasma bovine with the lowest level of 1 pg. Mycoplasma bovis LAMP detection method established in this experiment, only need to complete the reaction heat an hour at 61 degrees, after the completion of the reaction SYBR Green 1 color liquid can be directly according to the change of the color with the naked eye examination results, a simplified fit in the rapid detection of Mycoplasma bovis application of basic detection method.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.23

【相似文献】