鸡白色念珠菌鉴定及荧光定量PCR检测方法的建立
发布时间：2018-08-20 16:59
【摘要】：鸡白色念珠菌病的发病率呈现上升的趋势,危害性越来越大。目前鸡白色念珠菌病的诊断主要依赖临床症状和病理剖检,这些方法敏感性低,特异性差,耗时长,不能及时作出准确的诊断,以至于影响了该病的早期诊断和早期治疗。因此,研究鸡白色念珠菌的快速检测方法对鸡白色念珠菌的鉴别和流行病学调查具有重要意义。为鉴定分离疑似白色念珠菌,从患病鸡的嗉囊分离培养5株,经显色培养基培养,芽管形成,厚膜孢子诱导和ITS基因比对及分型等试验。结果发现该分离株在土豆培养基上,菌落为圆形,乳白色,背面没有色素沉着;在显微镜下,无论用甲苯胺蓝染色,还是荧光染色,菌体大小不一,为圆形或椭圆形的酵母样菌,革兰氏染色阳性;显色培养基上,白色念珠菌为无色,其他念珠菌为红色;在血清上培养形成芽管;玉米粉吐温培养基诱导出厚膜孢子。IST间区基因序列分析,分离株同源性为96.9~97.4%,与GenBank白色念珠菌的同源性为97.4%,而与其他念珠菌的同源性为94.6~76.92%。基因分型为A型和C型。结合形态特征和基因同源性分析,该分离株被鉴定为白色念珠菌。根据GenBank已发表的白色念珠菌rDNA内转录间区核苷酸序列设计一对目的扩增子长度为273 bp的特异引物,采用LightCycler实时PCR(LC-PCR)检测方法,以SYBR GreenⅠ为扩增产物荧光染色剂,对禽白色念珠菌疑似病例血液样本进行检测,并用临床常见的5种病原真菌对该方法的特异性进行检验。建立的鸡白色念珠菌检灵测方法敏度高,对白色念珠菌最低检出浓度为101CFU/mL;特异性强,与光滑念珠菌、克柔念珠菌、热带念珠菌、近平滑念珠菌、烟曲霉等病原真菌无交叉反应;耗时短,只需2 h即可完成整个试验过程。
[Abstract]:The incidence of Chicken Candida albicans is on the rise, and the harm is more and more serious. At present, the diagnosis of chicken Candida albicans mainly depends on clinical symptoms and pathological examination. These methods have low sensitivity, poor specificity, time consuming, and can not make accurate diagnosis in time, thus affecting the early diagnosis and early treatment of the disease. Therefore, the study of rapid detection of chicken Candida albicans is of great significance for identification and epidemiological investigation of chicken Candida albicans. In order to identify and isolate suspected Candida albicans, 5 strains were isolated from the crop of sick chicken, and were cultured in color-forming medium, bud tube formation, thick membrane spores induction, ITS gene comparison and typing. The results showed that the isolated strain on potato medium had round colony, milky white and no pigmentation on the back. Under the microscope, the bacterial size varied with toluidine blue staining or fluorescent staining. It is a round or oval yeast-like bacterium with Gram-positive staining; Candida albicans is colorless and other Candida is red on color medium; The results showed that the homology of the isolated strain was 96.9N 97.4m, 97.4% with GenBank candida albicans, 94.6% with other Candida albicans, and 94.6% with other Candida albicans. Genotype A and C were genotyped. The strain was identified as Candida albicans by morphological analysis and gene homology analysis. According to the published nucleotide sequence of rDNA in Candida albicans published by GenBank, a pair of primers were designed for the length of 273bp of the target amplifiers. LightCycler real-time PCR (LC-PCR) was used to detect the primers. SYBR Green 鈪，
本文编号：2194346
[Abstract]:The incidence of Chicken Candida albicans is on the rise, and the harm is more and more serious. At present, the diagnosis of chicken Candida albicans mainly depends on clinical symptoms and pathological examination. These methods have low sensitivity, poor specificity, time consuming, and can not make accurate diagnosis in time, thus affecting the early diagnosis and early treatment of the disease. Therefore, the study of rapid detection of chicken Candida albicans is of great significance for identification and epidemiological investigation of chicken Candida albicans. In order to identify and isolate suspected Candida albicans, 5 strains were isolated from the crop of sick chicken, and were cultured in color-forming medium, bud tube formation, thick membrane spores induction, ITS gene comparison and typing. The results showed that the isolated strain on potato medium had round colony, milky white and no pigmentation on the back. Under the microscope, the bacterial size varied with toluidine blue staining or fluorescent staining. It is a round or oval yeast-like bacterium with Gram-positive staining; Candida albicans is colorless and other Candida is red on color medium; The results showed that the homology of the isolated strain was 96.9N 97.4m, 97.4% with GenBank candida albicans, 94.6% with other Candida albicans, and 94.6% with other Candida albicans. Genotype A and C were genotyped. The strain was identified as Candida albicans by morphological analysis and gene homology analysis. According to the published nucleotide sequence of rDNA in Candida albicans published by GenBank, a pair of primers were designed for the length of 273bp of the target amplifiers. LightCycler real-time PCR (LC-PCR) was used to detect the primers. SYBR Green 鈪，
本文编号：2194346
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