雞白色念珠菌鑒定及熒光定量PCR檢測(cè)方法的建立
發(fā)布時(shí)間：2018-08-20 16:59
【摘要】：雞白色念珠菌病的發(fā)病率呈現(xiàn)上升的趨勢(shì),危害性越來(lái)越大。目前雞白色念珠菌病的診斷主要依賴臨床癥狀和病理剖檢,這些方法敏感性低,特異性差,耗時(shí)長(zhǎng),不能及時(shí)作出準(zhǔn)確的診斷,以至于影響了該病的早期診斷和早期治療。因此,研究雞白色念珠菌的快速檢測(cè)方法對(duì)雞白色念珠菌的鑒別和流行病學(xué)調(diào)查具有重要意義。為鑒定分離疑似白色念珠菌,從患病雞的嗉囊分離培養(yǎng)5株,經(jīng)顯色培養(yǎng)基培養(yǎng),芽管形成,厚膜孢子誘導(dǎo)和ITS基因比對(duì)及分型等試驗(yàn)。結(jié)果發(fā)現(xiàn)該分離株在土豆培養(yǎng)基上,菌落為圓形,乳白色,背面沒(méi)有色素沉著;在顯微鏡下,無(wú)論用甲苯胺藍(lán)染色,還是熒光染色,菌體大小不一,為圓形或橢圓形的酵母樣菌,革蘭氏染色陽(yáng)性;顯色培養(yǎng)基上,白色念珠菌為無(wú)色,其他念珠菌為紅色;在血清上培養(yǎng)形成芽管;玉米粉吐溫培養(yǎng)基誘導(dǎo)出厚膜孢子。IST間區(qū)基因序列分析,分離株同源性為96.9~97.4%,與GenBank白色念珠菌的同源性為97.4%,而與其他念珠菌的同源性為94.6~76.92%。基因分型為A型和C型。結(jié)合形態(tài)特征和基因同源性分析,該分離株被鑒定為白色念珠菌。根據(jù)GenBank已發(fā)表的白色念珠菌rDNA內(nèi)轉(zhuǎn)錄間區(qū)核苷酸序列設(shè)計(jì)一對(duì)目的擴(kuò)增子長(zhǎng)度為273 bp的特異引物,采用LightCycler實(shí)時(shí)PCR(LC-PCR)檢測(cè)方法,以SYBR GreenⅠ為擴(kuò)增產(chǎn)物熒光染色劑,對(duì)禽白色念珠菌疑似病例血液樣本進(jìn)行檢測(cè),并用臨床常見的5種病原真菌對(duì)該方法的特異性進(jìn)行檢驗(yàn)。建立的雞白色念珠菌檢靈測(cè)方法敏度高,對(duì)白色念珠菌最低檢出濃度為101CFU/mL;特異性強(qiáng),與光滑念珠菌、克柔念珠菌、熱帶念珠菌、近平滑念珠菌、煙曲霉等病原真菌無(wú)交叉反應(yīng);耗時(shí)短,只需2 h即可完成整個(gè)試驗(yàn)過(guò)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，
本文編號(hào)：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，
本文編號(hào)：2194346
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