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分子遗传学的方法识别和打字的致病性念珠菌酵母:复习一下

舒如提辛格*
助理教授,生物技术学系Mithibai大学,邪恶的说(W),孟买,印度
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国际创新研究期刊》的研究在科学、工程和技术

文摘

念珠菌属是一个医学上重要的真菌因其高频率作为共生体和病原微生物引起表面以及侵入性感染。念珠菌病的准确诊断仍然是困难的,因为快速和可靠的分析鉴定真菌病原体是至关重要的早期启蒙足够的抗真菌治疗和/或预防措施的介绍。新颖的分子遗传技术不断引入,他们在传染病管理中的作用也日益.Strain打字和物种的描述是必不可少的对于理解其生物学、流行病学与人口结构。广泛的分子技术已被用于此目的包括non-DNA-based方法(多位点酶电泳),传统的基于DNA的方法(电泳核型分析、随机扩增多态性DNA扩增片段长度多态性、限制性内切酶分析,没有杂交,rep-PCR)和基于DNA的方法叫做确切类型(基于PCR的方法、microstallite长度多态性茎序列类型,DNA微阵列),因为它们产生明确的和高度可再生的输入数据。今天,分子策略补充传统的方法和提供更准确和详细的见解。可以预期,未来的技术发展将提高他们的潜在此外。在本文中,我们提供一个评论在积极分子念珠菌鉴定方法和发酵领域打字和总结他们的优势和局限性对歧视性的权力、再现性、成本和易用性的性能。

关键字
念珠菌属、序列多态性、分子方法,应变打字,遗传多样性,

介绍
念珠菌是一种常见的共生的微生物在人类和一些温血动物。标准白念珠菌的基因组是二倍体和组成的八条染色体同源染色体,范围从约33到095 Mb [1]。白色念珠菌为主的无性繁殖方式。它是第一个医学上重要的致病真菌的完整基因组序列决心[2]。第一个基因组测序是SC5314临床孤立显示高度杂合性,包括超过55 700单核苷酸多态性(snp) 32 mb二倍体基因组[2]。尽管它的二倍性和克隆繁殖,白念珠菌能够实现高速率的遗传多样性在几个方面,包括复合、染色体多态性,基因替换和神秘的交配,反映了基因组的可塑性[1],[3]。在过去的三十年里,白念珠菌已成为医学上重要的病原体,负责表面以及深部感染。大多数感染机会取决于宿主的免疫防御和改变环境的酵母的有机体。事实上,自1980年代初以来,侵袭性念珠菌病,主要是由念珠菌引起的物种,已成为一个突出的问题[4],因为越来越多的免疫功能低下的患者和医疗的进步重症监护。
这凸显了需要实施适当的控制措施,这显然需要一个高的念珠菌属物种的生物学和流行病学知识包括白念珠菌,是公认的特别复杂。事实上,同样的健康的个体可以港口同样的菌株在不同的身体位置,或携带菌株在相同或不同的身体无关的网站[5],菌株可以取代在复发性感染[6],接受微进化(微小的变化在基因型相对较少的细胞代[7]和substrain洗牌(亚种群内个体随时间变化)从一个人转移到另一个[8],[9],[10],特定的菌株可能占主导地位在特定的地理区域和一些在一些医院和流行菌株可以接受微进化在医院设置。这凸显了需要高效的菌株类型和描述的方法。最早的打字方法白念珠菌和非白是基于表型特征包括血清型、生物型、形态类型、耐各种化学物质和毒素和抗真菌敏感性概要[11]。然而,表型技术很低程度的歧视和再现性,这显然构成限制进行可靠的流行病学分析。分子dna技术的出现彻底改变了白念珠菌的生物学和流行病学的知识。审查的目的是(我)来描述这些分子的原则方法,(2)强调他们的优点和缺点和(iii),讨论他们在输入不同类型的白念珠菌分子调查。

二世。相关工作
审查文件可用白念珠菌的分子生物学[7],[12],[13],[14],[15]解决当前推进白念珠菌分子遗传学的理解。此外,白念珠菌研究非常有用的WWW服务器(http://alces.med.umn.edu/candida.html)已由谢勒博士(美国明尼苏达大学、圣保罗、锰)的遗传信息,物理映射序列数据和其他有用的资源(方法论、代谢、形态等)对这个生物。[16]。血培养及表型方法相比,基于DNA的技术已经通过几个微生物实验室白念珠菌的快速和客观的识别。基于DNA的敏感性分析是依赖于样品制备,底漆和DNA目标选择,提取DNA,和放大效果徐出版社[17]发现和识别许多种类的真菌Clavariopsis水生,Tetracladium marchalianum植物鱼;Phyllosticta、Coleophoma Epicoccum, Godronia、链格孢属Pestalotia,通过DNA微阵列和帽状幼体动物;不同种类的念珠菌、新型隐球菌复杂,酿酒酵母,镰刀霉,f以上和Scedosporium prolificans人类太PCR方法。同样Spampinato茎等[18]使用输入(MLST)和微卫星序列长度多态性(MLP)分析依赖于DNA序列的内部区域的各种独立的管家基因,和类型的C。白色的和C。非白色的物种和告诉我们,这两种方法产生明确的和高度可再生的数据。几个技术,RAPD、RFLP和埃克被许多科学家使用最广泛的技术[19],[20],[21],[22],[23]白念珠菌的类型,其他念珠菌属物种(例如c . dubliniensis c . parapsilosis c . lusitaniae c . tropicalis c glabrata和c . krusei)和其他真菌(例如来自烟曲霉菌、黄曲霉、皮炎芽生菌新型隐球菌和)Skrzypek et .al[24]在他的论文中其他不感染念珠菌菌株的基因组序列,和完整的基因组序列为白念珠菌(WO-1), c . dubliniensis c . tropicalis c . parapsilosis念珠菌guilliermondii,念珠菌lusitaniae现在可用。假丝酵母基因组数据库(CGD)不断更新和其中几个被广泛用于研究团体(例如,http://genodb.pasteur.fr/cgi-bin/WebObjects CandidaDB; Genolevures at http://www.genolevures.org/yeastgenomes .html Sanger Institute at http://www.sagar.ac.uk/projects/ Fungi/ and Broad Fungal Genome Initiative at http://www. broad.mit.edu/node/304).This has undoubtedly opened enormous avenues to have comparative study of the genomes to identify the potential candidate genes responsible for virulence.[25].PCR based analysis offer a number of technical advantages over conventional typing methods including extremely high DP values and reproducibility, ease of use, and rapid reliable data.The selection of the technique depends on the purpose of the study, the accessibility of genotypic strains archives, the time available to complete the analysis, and the cost. Guiver et al [26] used TaqMan PCR for rapid identification of clinically important Candida species (C. albicans, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, and C. kefyr), and primers and probe sets have shown 100% of specificity for their respective species. Likewise, Selvarangan et al [27] observed that six Candida spp. (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, andC. lusitaniae) targeted by the real-time PCR approach using the LightCycler software were identified with 100% sensitivity and 100% specificity. Bougnoux et al [28] reported sensitive rolling-circle amplification (RCA)-based method using real-time PCR and Nested PCR for identification of clinically important Candida, Aspergillus, and Scedosporium species. The development of DNA sequence-based technologies led to a great progress in understanding the epidemiology of clinical isolates of Candida species.Here, in the review we focused to address a broad range of molecular techniques that have been used for identification and typing Candida species in detail.

一般考虑
基因分型念珠菌菌株允许以下:(i)院内念珠菌病调查,确定outbreakrelated菌株,区分流行性和地方性或零星的菌株和确定感染的起源,菌株的采集和传播的路线;(2)评估隔离在一个载体的多样性和复发性感染的调查识别特别是毒性菌株是否;(3)监测的耐药菌株的出现;(iv)研究的人口结构、物种的多样性和多变性。应变输入方法的选择取决于每个分子的性质和定义目标的调查。每个输入技术的性能应该评估方面的歧视性的权力,重现性和易用性表现和解释。歧视性的权力是指技术识别相同的应变的能力或高度相关的菌株在独立的隔离,评估微进化在相同的应变,集群组或适度相关菌株和区分无关的菌株[29],[30]。

三世念珠菌属物种的鉴定方法

一)Non-DNA-Based输入方法:1。多位点酶电泳(MLEE)
MLEE或同工酶类型特征酶蛋白质和评估他们的多态性通过分析后在凝胶电泳淌度特定酶染色程序。一种酶的迁移是受其分子大小和净电荷的影响。改变酶蛋白的流动反映其氨基酸序列的变化,因此,通过推理,编码DNA序列。因此,如果两个分离的酶带模式不同,这种差异是基于假定为DNA和遗传。MLEE是一种可靠的技术具有相对较高的歧视性权力无关的菌株之间的区别并显示良好的再现性[30],[31]。医学真菌学领域,同工酶类型在分类的研究已经显示出巨大的潜力,分类学,遗传学,进化的,和流行病学特征,尤其是酵母白念珠菌[32]。酶反应可以证明通过在凝胶电泳带的酶活性,表明同功酶或同功酶的存在。同功酶构成的多个分子结构相同的酶与个人都喜欢相同的底物,催化相同的细胞反应。发生基因的控制通过一个或多个等位基因或基因,位于一个或多个位点。同功酶控制通过等位基因从一个位点被称为alloenzymes或异型酶牵引,电泳迁移是由基于氨基酸序列的净电荷,因此,移动变异(electromorphs或alloenzymes)可以直接与相应的结构基因位点的等位基因。 For N meningitidis serogroup A, MEE has been used, in conjunction with outer membrane protein profiles, to divide 423 strains into four major families composed of 21 clones. Subsequently, MEE was used to detect the intercontinental spread of group A disease due to a clone (designated III-1) which caused outbreaks in Nepal, Saudi Arabia and Chad [33] It outperforms many of the DNA-based methods and remains a useful tool for molecular investigation of natural populations of C. albicans [34]. The major drawback of the MLEE is that it assays the genome indirectly and evaluates variations accumulated very slowly in the species. On the other hand, MLEE does not detect all variations at the nucleotide level as nucleotide substitutions do not necessarily lead to a change in the amino acid composition of the enzyme.

B)传统的基于dna的输入方法

1。Pulsed-field凝胶电泳(PEGE):电泳核型分析
pulsed-field凝胶电泳(但是脉冲场凝胶电泳的出现)技术的出现,在1984年彻底改变了研究真核生物的基因组的组织。这种技术及其变种(orthogonal-field替代凝胶电泳(求学),电场反转凝胶电泳(不要),contourclamped均匀电场(厨师)或横向交变场电泳(TAFE)],完整的DNA分子通过琼脂糖凝胶迁移矩阵的影响下脉冲字段,允许简单的几个megabases DNA分子的分离。白念珠菌染色体的大小范围从1到4 Mb,这种技术非常适合chromosomesized DNA分子的分离,染色体显带分析模式,称为电泳分析,发现在物种核型的变化。最早使用电泳核型分析(EK)证明的程度变化无关的白念珠菌分离株的分析[35],[36]。事实上,尽管一个标准的发生由八条同源染色体核型,变异分析临床分离株中非常普遍。乐队的数量差异发现和他们的移动模式被认为是由于chromosome-length多态性(不同大小的染色体同源染色体),染色体相互易位染色体或失踪[37]。艾克已经广泛用于指纹白念珠菌和其他念珠菌属物种(例如c . lusitaniae c . parapsilosis c tropicalis和c . glabrata) [14], [19]。它有一个温和的歧视性的权力当用于输入无关的白念珠菌分离株之间的歧视和不允许适度相关隔离和检测microevolutionary相同应变的变化。由于出色的再现性,电泳核型分析已经被广泛使用作为白念珠菌的分子标记方法显示更大的歧视性的力量相比,RFLP没有杂交,在独立隔离的化验。然而,EK显示良好的重现性,分析简单明了的解释[36]。

2。限制性内切酶分析(REA)
限制性内切酶分析(REA)是最早广泛应用于白念珠菌感染的流行病学调查[38],[39],[40],[41]。在这种技术中,总基因组DNA纯化和随后裂解频繁切限制性内切核酸酶(例如EcoRI, MspI、BglII HinF1或HindIII)产生大量的短片段导致顺序相依限制片段长度多态性(RFLP)。生成的片段使用常见的琼脂糖凝胶电泳分离和可视化与溴化乙锭染色后。不同的条带模式菌株之间的差异体现了这一点。这些变化发生的更改或删除限制网站删除和插入序列或DNA识别网站.REA之间的简单,快速和便宜。然而,它可能会导致复杂的模式的生成与大量的不平等的强度,从而使他们的目标解释和菌株的分化非常困难,无论是视觉上还是使用计算机辅助方法。简化条带模式获得的限制性内切核酸酶消化和提高辨别力的意图分析,生成的片段可以用特有的放射性标记或生物素化的DNA探针杂交后转移到硝基或尼龙膜(印迹南部)。探针识别重复序列分散在整个基因组的序列同源性。溴化乙锭染色法相比,杂交探针允许有限数量的碎片的选择性可视化并生成配置文件更容易解释,但与此同时,复杂到足以提供一个准确和敏感的隔离措施,反映了亲缘[42]。最常用的探针是相关(但不恒等的),27日包含重复的基因组序列和Ca3探测这些石头剪刀探针序列包含重复的元素和non-RPS白念珠菌的序列,因此杂交的很大一部分相同的乐队在印迹杂交检测。然而,这两个探针是不相同的。 The 27A probe contain sequences downstream from the RPS groups that hybridize with single bands, while the Ca3 probe contains sequences upstream from the RPS groups that hybridize with single bands [43]. For comparison, the Ca3 probe shows greater complexity than does 27A, and moreover, contains an additional repetitive sequence called sequence B. In general, its pattern also shows more complexity and satisfies the four requirement necessary for an effective DNA fingerprinting method .The Ca3 and 27A probes were also employed in various epidemiologic studies of C. albicans and the phylogenetically related species C. dubliniensis .When used to probe Southern blots of EcoRIdigested DNA, Ca3 identifies over 20 bands of various intensities that include invariable (monomorphic), moderately variable and hypervariable bands [44]. The Ca3 probe has been demonstrated to be highly effective in the analysis of C. albicans populations and has identified five major clades (named I, II, III, SA and E) of closely related strain types [45],[46].In addition, Southern blot hybridization with probe 27A also shows greater discriminatory power in clinical strains of C. albicans when compared to results from EK, REA with Not I and PCR fingerprinting .Inherent drawbacks of Southern hybridization techniques include several laborious and time-consuming steps and the need to use DNA probes, which requires a transfer procedure to a solid support as well as adequate detection systems making implementation of the method in a medical laboratory analysis difficult. In addition, fingerprint data do not lend themselves to inter laboratory data exchange.

3所示。随机扩增多态性DNA (RAPD)
RAPD技术是基于基因组DNA的放大与单一短(通常10个基点)任意序列的引物。随机引物绑定到目标DNA片段的扩增未知序列。放大反应进行低紧缩的条件下(通常是35 - 40°C, 25更易与l MgCl2)。放大产品琼脂糖凝胶上分离和溴化乙锭染色。RAPD的解释模式是基于数量和大小的扩增片段。总的来说,RAPD分析生成相对复杂的模式,极大地改变隔离无关。RAPD已广泛用于打字的白念珠菌、其他念珠菌属物种(例如C . dubliniensis C . parapsilosis C . lusitaniae C。tropicalis, c . glabrata和c . krusei)[20],[21]和其他真菌(例如来自烟曲霉菌、黄曲霉、皮炎芽生菌新型隐球菌和)[22],[23],因为它提供了许多优点,包括简单,速度和costeffectiveness不需要任何DNA序列信息[20]。RAPD的歧视性的力量是温和的;然而,如果需要更大程度的歧视,不同引物可用于独立运行和最后的数据结合分析(DNA复合类型)。This approach allows the discrimination between unrelatedisolates, clustering of related isolates, identification of dentical or highly related isolates and assessment of microevolution within the same strain. Indeed, Pujol et al. demonstrated a high concordance between RAPD analysis, MLEE and Southern blotting using the Ca3 probe. The Ca3 probe exhibited the greatest resolving power mainly in assessing microevolution within the strain [47]. Although the RAPD technique has been used successfully in several clinical studies, it raises the well-known problem of reproducibility and data comparison between laboratories. Indeed, banding patterns are easily affected by slight changes in the experimental conditions (Mg2+ concentration, primer-to-template–concentration ratio,Taq polymerase concentration and source, the model of thermal cycler, thermal cycling parameters, etc.) [48].Nevertheless, a good interassay reproducibility can be achieved under rigidly controlled conditions.

4所示。扩增片段长度多态性(妊娠)
在1990年代中期妊娠开发[49]。简单地说,它涉及基因组DNA和两个限制性内切酶消化(通常是一个频繁的刀和一种罕见的铣刀)其次是结扎的寡核苷酸适配器的粘性末端限制性片段[50]。适配器与网站限制序列作为引物退火的目标,然后结扎产品高紧缩条件下放大。这个过程允许限制片段的一个子集的选择。通常,50 - 100放大产生碎片。可视化,这些片段需要在高分辨率的电泳分离系统(变性聚丙烯酰胺凝胶)。荧光dye-labelled引物可用于允许放大碎片的检测凝胶或毛细管DNA测序仪。这种变体技术被称为荧光扩增片段长度多态性(FAFLP)允许的最高分辨率不同的碎片大小[51],[52]。妊娠通常涉及两个PCR步骤。第一个由预选式放大利用未标记的引物用单一选择性核苷酸引物。 The reaction product is then diluted to obtain the adequate template concentration for the second PCR amplification in which additional selective nucleotides are added in the primer to improve specificity. During this latter amplification, the labelled primers are used. AFLP is a highly resolutive typing technique. Like RAPD, AFLP is a universal and multilocus marker technique that can be applied to genomes of any source without requiring any prior sequence information. However, AFLP is more reproducible than RAPD as it uses specific primers, and amplification is achieved under high stringency conditions [49], [50]. Although AFLP has proved to be reliable and reproducible as a genotyping method, it has been rarely used for C. albicans typing mainly because it is multiple-step, fairly expensive and requires a relatively high level of expertise [50],[52],[53].

C)的基于dna的打字方法。1。pcr方法
PCR的发明是一个具有里程碑意义的进展的分子微生物学和已经在传染病的诊断产生重大影响。这些技术的主要优点在于放大和检测微量微生物核酸的宿主DNA的背景。

1.1目标和引物的选择
一般来说,两种策略的PCR可以采用目标选择。如果种特异的序列是选为primerannealing网站,PCR将使高度特定检测致病性酵母菌种。另一方面,当万能锅真菌序列是有针对性的,聚合酶链反应将导致扩增子,以防任何真菌DNA存在于样品。Candida-genus特定序列也可以针对假丝酵母检测所有物种。如果目标物种更广泛的光谱,post-PCR分析对于后续的物种鉴定是必要的。确保PCR检测的高灵敏度,引物应优先目标multicopy基因。同时,专门针对序列应该获得高特异性发现,只有在感兴趣的病原体。核糖体RNA (rRNA)基因似乎满足这两个条件。一系列串联的50到100的副本的单倍体基因组核糖体rna基因可以找到所有的真菌。这个由小亚基rRNA基因(18岁),5.8 s基因和大亚基rRNA(25)基因,由内部转录间隔区地区,ITS1和ITS2。 While rRNA genes are highly conserved in fungi, ITS regions involve both highly variable and highly conserved areas [54], thus allowing the generation of species,genus or fungus specific primer.

1.2嵌套PCR
嵌套PCR可用于提高PCR检测的敏感性和特异性。在这种方法中,两轮PCR。在第一轮,外部引物目标放大的较大的地区。从这一轮扩增子然后作为模板添加到第二轮反应混合物,在第一轮的内在目标片段引物扩增子。特异性试验的增加,因为四个引物退火安排的方式而不是单一PCR一分之二。敏感性增加,因为增加新鲜的试剂和稀释的第一轮扩增子在第二轮混合使额外的扩增片段的扩增子从第一轮混合物。两轮的嵌套PCR也可以用来结合广泛的优点和导致的酵母的目标序列。外部引物可以针对通用序列导致扩增子在广泛的酵母菌种,其次是几个平行的第二轮与特有的内在反应引物对。当第二轮引物是精心设计,防止干扰,底漆可以使用混合在一个常见的反应混合物降低成本,在一个名为多重PCR的方法。嵌套PCR方法是用于念珠菌属物种的检测[55],[29]另一方面,嵌套PCR结果的极端敏感性的主要缺点,假阳性结果的出现主要是由于之前放大产品的交叉污染,同时由于污染与环境微生物,甚至污染商用试剂。 To avoid this pitfall, laboratories must follow stringent precautions such as establishing separate rooms and equipment for each step of the PCR and other procedures [56], [57].

1.3。实时聚合酶链反应
实时聚合酶链反应使用荧光记者分子可视化的生产PCR的扩增子在每个周期的反应。这与端点检测在常规PCR,扩增子在哪里检测完成后只放大。实时监测荧光放大的基础上增加的记者使量化目标DNA的分子,因为这个时间点的扩增子达到一个特定的荧光水平在自行车与起始目标基因的数量。这种相关性是不可能在常规PCR,扩增子的金额都达到统一水平由于抑制反应的高原期进一步放大。放大的过程中可以使用标记探针监测特别是新成立的扩增子的分子杂交,或通过与非特异性染色新形成的双链DNA分子dsDNA绑定染料(如SYBR绿色我,BEBO, LC绿色或BOXTO)。使用探针增加PCR的特异性,因为额外的序列之间的同源性扩增子和放大探测器是成功所必需的报告。当dsDNA绑定染料代替特定的探测,融化的扩增子的分析必须随后执行验证扩增子的身份。有时,明确区分特定和非特异性的产品可能会有问题。另一方面,融化的分析可以提供额外的有用的信息放大序列。使用一个集成的thermocycler /荧光计与高效热交换机制大大缩短实时PCR的周转时间。Both amplification and detection take place in the same closed vessel, reducing post-amplification manipulation steps and dramatically decreasing the risk of false-positive results. Despite greater start-up expense and the lack of standardization, the oncoming explosion of new chemistries and instrumentation, sensitivity, reproducibility and potential for high-throughput, will nevertheless make the real-time PCR attractive and indispensable for future diagnostic mycology.Several studies have reported the identification of Candida species by targeting the rRNA gene complex using real-time PCR.[58],[26],[59]

1.4。Post-PCR分析
无论是传统还是使用实时PCR, post-PCR分析几个选项可用于描述在其物种特异性扩增子和结论可以得出,特别是通用序列是针对放大。显然,唯一的终极post-PCR分析和最准确的方法是直接测序[60]。然而,替代测序技术,如焦磷酸测序,正在不断发展并承诺在未来进一步降低成本。所有的其他技术扩增子post-PCR分析在某种程度上依赖sequence-related可变性的特征。扩增子的长度可以大致估计通过琼脂糖凝胶电泳,post-PCR代表最简单和传统技术分析,也称为放大产品长度多态性(APLP)。更准确的描述长度的扩增子可以通过聚丙烯酰胺凝胶电泳,可在要自动化分析仪[61],[62]。限制分析扩增子代表了一种廉价和优雅但费力而更耗时的技术。同样,singlestrand构象多态性(SSCP)可以用来评估序列扩增子的特点[63]但不是因为特殊的专业知识和广泛使用的劳动力需要正确的性能。避免了费时和费力电泳一步,传统上用于上述技术,可以应用两种方法。微量滴定板酶免疫分析法(PCR-EIA)可以利用一个用户友好的替代,也可以提高检测灵敏度。

2。基于核酸序列的扩增(NASBA)
NASBA是一个特定的和非常敏感的核糖核酸扩增技术,它利用三种酶的作用,即逆转录酶、核糖核酸酶H和T7 RNA聚合酶,在等温扩增过程cDNA作为中间[64],[65]。在医学上重要的真菌,保守地区的18 s rRNA基因可以针对性的放大。然后标记寡核苷酸探针杂交假丝酵母内部特定序列的物种。放大和检测可以在几个小时内完成和分析表明1 CFU的检出限。NASBA已经评估检测六各种念珠菌属物种[66]。NASBA PCR相比的主要优点是不需要热循环仪和具体检测活酵母细胞,因为RNA与DNA细胞外迅速退化。主要的缺点,从而防止NASBA的使用越来越广泛,高价格的三个酶混合物。

3所示。荧光原位杂交鉴定(鱼)
荧光原位杂交(FISH)与fluorescein-labelled寡核苷酸探针是一种方便的方法来检测酵母没有纯培养的需要。小说的就业机构(肽核酸)探针结合了他们的高亲和性和优势的目标高度结构化rRNA地区扩展这种方法的潜力。短暂,探针杂交,涂片直接从血培养瓶在幻灯片的内容,non-hybridised探针都冲毁,幻灯片是由荧光显微镜检查揭示生物体的存在。方法的灵敏度据估计至少类似于大多数结果通过pcr检测[67]由于一个简单的技术协议排除DNA提取,整个机构鱼只需要2.5小时后血培养是指定正通过一个自动化的血培养系统,整个过程适用于自动化。鱼包括探测特定的念珠菌属物种已被证明是一个合理的诊断工具对于物种识别[68]。机构鱼了白念珠菌与nonalbicans念珠菌属物种[69]评估一项多中心研究及其实现医院减少抗真菌药物费用相同的小组还进行了机构鱼测定白念珠菌与c Dubliniensis [70]
上面描述的大部分技术已被证明是有效的和可靠的打字方法为白念珠菌流行病学的调查在当地的水平。这些技术适用于临床研究在个别实验室设置,但实验室之间的数据交换和比较困难,如果不是不可能的。目前,只有两种技术就有这种交流因为他们产生明确结果具有良好的重现性。出于这个原因,对白色念珠菌分离株做基因分型,最近出现了两个pcr方法:精确的基于dna的打字方法调用。它们包括微卫星序列长度多态性(MLP)和茎类型(MLST)

4所示。微卫星长度多态性(MLP)
延时输入是一个pcr系统,利用重复的高可变性的微卫星序列,定义为衔接着重复的2 - 6个核苷酸。微卫星标记由一对引物定义在一个特定的侧面在基因组微卫星区域。PCR扩增片段长度根据不同的数量的微卫星重复。微卫星显示高多态性水平和孟德尔共显性的继承,因此可以作为优秀的候选基因分析。对于每个隔离,延时输入标识的存在一个(纯合子)或两个(杂合的)不同的片段,或等位基因,在给定的轨迹。微卫星是高度可变的序列,一个关注MLP方法而不是继承是等位基因可能是相同的突变,导致类似[71],[72]。荧光标记引物用于放大特定位点等位基因的长度是衡量在高分辨率的凝胶电泳迁移的PCR产品通过自动测序仪[73]。等位基因的长度数值数据,可以很容易地比较。
延时是容易执行,快速,适合自动化和高吞吐量。中长期规划总体而言,是一种最歧视白念珠菌的方法类型。然而,它的分辨率取决于所使用的微卫星标记。几个多态性微卫星位点(例如EF3, CDC3 HIS3, ERK1, 2 nf1 CCN2, CPH2, EFG1, CAI和CAIII CAVII)已确定在白念珠菌基因组中,展示一个不平等的歧视介于057年(CAV)和097 (CAI) [73], [74]。相比传统技术在测试标本的数量相同的运行受到传统电泳的约束,简要分析被证明是非常适合应用在大规模流行病学研究多达96个样本可以分析一个运行。[74]。延时是一个健壮的技术和强烈推荐的流行病学研究白念珠菌等真菌新生隐球菌[75],如来自烟和[76]。然而,固有延时的缺点包括专用设备的高成本和实现技术在常规使用的难度,特别是在发展中国家。为了克服这个困难,李、白描述单链构象多态性(SSCP)微卫星CAI和显示SSCP分析的微卫星标记是一个强大的(0993年的)和具有成本效益的方法,用于快速应变类型的白念珠菌在临床实验室,尤其是microevolutionary变化的检测。这种技术后来被用于外阴阴道炎的准确输入白念珠菌分离株[77]

5。茎序列输入(MLST)
MLST通常是基于分析核苷酸序列多态性在内部六到八个独立的基因片段的序列(位点)。基因选择MLST分析通常是那些管家功能,稳定的选择。此外,选择位点必须提供尽可能多的序列多样性,使高水平的等位基因歧视[15]。MLST涉及DNA片段的扩增PCR(400 - 500个基点)的DNA测序紧随其后。对于每一个管家轨迹,不同序列视为不同的等位基因。每个隔离因此表现为一系列的等位基因在茎的不同位点对应序列类型。由DNA序列分析是明确的和生成的数据可以存储和可存取的数据库[15],[78]。中长期规划相比,MLST被证明是高度可再生的实验室之间,从而易于标准化和可移植性[78]。它允许交换基因分型数据和建设的国际数据库可以通过互联网访问。目前在线全球数据库对许多微生物可以在www.mlst.net,从流行病学研究包括数据在全球范围内进行。 This permits global epidemiological and population analysis. MLST methodology was first developed and used for typing pathogenic bacteria and later some pathogenic fungi (e.g. Cryptococcus gatti, Fusarium solani and Batrachochytrium dendrobatidis) [79], [80]. The technique was applied to C. albicans in the early 2000s. The first protocol,based on the analysis of six loci and a second one, based on the analysis of eight loci, were developed by Bougnoux et al. in 2002 [15] and Tavanti et al. in 2003 [81], respectively. Later, an optimized protocol based on a set of seven loci was proposed as an international consensus unifying the MLST scheme for C. albicans [82]. Results from strain typing using this system are shared through public Internet-linked database (http://calbicans.mlst.net/.) where data have been accumulated from different geographical locations. This database provides an interesting source to evaluate the worldwide diversity of C. albicans and the relationships of isolates identified at various locations. It is worth mentioning that MLST is the only typing method that has a public database, not only for C. albicans but also for C. tropicalis, C. glabrata, C. dubliniensis and C. krusei. In contrast, C. parapsilosis shows too little sequence diversity to be typeable by MLST [78]. Because C. albicans is diploid, nucleotide sequences generated by the MLST analysis are likely to show heterozygosity at polymorphic sites, and therefore,strains are unambiguously characterized by a diploid sequence type (DST) .MLST analysis has been successfully applied to population genetics and molecular phylogeny studies of C. albicans [6],[15],[78]. In population genetic studies, MLST data have confirmed previous studies in the field and, in some instances, refined our understanding of the epidemiology of C. albicans [6].Population genetic analysis of C. albicans MLST data concerning isolates obtained from separate sources showed that the species could be divided into a large number of clades and that clades differed in the proportions of isolates they included according to the geographical origin and the anatomical sources.
白色念珠菌人口结构由MLST类型表现出良好的相关性与Ca3指纹识别获得的聚类,与演化支I, II, III和SA分隔Ca3调查。然而,进化枝E为被Ca3指纹已经被MLST分析分离成不同的演化支(。MLST的局限性的特征无关的菌株可能解释这一事实(i)方法分析了只有7 300 - 400 bp位点序列,以便隔离与相同的数据可以通过大型基因组重组显著不同的地区不包括测序基因座和(2)的二倍体性质白念珠菌会导致两种收益率相同的数据,即使他们可能不同的杂合的基地组织的多态网站[78],[82]。额外的问题提出的白念珠菌MLST分析是三白念珠菌的染色体(3、5和7)并不代表一致的方案。洛特和斯卡伯勒发达SNP分析微阵列来补充MLST打字和延长酵母染色体SNP。阵列由多个复制79个snp,来自19个位点位于所有八个染色体,包括七个基因组成MLST共识(57个snp)计划。剩下的22个snp是来自12个额外染色体位点位于其余[9]。还需要进一步的研究来评估系统的输入在白念珠菌的流行病学和种群遗传学研究。延时和MLST满足若干标准基因型白念珠菌分离株的广泛的有用的工具。首先,所有的隔离typeable两种方法。 Second, the variability within the selected sequence was sufficient to differentiate 50 unrelated isolates and the discriminatory power was >0.99 for both methods. Third, the resulting data can be stored in digital form for subsequent analysis with the aid of specialized software. A theoretical disadvantage of both methods is that only very small regions of the C. albicans genome are analyzed, in contrast to techniques such as RAPD, which potentially examines the entire genome.

6。dna芯片
芯片系统提供一个有吸引力的前景不仅对未来的应变类型。他们提供高水平的敏感性,特异性和吞吐量能力,不需要先验知识的特定序列。芯片或芯片高密度微观的寡核苷酸探针固定在固体表面上,以核酸样品杂交。完全匹配的序列样本杂交效率相应的寡聚物的数组,给信号比不匹配的序列。最后一个信号是高分辨率荧光扫描和探测到由计算机软件分析,从而使自动化和标准化。[83]容易管理庞大的数据生成和减少dna芯片的成本只是一个时间问题。然后,微阵列肯定将从临床实践的研究领域。用于输入,微阵列可以直接识别的存在和数量不同序列变异的特定基因或地区,e.g.rRNA基因,内部转录间隔器(同期)。在致病性酵母菌进行测序项目也将很快使很简单的设计全基因组DNA微阵列[84]。微阵列的使用微生物指纹已经报道沙门氏菌分离株,为密切相关、黄pathovars和分枝杆菌物种[85],[86]。快速自动化性能的成千上万的杂交分析微型芯片是最有力的技术。

四。结论
传统的基于dna的方法和准确的基于dna的方法提供了有益的见解的流行病学和人口结构与其他酵母白念珠菌和非白的物种。传统的方法的主要缺点在于缺乏标准化,减少潜在的多个实验室的比较,因此全球人口研究;但这些方法非常适合调查流行病学趋势在当地的水平。精确的基于dna的方法,包括基于PCR的方法,简要MLST,已成为非常有效的输入工具。这些方法的主要优势是生成的数据明确的和高度可再生的和可以存储在数据库提供前所未有的可移植性和可访问性对所有感兴趣的用户。这种技术更适合全球流行病学。目前,MLST是唯一的输入方法,有一个公共数据库和代表最强大的系统发生学方法白念珠菌,而延时分析需要进一步标准化。

诉确认
我想表达我衷心的感谢我的父母,他们的祝福,我的同事们的帮助和祝福,成功完成了这篇评论文章。我还想感谢博士D。令Kamat V(头),微生物学系Mithibai大学,邪恶的说(W),孟买。

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