ITS基因测序分析对89株病原真菌鉴定的应用评价

摘要：目的：探讨内转录间隔区（ITS）基因扩增测序技术在病原真菌鉴定中应用价值。 方法：应用通用引物ITS1和ITS4对收集的89株真菌（包括酵母菌标准菌株6株,室间质评菌7株,临床分离的酵母样真菌42株和丝状真菌34株）进行PCR扩增和基因测序。测序成功的ITS序列在CBS数据库(http://www.cbs.knaw.nl/Collections)进行序列比对,结合Mycobank(http://cn.mycobank.org/)分类信息以获得其菌种鉴定信息。 结果：89株不同种类的真菌均成功扩增到ITS靶基因,经测序,89株真菌均获得扩增的全序列。序列比对结果显示,6株酵母菌标准株中,5株鉴定到种,1株鉴定到属;7株室间质评株中,1株酵母菌鉴定到种,6株丝状真菌中2株鉴定到种,1株鉴定到属,3株鉴定到复合群;42株临床分离的酵母样真菌中,39株鉴定到种,3株鉴定到属;34株临床分离的丝状真菌中,10株鉴定到种,18株鉴定到属,6株鉴定为复合群。 结论：通用引物ITS1和ITS4广谱性强。ITS基因序列扩增和测序分析可快速、有效鉴定临床疑难真菌,但仍存在数据分析过程烦琐和结果解释困难的问题。     

急性淋巴细胞白血病患者血液中草螺菌的鉴定

目的 分离鉴定源于急性淋巴细胞白血病（ALL）患者血液中的未知细菌.方法 采用常规及VITEK32微生物GNI鉴定卡鉴定细菌,K-B法进行药敏试验,PCR法扩增细菌的16S rRNA基因,通过测序并与GenBank中相关序列进行比对.结果 常规生化反应和微生物自动化鉴定系统不能鉴定.16S rRNA PCR扩增产物经测序与GenBank中序列比对,发现与草螺菌属有99%同源性.结论 来源于ALL患者血液中的未知细菌是草螺菌.     

rDNA-ITS序列分析对临床少见丝状真菌鉴定作用的评估

目的评价内转录间隔区（ITS）的多态性序列分析（rDNA-ITS序列分析）对临床少见丝状真菌的鉴定作用,以形态学鉴定方法加以补充验证。方法将3株待检真菌通过聚合酶链反应（PCR）扩增和基因测序方法分析rDNA-ITS序列,从GenBank获取相似序列,使用BLAST工具对rDNA-ITS序列进行对比分析,利用GenBank中的系统发育软件自动生成系统,构建系统发育树,根据系统发育树并结合对比指标确定距离与同源性均有较大鉴定意义的对比序列,并与真菌ITS1、ITS2、26S rDNA D1/D2片段的序列分析结果及形态学鉴定结果进行比较。结果 2株菌株能通过rDNA-ITS序列分析的方法鉴定到种,另1株由于相似序列较多,需要形态学方法加以配合鉴定。rDNA-ITS序列分析相对真菌ITS1、ITS2、26S rDNA D1/D2片段有更好的真菌鉴定作用。结论相对于传统形态学方法,利用rDNA-ITS序列分析鉴定丝状真菌不受检验人员经验水平影响,较为客观,同时由于其包含的信息量相对丰富,因此,较其他靶序列具有更好的真菌鉴定作用。但该方法也存在一定局限,因此,仍需结合形态学方法进行鉴定。     

海南省类鼻疽临床分离株核糖体16S-23S内转录间隔区序列多态性分析

目的 研究海南省类鼻疽伯克霍尔德菌核糖体16S-23S内转录间隔区（ITS）序列多态性及基因型特征,了解该省与其他类鼻疽流行区菌株间的遗传背景差异。方法 PCR扩增272株类鼻疽伯克菌的ITS片段并通过毛细管凝胶电泳检测产物长度;通过DNA测序及序列比对确认不同长度ITS的基因型及序列一致性;通过2检验分析不同流行区ITS型别的分布差异。结果 经毛细管凝胶电泳及DNA测序确认,272株类鼻疽伯克菌中发现C、E、CE、G 4种ITS基因型:C型64株（23.53%）,E型144株（52.94%）,CE型56株（20.59%）,G型8株（2.94%）。序列比对证实,海南省类鼻疽伯克菌中各型别（C、E、G）ITS序列高度保守,不同型别ITS的长度变化由其主要变异区的序列差异引起。我国海南省与泰国流行区的ITS型别分布差异有统计学意义（2=8.296,P0.05）,与澳大利亚流行区分布差异无统计学意义（2=5.521,P0.05）。结论 海南省类鼻疽伯克菌临床菌株中存在C、E、CE、G 4种ITS基因型,C、E、CE为优势型别;与泰国、澳大利亚等传统流行区相比,其G型菌株比例较高。     

大肠埃希菌的qepA基因检测及菌株同源性分析

摘要：目的：检测质粒介导的喹诺酮类外排基因qepA在临床分离的产超广谱β-内酰胺酶(ESBLs)的大肠埃希菌中的分布情况,并对阳性菌株进行同源性分析。 方法：用Vitek2 Compact系统对57株大肠埃希菌进行鉴定和药敏实验,用PCR法检测qepA基因并进行基因测序及序列比对。用细菌基因组重复序列PCR技术（rep-PCR）和芯片分析技术对qepA阳性菌株进行基因分型及同源性分析。 结果：57株大肠埃希菌对环丙沙星、左氧氟沙星、庆大霉素、妥布霉素及阿米卡星的耐药率分别为86.0%（49/57）、82.5%（47/57）、70.2%（40/57）、26.3%（15/57）和8.8%（5/57）。6株大肠埃希菌检测到qepA基因,检出率为10.5%。Diversilab分析结果表明,含qepA的大肠埃希菌相似性为70.4%~97.2%,未发现高度同源的菌株。 结论：临床分离大肠埃希菌菌株可检出质粒介导的喹诺酮类外排基因qepA。     

云南省西北地区家畜体表蜱类形态学与分子生物学鉴定

目的 调查云南省西北地区家畜体表蜱的种类及其种群遗传进化情况。方法 采集家畜体表寄生的蜱虫,经形态学鉴定后,用PCR法扩增蜱样本的16S rDNA和ITS2基因片段,测序后进行序列分析。结果 共采集成蜱样本1 275只,经形态学鉴定为1科3属4种,其中微小扇头蜱1 263只（占99.1%）,卵形硬蜱7只（占0.6%）,锐跗硬蜱4只（占0.3%）,未知血蜱1只（占0.1%）。样品分子鉴定结果与形态学鉴定结果一致。16S rDNA序列分析显示,蜱P6与印度微小扇头蜱（EU918188）的相似性最高为99.8%,与中国云南微小扇头蜱（JX051062）的相似性99.4%;蜱P2与美国卵形硬蜱（U95900）的相似性最高为93.8%;蜱P1与日本锐跗硬蜱（AB105167）的相似性最高为95.9%;蜱P4与澳大利亚parva血蜱（JX573136）的相似性为90.5%,与中国云南长角血蜱（JX051064）的相似性为88.7%。ITS2序列分析显示,蜱P6与来自老挝（KC503276）、中国云南（KC203364）的微小扇头蜱的相似性均为99.9%;蜱P2与日本卵形硬蜱（D88857）的相似性最高为96.1%;蜱P1与日本锐跗硬蜱（AB605168）的相似性最高为95.3%;蜱P4与罗马尼亚Haemaphysalis parva血蜱（FN296282）的相似性最高为91.0%。结论 云南地区家畜体表存在微小扇头蜱、卵形硬蜱、锐跗硬蜱和一种与parva血蜱相关的新型血蜱。     

复发性外阴阴道念珠菌病菌种的26S rDNA序列分析

目的探讨基于核糖体基因26SrDNA D1/D2区序列分析法在临床酵母菌菌种鉴定中的应用。方法收集来源于复发性外阴阴道念珠菌病分泌物标本93株,PCR扩增其26SrDNA D1/D2区,对扩增产物进行序列测定和分析,并与基因库中的基因序列进行同源性比对。结果所有菌株均鉴定到种,同源性达99%和100%,同属于真菌双核亚界、子囊菌门、酵母菌科的3个属,89株为candida,3株为Kodamaea,1株为Pichia。其中candida中有7个种,38株candida glabrata,23株candida albicans,16株candida parapsolisis,9株candida metapdilosis,1株candida orthop-silisis,1株candida tropicalis,1株candida nivariensis;3株Kodamaea ohmeri;1株Pichia kudriavzevii。结论复发性外阴阴道念珠菌病的病原体主要为candida属的candida glabrata、candida albicans和candida parapsolisis,非candidaalbicans占75.27%是其特征;26SrDNA D1/D2区序列分析为临床酵母菌的分子水平鉴定提供了一种准确、可行的方法。     

ITS序列鉴定真菌性鼻窦炎病原的方法评价

目的 建立ITS(包括ITSI-5.8 rRNA-ITS2)测序鉴定真菌性鼻窦炎病原的方法.方法 收集北京同仁医院2006-2008年,经临床与CT诊断为真菌性鼻窦炎,并行鼻内镜手术切除的组织标本270份.所有标本分别进行组织病理检查、压片直接镜检、真菌培养鉴定和核糖体RNA转录间隔区测序分析,通过方法比较,评价序列分析直接鉴定病原真菌的可行性,同时分析真菌性鼻窦炎病原学特征.结果 在270份标本中,组织病理阳性率为80.0%(216/270),压片阳性率为80.0%(216/270),真菌培养阳性率为53.0%(143/270),ITS测序阳性率为63.0%(170/270).经培养得到22个种,6个属.ITS测序鉴定32个种.培养与ITS序列种水平符合率为76.1%(102/143).结论 ITS测序可成为真菌鉴定的辅助工具.     

临床常见产AmpC β-内酰胺酶菌株中染色质ampC共同序列的研究

目的分析临床常见AmpC β-内酰胺酶（简称AmpC酶）产酶菌株中染色质ampC的基因序列,从而为AmpC酶的分子生物学检测以及其调控机制研究提供理论依据。方法 57株临床常见AmpC酶产酶菌株分离自医院感染患者样本,抽提细菌染色质DNA,聚合酶链反应（PCR）扩增ampC基因并连接入pMD19-T载体,双链测序后比对同种细菌之间和不同种细菌之间染色质ampC基因的同源性和共同序列。根据共同序列设计引物,进一步利用该引物检测染色质ampC。结果 57株细菌的基因组中,使用PCR扩增出染色质ampC基因41株,并成功测定了其ampC基因的序列。比对后发现大肠埃希菌、阴沟肠杆菌、鲍曼不动杆菌和产气肠杆菌的染色质ampC菌种内有很高的同源性,但细菌之间的同源性较低。根据共同序列设计出菌种特异性PCR引物,能够有效的鉴定出染色质ampC基因。结论大肠埃希菌、阴沟肠杆菌、鲍曼不动杆菌和产气肠杆菌各自染色质ampC具有高度同源性,其菌种特异性的ampC引物可用来检测其染色质ampC的存在。     

云南省不明原因发热患者恙虫病东方体实验室检测分析

目的 对云南省不明原因发热患者进行恙虫病东方体检测与分析。方法 应用间接免疫荧光方法（IFA）和PCR（nPCR）法对不明原因发热患者分别进行恙虫病东方体（Ot） IgG抗体和sta56基因检测及其基因序列测定分析。结果 在79例发热患者中, Ot IgG抗体阳性率48.10%, Ot核酸阳性率3.80%。基因序列分析显示序列12-17与日本O3株同源性最高为99.8%,序列12-9与中国台湾KM05、TW45R等株的同源性最高均为99.8%,序列11-6与中国台湾KM05、TW45R等株的同源性最高均为100%。进化树分析显示序列12-17、12-9和11-6均为Karp型Ot。结论 云南省恙虫病患者中存在Karp型Ot感染,其基因进化来源可能复杂。     

DNA sequence analysis of the ribosomal DNA ITS2 region for the Anopheles punctulatus group of mosquitoes

The internal transcribed spacer 2 (ITS2) from the ribosomal DNA was sequenced and characterized for ten cryptic species in the Anopheles punctulatus group, the members of which are major vectors of malaria and filariasis in the south-west Pacific. The length of the ITS2 ranged from 549 bp to 565 bp and displayed levels of sequence variation ranging from 2.3% to 24.3% due mainly to indels of simple sequences. The GC content varied from 61.3% to 70.9%. These values were higher than those found in other cryptic species of mosquitoes and comparable only to members of the An. dirus complex suggesting a possible link between this group of Asian mosquitoes and the An. punctulatus group. Optimal and suboptimal secondary structures were investigated and revealed structures where the 5' region folded independently of the 3' region. Due to the large level of sequence variation between species, the ITS2 region proved unsuitable for phylogenetic analysis.     

Ribosomal DNA internal transcribed spacer (ITS2) sequences differentiate Anopheles funestus and An. rivulorum, and uncover a cryptic taxon

Differentiation among the closely related Afrotropical species comprising the Funestus Group is difficult by traditional taxonomic measures. Anopheles rivulorum is the second most abundant and widespread species in the Funestus Group, and is occasionally collected indoors along with the dominant member and major malaria vector, An. funestus. The prospect of misidentification of An. rivulorum as An. funestus prompted the development of a rapid, polymerase chain reaction (PCR)-based method for identifying these two species. The ribosomal internal transcribed spacer 2 (ITS2) was amplified from thirty-five specimens of An. rivulorum collected from the extremes of its range: Eastern Africa (Kenya), Southern Africa (South Africa) and Western Africa (Burkina Faso). The ITS2 region of An. rivulorum ( approximately 380 bp) is sufficiently different in size from the ITS2 of An. funestus ( approximately 700 bp) that these species can be distinguished by agarose gel electrophoresis of PCR products without further manipulation. Comparison of the An. rivulorum and An. funestus ITS2 nucleotide sequences revealed such extensive divergence that meaningful alignment was impossible, except for a 25 bp island near the 5' end. Intraspecific sequence comparisons revealed no variation among An. rivulorum individuals collected from the same country. However, sequence divergence was 2% between specimens from South Africa and Kenya, and nearly tenfold higher ( approximately 19%) between specimens from Burkina Faso and either South Africa or Kenya, an unprecedented level of intraspecific ITS2 divergence in Anopheles. Taken together, these data suggest that the Burkina Faso sample is not An. rivulorum, but rather a cryptic taxon within the Funestus Group.     

Detection and identification of Candida spp. in human serum by LightCycler real-time polymerase chain reaction

The aim of this work was to develop LightCycler real-time polymerase chain reaction method to allow rapid detection and identification of Candida spp. in human serum with panfungal primers (internal transcribed spacer [ITS] and L18). Melting-curve analysis of the ITS sequences showed that each amplicon presented a specific melting point and enabled identification of 5 Candida spp. After parameters optimization, 58 sera were preliminary analyzed from 23 patients. For L18 primers, the LightCycler system enabled detection of DNA in 92% of patients with positive blood culture. These primers were not able to differentiate between species of Candida. By using ITS primers, the LightCycler system enabled detection of DNA in sera from 76.9% of patients with positive blood culture. With ITS primers, the species responsible for the infection was identified for 11 patients. These data revealed the LightCycler as a potential tool for early detection and identification of Candida.     

Morphological and Molecular Characterization of Ectomycorrhizal <Emphasis Type="Italic">Amanita</Emphasis> Species Associated with <Emphasis Type="Italic">Pinus wallichiana</Emphasis> A. B. Jacks.

Worldwide genus Amanita is represented by more than 500 ectomycorrhizal (ECM) species. These species form mutualistic associations with a number of host trees among which the conifers predominate. Set out to explore the ECM associations of Amanita in Kashmir Himalayas, Amanita calyptroderma, Amanita pantherina and Amanita vaginata show symbiotic associations with the roots of Pinus wallichiana. Besides morphological characterization of mycorrhizal roots and sporocarps, molecular characterization of rDNA ITS region was performed for identification of species. ITS-rDNA was used for molecular analysis. The target region of rDNA was amplified using universal fungal primers (ITS1 and ITS4). The sequencing of amplified products and their subsequent blast analysis confirmed the identification of species by comparing the sequences of these species with sequences present in GenBank. Phylogenetic analysis also confirmed the identification of species.     

Intragenomic variation in ITS2 rDNA in the louse of humans,Pediculus humanus: ITS2 is not a suitable marker for population studies in this species

The two internal transcribed spacers (ITS) of ribosomal DNA are often used as markers of populations of insects. We studied the ITS2 of the head lice and body lice of humans, to determine whether this gene is a suitable marker of populations of these insects. ITS2 sequences were amplified by PCR from lice from four different countries: Australia, China, Japan and the USA. Direct cycle-sequencing of some of these PCR products gave equivocal nucleotide chromatograms. This indicated that some lice had more than one ITS2 sequence, so we cloned PCR products from these lice. Temperature gradient gel electrophoresis (TGGE) revealed that 50 of the 67 clones we screened had different nucleotide sequences. All lice had several ITS2 types, including those with unequivocal chromatograms. A phylogenetic tree of 15 different ITS2 sequences showed that the sequences from individual lice were not monophyletic. We conclude that the ITS2 is not a useful marker of populations for Pediculus humanus.     

Sequence and secondary structure of the rDNA second internal transcribed spacer in the sibling species Culex pipiens L. and Cx. quinquefasciatus Say (Diptera: Culicidae)

The primary and secondary structure of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) of two members of the Cx. pipiens complex, Cx. pipiens and Cx. quinquefasciafus , were examined in order to better understand the relationships between these two sibling mosquito species. The length of the sequenced rDNA fragments was 512 bp ( Cx. pipiens ) and 513 bp ( Cx. quinquefasciatus ), including the ITS2 regions and flanking 5.8S-28S coding regions. The ITS2 sequences of Cx. pipiens and Cx. quinquefasciatus were 297 and 298 bp in length respectively and showed a 97% identity. In fact, they had identical G + C content (58%) and the only differences observed are six mismatches (three transitionslthree transversions), six single-base and one triple-base deletions/insertions. The observed ITS2 secondary structures of Cx. pipiens and Cx. quinquefasciafus were very similar. Furthermore, the ITS2 sequences of specimens belonging to three populations of Cx. pipiens from Italy and four populations of Cx. quinquefasciatus (three from Africa and one from North America) were enalysed in order to detect the presence of potential species-specific diagnostic restriction sites.     

Ribosomal DNA spacer genotypes of the Anopheles bancroftii group (Diptera: Culicidae) from Australia and Papua New Guinea

Mosquitoes of the Anopheles bancroftii group collected from Northern Australia and Papua New Guinea (PNG) were investigated for sequence variation within the ribosomal DNA ITS2. Wing fringe morphology originally used to identify members of this group was compared to genotypes identified by restriction fragment length polymorphism analysis (RFLP) and heteroduplex analysis (HDA) of the rDNA ITS2. Members of this group separated into four RFLP genotypes (A, B, C and D) with some genotypes displaying wing fringe polymorphisms. Heteroduplex analysis of the ITS2 within and between populations identified genotype A as containing two geographically separate ITS2 sequences: A1 from the Northern Territory of Australia and A2 from Queensland and the Western Province of PNG. Genotypes B and C and genotypes C and D were found sympatric and appeared to be evolving independently suggesting the possibility of cryptic species. Genotype C contained two ITS2 sequence types within the genome.     

DNA Barcodes Distinguish <Emphasis Type="Italic">Withania somnifera</Emphasis> and <Emphasis Type="Italic">Withania ashwagandha</Emphasis>

On the basis of morphology, chemical profiling, crossing ability, amplified fragment length polymorphism and comparison of nucleotide sequences of nuclear ribosomal internal transcribed spacer (ITS), the cultivated form of Withania somnifera, a species of therapeutic value, has been circumscribed as a new species, Withania ashwagandha. The present study was undertaken to ascertain whether the two species can be distinguished on the basis of DNA barcoding. Six barcode loci, ITS, ITS2, matK (maturase K), rbcL (ribulose-bisphosphate carboxylase/oxygenase, large subunit), rpoC1 (RNA polymerase-β′ subunit, main catalytic subunit) and trnH-psbA spacer (transfer RNA for histidine-photosystem II protein D1 spacer) from W. somnifera, W. ashwagandha, their hybrid, and ‘ashwagandha’ market samples were amplified, sequenced, and compared. ITS, ITS2 and matK distinguished two species on the basis of phylogenetic tree method. Likewise, BLAST 1 analysis based on ITS, ITS2, matK, and rbcL individually discriminated two species. However, on the basis of Kimura 2 Parameter distances, two species could not be distinguished as the requirement of a distinct barcode gap—the highest intraspecific distance being lower than the lowest interspecific distance—was not met by any of the loci. If compared by character-based method, ITS, ITS2 and matK sequences of the two species had distinct diagnostic nucleotides (pure character attributes) at nine, four and one positions, respectively. Interestingly, all market samples co-segregated and shared character attributes with W. ashwagandha.     

ITS和β-微管蛋白基因序列鉴定曲霉菌的临床应用

目的 研究ITS序列分析和β-微管蛋白基因序列分析在曲霉菌鉴定中的临床应用价值.方法 收集2007年7月至2010年1月,首都医科大学附属北京同仁医院真菌性鼻窦炎病原菌株中曲霉菌124株,分别对其进行形态学和分子鉴定.形态学包括传统培养方法、玻片培养和乳酸酚棉蓝染色及KOH消化后显微镜镜检.将菌株的PCR扩增产物进行ITS序列分析和β-微管蛋白基因序列分析,其测序结果与GenBank、European Molecular Biology Laboratory和DNA Data Bank of Japan 3个数据库比对,得到分子鉴定结果.结果 形态学鉴定为黄曲霉的56株曲霉中,经ITS序列分析鉴定为黄曲霉55株,寄生曲霉1株,β-微管蛋白基因序列分析结果与ITS序列分析相同;形态学鉴定为烟曲霉的37株曲霉中,ITS序列分析鉴定为37株烟曲霉复合种,β-微管蛋白基因序列分析鉴定为烟曲霉35株和仑图卢斯曲霉2株;形态学鉴定为杂色曲霉的21株曲霉中,ITS序列分析鉴定为杂色曲霉16株和未鉴定到种的曲霉5株,β-微管蛋白基因序列分析鉴定为杂色曲霉16株和聚多曲霉5株;形态学鉴定为构巢曲霉的10株曲霉中,ITS序列分析和β-微管蛋白基因序列分析均鉴定为构巢曲霉.结论 β-微管蛋白基因序列分析曲霉的分辨率较ITS序列分析高,可以将曲霉准确鉴定到种,ITS序列可以分析到曲霉复合种.

Abstract：

Objective To study the clinical application of the ITS and β-tubulin gene regions in identification of Aspergillus spp. Methods One hundred and twenty-four Aspergillus strains that isolated from fungal rhino-sinusitis specimens were collected in Beijing Tongren Hospital, Capital Medical University from July 2007 to January 2010. They were identified by morphological and molecular methods. The first one included traditional culture, slide culture, and microscopic examination after lactophenol cotton blue stain and KOH digestion. The second one was amplifying and sequencing the part of ITS and β-tubulin gene and aligned all the sequences in the GenBank, European Molecular Biology Laboratory nucleotide sequence database, and DNA Data Bank of Japan. Results Of the 56 Aspergillus flavus identified by morphological features, fifty-five isolates were identified as Aspergillus flavus and 1 isolates was Aspergillus parasiticus by the ITS and β-tubulin gene region sequence analysis. In the 37 Aspergillus fumigatus identified by morphological method, and all the 37 isolates were identified as species complex of Aspergillus fumigatus by the ITS region sequence analysis, but through the sequence analysis of β-tubulin gene region, thirty-five isolates were identified as Aspergillus. fumigatus and 2 were Aspergillus lentulus. Twenty-one isolates were identified as Aspergillus versicolor by morphological method, but 16 of them were identified as Aspergillus. versicolor and 5 can not be identified to species level by the ITS region sequence. And by comparative-sequence analysis of β-tubulin gene region, the 5 isolates were identified as Aspergillus sydowii,the other 16 isolates were Aspergillus. versilcolor. Ten isolates were identified as Aspergillus nidulans by morphological features, the ITS and β-tubulin gene region sequence analysis. Conclusions β-tubulin gene sequencing is more suitable for identifying Aspergillus, and could identify Aspergillus spp. to species level Sequences of ITS region could only identify Aspergillus spp. to species complex.