国内首见三株棘状外瓶霉的研究

作者研究了国内首见的三株棘状外瓶霉[Exophiala spinifera(Nielsen et Conant)McGinms],观察了菌落形态,研究了此菌在光学显微镜下和扫描电镜下的特点,并作了外抗原试验。在光学显微镜下,本菌有暗色长棘状的分生孢子梗。在扫描电镜下,有很长的环痕梗,其尖端的环痕数目可达30个以上。有的环痕梗和甄氏外瓶霉[Exophiala jeanselmei(Langeron)McGinnis et Padhye]的环痕梗很相似。有些酵母样细胞上也可产生短的环痕产孢尖端。有一株菌的产孢细胞顶端有几个突起的环痕产孢尖端,呈假单轴性排列。另一株菌产生瓶梗。因此考虑此菌是一种多形性真菌。外抗原试验,一株菌符合棘状外瓶霉,另二株菌符合外瓶霉。     

长白山分解纤维素的瓶霉属和外瓶霉属真菌

瓶霉属、外瓶霉属真菌在自然界中广泛分布,是有着重要经济意义的一类真菌。迄今,我国已报道疣状瓶霉（Ｐｈｉａｌｏｐｈｏｒａ ｖｅｒｒｕｃｏｓａ）。裴氏瓶霉（Ｐｈ．ｐｅｄｒｏｓｏｉ）,棘状外瓶霉（Ｅｘｏｐｈｉａｌａ ｓｐｉｎｉｆｅｒａ）、皮炎外瓶霉（Ｅ．ｄｅｒｍａｔｉｔｉｄｉｓ）和甄氏外瓶霉（Ｅ．ｊｅａｎｓｅｌｍｅｉ＝Ｐｈ．ｇｏｕｇｅｒｏｔｉｉ）５种,均是分离自人体的病原真菌。在长白山自然保护区的原始林     

长白山分解纤维素的瓶霉属和外瓶霉属真菌

瓶霉属、外瓶霉属真菌在自然界中广泛分布,是有着重要经济意义的一类真菌。迄今,我国已报道疣状瓶霉（Phialophoraverrucosa）、裴氏瓶霉（Ph.pedrosoi）、棘状外瓶霉（Exophialaspinifera）、皮炎外瓶霉（E.dermatitidis）和甄氏外瓶霉（E.jeanselmei＝Ph．gougerotii）5种,均是分离自人体的病原真菌。在长白山自然保护区的原始林中,用生长锥取样器随机钻取腐朽林木髓心,在实验室进行分离培养。共鉴定出瓶霉属真菌2种,外瓶霉属真菌3种,其中美州瓶霉（Ph.americana、烂木瓶霉（Ph．richardsiae）和鲑外瓶霉（E.salmonis）为国内新记录种。     

临床皮炎外瓶霉荧光定量PCR检测方法的建立

目的 建立基于TaqMan探针技术的皮炎外瓶霉荧光定量PCR检测方法.方法 通过对皮炎外瓶霉ITS区域基因组序列（GenBank：JN675373.1）进行分析,设计合成特异性引物和荧光标记探针,优化荧光定量PCR反应条件.以临床标本中分离的皮炎外瓶霉为阳性菌株,及其他种类真菌和细菌作为阴性对照菌株,从特异性、敏感性及重复性方面对该方法检测效果进行评价.结果 该研究设计的引物和探针能扩增皮炎外瓶霉特异性序列.临床分离得到的皮炎外瓶霉在反应中有明显扩增曲线,而甄氏外瓶霉、棘状外瓶霉、烟曲霉、白色念珠菌、新生隐球菌、马内菲青霉等20株菌在CT值≤38范围内均未有扩增;利用基因重组构建的标准品完成了标准曲线的绘制,在1.0×103～1.0×107拷贝数（Cp）内具有良好的线性关系（R2=1.000）,最低可检出量为10 Cp/μL.结论 成功建立了荧光定量PCR检测皮炎外瓶霉方法,该法特异度强、敏感度高、重复性好,将有助于临床皮炎外瓶霉感染的早期诊断和针对性治疗.     

外瓶霉分类鉴定研究进展

外瓶霉是一组以环痕产孢为主要产孢方式的暗色真菌,可致皮肤,皮下组织及系统性感染,重者可危及生命。由于该属内各种差异性小,单纯依靠形态学难以将其准确分类,营养生理学方法、流式细胞计数法、泛醌系统分类法、免疫学方法及分子生物学方法的应用,使外瓶霉分类日臻完善,本文综述了上述几种方法在外瓶霉分类鉴定中的应用。     

核糖体基因ITS作为苎麻疫霉,恶疫霉分类辅助性状的研究

以２个雄器大多围生、少数侧生的苎麻疫霉菌株与１个雄器侧生、偶有围生的恶疫霉菌株为材料,采用真菌核糖体基因转录间隔区（ＩＴＳ）通用引物,ＰＣＲ扩增３个供试菌株核糖体基因的ＩＴＳ１和ＩＴＳ２,并对ＰＣＲ产物进行了克隆和序列分析。结果是苎麻疫霉的ＩＴＳ１和ＩＴＳ２分别由２０６和４５３个碱基组成,而恶疫霉则分别由２１８和４１５个碱基组成。２个供试苎麻疫霉菌株的ＩＴＳ１和ＩＴＳ２的碱基序列同源性均分别为１０     

外瓶霉属真菌耐药性和降解能力的研究

外瓶霉属真菌对五氯酚和杂酚油都有很强的耐药性,其中以菌株Ｐ－３１８２对五氯酚和菌株Ｐ－２８３０、Ｐ－３１８２对杂酚油的耐药性最强。外瓶霉属真菌不但能在五氯酚和杂酚油溶液中生长,而且具有降解这两种防腐剂的能力。而在砷铬酸铜溶液中不能生长,对环烷酸铜和砷铬酸铜的耐药性与对照菌种相似或稍高。     

核糖体基因ITS作为苎麻疫霉、恶疫霉分类辅助性状的研究

以2个雄器大多围生、少数侧生的苎麻疫霉菌株与1个雄器侧生、偶有围生的恶疫霉菌株为材料,采用真菌核糖体基因转录间隔区（ITS）通用引物,PCR扩增3个供试菌株核糖体基因的ITS1和ITS2,并对PCR产物进行了克隆和序列分析。结果是苎麻疫霉的ITS1和ITS2分别由206和453个碱基组成,而恶疫霉则分别由218和415个碱基组成。2个供试苎麻疫霉菌株的ITS1和ITS2的碱基序列同源性均分别为100%。苎麻疫霉和恶疫霉ITS1同源性为74.9%,其中中间区域40bp-164bp之间在两种间变异丰富,同源性只有59.4%,而1bp-39bp和165bp-239bp两区域的同源性分别为92.3%和92.1%;ITS2在两种疫霉菌间的同源性为71.0%。结果表明苎麻疫霉和恶疫霉ITS的碱基序列有明显差异。上述结果提示,ITS区域碱基序列可区分苎麻疫霉和恶疫霉。     

全球皮炎外瓶霉感染流行现状的回顾性分析

目的分析皮炎外瓶霉感染的流行病学和临床特征,为提高皮炎外瓶霉感染的诊治水平提供科学依据。方法采用文献回顾和荟萃分析的方法,分析全球范围内已报道的皮炎外瓶霉感染病例的国籍、性别、年龄分布、危险因素、发病部位、临床表现、诊疗方法及预后等流行病学和临床特征。结果皮炎外瓶霉感染在免疫功能正常和免疫功能缺陷患者中均可发生,患者的男女性别比为1.10∶1.00,最常发病年龄段为51~60岁,肺(27.90%,17/61)为最常受累的器官,但不同地域的病例感染器官存在差异。约半数(47.54%,29/61)病例伴有各种免疫抑制的基础疾病或危险因素。皮炎外瓶霉感染的临床确诊主要依赖培养和分子鉴定,系统感染患者推荐联合伊曲康唑和特比萘芬作为抗真菌治疗方案。结论近年来皮炎外瓶霉感染的发病率在全球范围内呈上升趋势,肺部为系统感染患者最常受累的器官,诊断主要依赖于真菌培养。加强皮炎外瓶霉菌株的药敏监测和分子流行病学研究,对于提高皮炎外瓶霉感染的临床诊治水平非常必要。     

核糖体基因ITS作为苎麻疫霉、恶疫霉分类辅助性状的研究*

以2个雄器大多围生、少数侧生的苎麻疫霉菌株与1个雄器侧生、偶有围生的恶疫霉菌株为材料,采用真菌核糖体基因转录间隔区（ITS）通用引物,PCR扩增3个供试菌株核糖体基因的ITS1和ITS2,并对PCR产物进行了克隆和序列分析。结果是苎麻疫霉的ITS1和ITS2分别由206和453个碱基组成, 而恶疫霉则分别由218和415个碱基组成。2个供试苎麻疫霉菌株的ITS1和ITS2的碱基序列同源性均分别为100%。苎麻疫霉和恶疫霉ITS1同源性为74.9%,其中中间区域40bp-164bp之间在两种间变异丰富,同源性只有59.4%,而1bp-39bp和165bp-239bp两区域的同源性分别为92.3%和92.1%; ITS2在两种疫霉菌间的同源性为71.0%。结果表明苎麻疫霉和恶疫霉ITS的碱基序列有明显差异。上述结果提示,ITS区域碱基序列可区分苎麻疫霉和恶疫霉。     

Mitochondrial DNA analysis of Exophiala jeanselmei and Exophiala dermatitidis

Mitochondrial DNA (mtDNA) analysis with Hae III, Hind. III and Msp I was performed in 45 Exophiala jeanselmei strains (30 Phialophora jeanselmei and 15 Phialophora gougerotii strains) and 31 Exophiala dermatitidis strains. The results were as follows, 1) P. jeanselmei and P. gougerotii are identical, 2) E. jeanselmei is classified into 18 types based on restriction profiles, 3) two strains of E. jeanselmei CBS 577.76 and CBS 578.76 are identified as E. dermatitidis, 4) E. dermatitidis has no intraspecific variation and is definitely distinct from E. jeanselmei, 5) E. jeanselmei is suggested to be a complex organism because of extensive mtDNA polymorphism.     

核糖体DNA的内转录间隔区序列标记在真菌分类鉴定中的应用

传统的真菌分类主要根据真菌菌株的形态特征、生长特性与生理生化指标进行,而分子生物学技术的发展提升了真菌分类鉴定研究的手段。真菌核糖体DNA内转录间隔区(ITS)在进化上比编码区快,种内的不同菌株之间高度保守,但在种间变化极大,故可为真菌学的研究提供丰富的遗传信息。简要综述了ITS序列分析技术在真菌分类鉴定中的应用现状、相关问题及前景。     

Phylogeny of the GenusAgaricusInferred from Restriction Analysis of Enzymatically Amplified Ribosomal DNA

Bunyard, B. A., Nicholson, M. S., and Royse, D. J. 1996. Phylogeny of the genusAgaricusinferred from restriction analysis of enzymatically amplified ribosomal DNA.Fungal Genetics and Biology20,243–253. The 26S and 5S ribosomal RNA genes and the intergenic region between the 26S and the 5S rRNA genes of the ribosomal DNA repeat of 21 species ofAgaricuswere amplified using PCR and then digested with 10 restriction enzymes. Restriction fragment length polymorphisms were found among the 21 putative species ofAgaricusinvestigated and used to develop a phylogenetic tree of the evolutionary history ofA. bisporus.The 5′ end of the 26S gene showed more variability than the 3′ end.A. excellens, A. chionodermus,andA. carolirepresented the species most distantly related toA. bisporus.We present here the first comprehensive attempt at systematically resolving the entire genusAgaricususing modern techniques for molecular genetic analysis. Our data indicate that previous taxonomic schemes, based on morphological characters, are in need of revision.     

Mitochondrial DNA analysis of Exophiala jeanselmei var. lecanii-corni and Exophiala castellanii

A Japanese clinical isolate (KU-A-0094) which was identified by de Hoog et al. as Exophiala jeanselmei var. lecanii-corni with difficulty, was compared with 5 strains including the type cultures of E. jeanselmei var. lecanii-corni, var. jeanselmei and E. castellanii using RFLP (restriction fragment length polymorphism) patterns of mtDNA (mitochondrial DNA). RFLP patterns of KUA-0094 were identical with those of E. jeanselmei var. lecanii-corni and different from those of E. castellanii with restriction enzymes of HaeIII, MspI and hindIII. Therefore, de Hoog et al.'s identification of KU-A-0094 was confirmed. Additionally, mtDNA-RFLP patterns of E. jeanselmei var. lecanii-corni and E. jeanselmei var. jeanselmei were also different from each other. Consequently E. jeanselmei var. lecanii-corni seem to be a species in its own right rather than a variant of E. jeanselmei. This revised version was published online in June 2006 with corrections to the Cover Date.     

First successful amplification of 18S ribosomal DNA ofCordyceps spp. by the PCR method

The 18S ribosomal DNAs ofCordyceps spp. were amplified for the first time by the PCR method. New primers were designed based on the sequence of the 18S ribosomal DNA ofSclerotinia sclerotiorum.     

哈尔滨地区假丝酵母菌DNA异质性及药物敏感性分析

研究假丝酵母菌的DNA异质性及药物敏感性,为预防和监控院内假丝酵母菌感染奠定基础。将临床分离的假丝酵母菌菌株,用科玛嘉显色培养基鉴定菌种,经纸片扩散法进行药敏试验,应用随机扩增多态性DNA（RAPD）技术对这些菌株进行基因分型。结果显示：93株假丝酵母菌中白假丝酵母菌68株,非白假丝酵母菌25株,所有菌株对制霉菌素,两性霉素B两种药物的敏感率最高（100％）,酮康唑其次（70．9％）,氟康唑的敏感率最低（50．5％）,引物1和引物2将来源不同的68株白假丝酵母菌分别分成4型（A1、B1、C1、D1）和6型（A2、B2、C2、D2、E2、F2）。哈尔滨地区的假丝酵母菌感染以白假丝酵母菌为主,且主要为A1、B1型（引物1）或A2、B2型（引物2）;基因型与药敏谱无明显相关性。     

Ribosomal DNA in Drosophila melanogaster. II. Heteroduplex mapping of cloned and uncloned rDNA.

Sequences in the cloned Drosophila melanogaster rDNA fragments described by Dawid et al. (1978) were compared by heteroduplex mapping. The nontranscribed spacer regions in all fragments are homologous but vary in length. Deletion loops were observed at variable positions in the spacer region suggesting that spacers are internally repetitious.Many rDNA repeats in D. melanogaster have a 28 S gene interrupted by a region named the ribosomal insertion. Insertions of 0.5, 1 and 5 kb were found in repeat-length EcoRI fragments. These DNA regions, named type 1 insertions, are homologous at their right ends. Although 1 kb insertions are quite precisely twice as large as 0.5 kb insertions they do not represent a duplication of the shorter sequence. Some insertions have at least one EcoRI site and therefore yield EcoRI fragments which are only part of a repeat. The sequences in two cloned right-hand partial insertion sequences are homologous, but the sequences in two lefthand partial insertions are not. None of the EcoRI-restrictable insertion sequences has any homology to any part of type 1 insertions; they are thus grouped together as type 2. Evidence for insertion sequences of at least two types in uncloned rDNA was obtained by annealing a cloned fragment with a 1 kb insertion to genomic rDNA. About 15% of the rDNA repeats show substitution type loops between the 1 kb type 1 insertion derived from the cloned fragment and type 2 insertions in the rDNA.     

Ribosomal repeat in cell free DNA as a marker for cell death

A novel method for in vivo evaluation of cell death in patients with acute and/or chronic heart diseases, accompanied by apoptosis or cell necrosis has been developed. The method is based on the analysis of cell free DNA (cfDNA) in the blood serum (plasma). It includes estimation of concentration of serum ribosomal repeat (rDNA), content of rDNA in total cfDNA, as well as factors of cfDNA elimination, such as nuclease activity and anti-DNA antibody. We have found a fivefold increase in serum cfDNA concentration and a 12-fold increase of serum rDNA concentration in patients with acute myocardial infarction compared with healthy individuals. In chronic coronary ischemia serum cfDNA concentration was nearly normal, but the content of rDNA in cfDNA was 4.8-fold higher, and the serum rDNA concentration was increased sevenfold. We hypothesize that putative reason for accumulation of rDNA within cfDNA might be attributed to the previously reported resistance of rDNA to the ds-fragmentation by serum endonucleases. In acute and chronic coronary disease serum nuclease activity and the titer of anti-DNA antibodies (which are mainly bound to the cfDNA) was substantially higher than in the healthy controls. It is suggested that release of rDNA fragments into blood not only reflects cellular death in the body but also determines the response of the organism to the disease-associated stress.