Construction and use of PCR primers from a 65 kDa mannoprotein gene for identification of C. albicans

A method for detection of Candida albicans in biological samples (blood, serum, urine) was developed by the use of polymerase chain reaction (PCR) amplification of a DNA fragment of a gene coding for a 65 kDa mannoprotein of C. albicans (CaMP65). The PCR amplifies a 220 bp fragments whose specificity for C. albicans was demonstrated by Southern blot with a non-radioactive probe, leading to the differentiation from all other yeast species or human and bacterial DNA. The sensitivity of this assay was 5-10 C. albicans cells per milliliter of biological sample.     

Use of 65 kDa mannoprotein gene primers in PCR methods for the identification of five medically important Candida species

We have developed PCR and Multiplex PCR assays for the detection of medically important Candida spp. using different species and genus-specific PCR primers selected within the MP65 gene, a recently cloned gene encoding a mannoprotein adhesin. The genus-specific PCR primers were able to amplify Candida species DNA (100% positivity) whereas DNA from all other isolates tested, belonging to other fungal genera, was not amplified. The species-specific PCR primers allowed differentiation of each of five Candida species by the amplicon length produced. No amplicons were detected using species- or genus-specific primers in several bacterial or human DNA templates. The methods described in this study are reproducible, simple and specific. The total time required for each PCR method was less than 4 h from the extraction to the visualized amplicons after PCR. In conclusion, we developed PCR methods to differentiate the five most medically important Candida species using primers directed to the MP65 gene.     

白假丝酵母菌致食物中毒的病原学鉴定及药敏试验结果的报告

目的明确白假丝酵母菌的鉴定过程,探寻治疗白假丝酵母菌致食物中毒的药物。方法收集16例食物中毒者吐、泻物标本及剩余饭菜,酱油。白假丝酵母菌的鉴定用快速微生物鉴定系统(API)20C Aux V3.0;药敏试验用FUNGUS-11药敏最低抑菌浓度(MIC)测定试验条。结果检样呕吐物、水样便、剩菜、酱油中检出白假丝酵母菌,食物中毒主要是白假丝酵母菌引起的,白假丝酵母菌对两性霉素B和制霉菌素敏感。结论综合流行病学调查、临床症状、实验室鉴定结果,确定该次食物中毒由白假丝酵母菌引起;首选治疗药物为两性霉素B和制霉菌素。     

Effect of matrix metalloprotease inhibitors on the 95 kDa metallopeptidase of Candida albicans

A 95 kDa metallopeptidase of Candida albicans could be involved in the process of dissemination of the yeast. Matrix metalloproteases (MMPs) are also responsible for collagen breakdown in inflammatory and malignant processes. We tested six compounds on the C. albicans enzyme. Doxycycline, gentamicin, cefalothin, galardin, and elaidic and oleic acids are known for their capacity to inhibit some MMPs. Amongst these agents, only oleic acid was able to markedly inhibit the purified metallopeptidase at very low concentrations. Moreover, this fatty acid inhibited the secretion of the enzyme in the culture medium without altering the yeast viability.     

Rapid identification of Candida albicans using 4-methylumbelliferyl N-acetyl-beta-galactosaminide

Rapid identification of Candida albicans is performed mainly by the germ-tube test. However, recent reports have suggested that up to 5% of C. albicans species can give false negative results. We describe the use of 4-Methylumbelliferyl N-acetyl-beta-D-galactosaminide (4-MAG) conjugate as an alternative to the germ-tube test. Our results indicate that, in comparison to the germ-tube test, the 4-MAG test has a sensitivity of 100% and a specificity of 92%. Candida tropicalis can give false-positive results, and that a further screening test is required to identify this species. Problems reading end-points were not encountered.     

白念珠菌烯醇化酶N端肽段ENO1-319P的原核表达和鉴定

目的 制备白念珠菌烯醇化酶N端基因重组肤段EN01-319P并检测其抗原性.方法 选择与人烯醉化酶蛋白a-ENO同源性低的白念珠菌烯醇化酶肤段ENOI-319P,将其编码DNA序列克隆到表达载体pET28a(+),并在大肠埃希菌BL21(DE3)中用IPTG诱导带有His标签的重组肽段的表达.用SDS-PAGE电泳分析...     

白念珠菌果糖二磷酸醛缩酶重组蛋白的制备及鉴定

摘要：目的：制备有免疫原性的白念珠菌果糖二磷酸醛缩酶（fructosebisphosphate aldolase, Fba1)重组蛋白。 方法：以白念珠菌C1标准株基因组DNA为模板,用PCR法扩增Fba1 DNA序列,与克隆载体pMD18-T连接、测序,再与原核表达载体pET28a(+)连接,构建成重组表达质粒pET28a(+)/Fba1,转化大肠埃希菌BL21(DE3),IPTG诱导重组融合蛋白表达,亲和层析柱纯化重组蛋白。用抗His标签的单克隆抗体鉴定融合蛋白,并用确诊为侵袭性白念珠菌病（ICAI）、且抗白念珠菌烯醇化酶抗体阳性的患者血清进行抗原性鉴定。 结果：Fba1 DNA序列与GenBank中的序列一致。在大肠埃希菌中获得白念珠菌Fab1重组蛋白的高效表达,表达产物以可溶性蛋白为主,最终蛋白质得率为7.6 mg/g湿菌。经免疫印迹鉴定,重组蛋白与抗His标签的单克隆抗体和确诊ICAI患者血清呈特异性反应,显示出良好的抗原反应性。 结论：成功制备了具有良好抗原反应性的重组蛋白,为建立特异性诊断ICAI的新方法打下基础。     

白念珠菌烯醇化酶N端肽段ENO1 319P的原核表达和鉴定

摘要：目的:制备白念珠菌烯醇化酶N端基因重组肽段ENO1-319P并检测其抗原性。 方法:选择与人烯醇化酶蛋白α-ENO同源性低的白念珠菌烯醇化酶肽段ENO1-319P,将其编码DNA序列克隆到表达载体pET28a(+),并在大肠埃希菌BL21(DE3)中用IPTG诱导带有His标签的重组肽段的表达。用SDS-PAGE电泳分析,TALON金属亲和层析柱纯化重组蛋白,其抗原性用侵袭性白念珠菌感染（ICAI）患者血清进行western blot评估。 结果:成功表达并纯化了目的肽段,该蛋白可与ICAI患者血清发生特异性反应。 结论:获得了白念珠菌烯醇化酶N端肽段ENO1-319P的基因表达工程菌株,为将此重组肽段用于制备特异性更高的抗体打下基础。     

PCR primers for the amplification of four insect mitochondrial gene fragments

Insect mitochondrial genome (mtDNA) analysis is a powerful tool for the study of population genetics and phylogenetics. In the past few years primer sequences for the PCR amplification of various insect mtDNA genes have been published. The objectives of this study were (1) present new primer sequences for six insect mitochondrial genes and (2) test primers designed in our laboratory and some previously published primers on a wide range of insects to determine if amplification of the target fragment could be obtained. The primers for the amplification of the two ribosomal RNA gene (16S and 12S rRNA) fragments are universal for insects and related groups; the primers for NADH5 and NADH4 dehydrogenase gene fragments and cytochrome c oxidase I gene fragment are applicable broadly.     

Use of novel species-specific PCR primers targeted to DNA gyrase subunit B (gyrB) gene for species identification of the Cronobacter sakazakii and Cronobacter dublinensis

Cronobacter sakazakii and its phylogenetically closest species are considered to be an opportunistic pathogens associated with food-borne disease in neonates and infants. Neither phenotypic nor genotypic (16S ribosomal DNA sequence analysis) techniques can provide sufficient resolutions for accurately and rapidly identification of these species. The objective of this study was to develop species-specific PCR based on the gyrB gene sequence for direct species identification of the C. sakazakii and Cronobacter dublinensis within the C. sakazakii group. Two pair of species-specific primers were designed and used to specifically identify C. sakazakii and C. dublinensis, but none of the other C. sakazakii group strains. Our data indicate that the novel species-specific primers could be used to rapidly and accurately identify the species of C. sakazakii and C. dublinensis from C. sakazakii group by the PCR based assays.     

Multidrug resistance in Candida albicans: disruption of the BENr gene.

The BENr gene of Candida albicans, which confers resistance on susceptible strains of Saccharomyces cerevisiae to six structurally and functionally unrelated drugs, was described recently (R. Ben-Yaacov, S. Knoller, G. Caldwell, J. M. Becker, and Y. Koltin, Antimicrob. Agents Chemother. 38:648-652, 1994). This gene bears similarity to membrane proteins encoding antibiotic resistance in prokaryotes and eukaryotes. The effect of disruption of this gene on viability and drug susceptibility was determined. The results indicate that the gene is not essential but its inactivation leads to susceptibility to three of the four drugs tested. Inactivation of this gene did not increase the susceptibility of the mutant to benomyl, suggesting that C. albicans has other mechanisms of resistance, some of which may be additional efflux pumps that confer resistance to this tubulin-destabilizing agent.     

实时荧光PCR方法快速检测KI多瘤病毒

目的采用实时荧光PCR方法快速检测KI多瘤病毒(KIPy V),并与巢式PCR扩增法进行比较。方法收集2007年11月至2015年1月在福州市一家妇幼保健院因呼吸道感染住在儿科重症监护病房(PICU)的259例小儿鼻咽抽取物标本和另两家综合医院146例因严重急性呼吸道感染(SARI)住院的成年患者咽拭子标本,通过实时荧光PCR方法对KIPy V的调节区的基因片段进行检测。结果在小儿鼻咽抽取物标本中检测出3例KIPy V感染阳性病例,检测阳性率约为1.2%,而用巢式PCR扩增法只检测出1例,这例的Ct值较低,约为23,并且扩增曲线呈S型。其它两例的Ct值较高,分别约为33和35。在成年患者咽拭子标本中,用两种方法都未检测出KIPy V感染。在两例Ct值较高的小儿病例中,检测出混合有呼吸道合胞病毒(RSV)感染。结论确立的实时荧光PCR方法可以快速检测KIPy V,且特异性较高。     

Detection and identification of Candida species associated with Candida vaginitis by real-time PCR and pyrosequencing

Real-time polymerase chain reaction (PCR) is currently considered the most sensitive method to detect low abundance DNA of pathogens in clinical samples. Furthermore, obtaining DNA sequence is the 'gold standard' of precise molecular detection. Here we combine species-specific real-time PCR and pyrosequencing to rapidly amplify and sequence ribosomal DNA from Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis, which are commonly associated with candida vaginitis (CV). A standard curve was developed from plasmids containing the target DNA for each of the Candida species. A minimum real-time PCR and pyrosequencing detection limit of 100 copies per reaction was achieved. The combined technique was applied to the identification of the four Candida species in DNA extracts from vaginal samples. The results from 231 samples were compared with conventional PCR methods of identification. The results of both methods agreed on all but two samples, which were determined by both methods to contain C. albicans, but real-time PCR and pyrosequencing identified a second species that went undetected by conventional PCR. This is the first application of real-time PCR and pyrosequencing to DNA from vaginal samples for identification of four Candida species associated with CV, without the need for time-consuming culture methods.     

改进的多重PCR技术鉴定白色念珠菌、新生隐球菌及烟曲霉的实验研究

[目的]建立一种快速而简便的鉴定白色念珠菌、新生隐球菌及烟曲霉的分子生物学方法。[方法]应用通用引物ITS1与ITS4，PCR扩增真菌的内转录间隔区（包含5．8SrDNA），然后将白色念珠菌、新生隐球菌、烟曲霉的单条种特异性异物与引物ITS1结合，对通用引物的扩增产物进行多重PCR扩增，达到鉴定上述菌株的目的。[结果]实验所采用的所有致病真菌，经通用引物的PCR扩增均能扩增出特异性的条带，而非真菌菌株扩增为阴性；白色念珠菌、新生隐球菌、烟曲霉的3种种特异性引物与ITS1结合进行的多重PCR扩增，只对目的菌株进行了有效扩增，其他菌株扩增为阴性。应用此方法对临床分离的菌株，白色念珠菌，新生隐球菌，烟曲霉进行鉴定，也得到特异性扩增条带。[结论]改进的多重PCR技术，更加经济，简单，快速，为致病真菌菌种的鉴定提供了有效手段。     

白念珠菌果糖二磷酸醛缩酶单克隆抗体的制备及鉴定

目的制备抗白念珠菌果糖二磷酸醛缩酶(Fba1)单克隆抗体,用以研究胞壁蛋白Fba1在侵袭性念珠菌感染(IC)中的致病作用。方法取重组白念珠菌Fba1蛋白免疫BALB/c小鼠的脾细胞与骨髓瘤细胞Sp2/0融合,筛选分泌抗体的杂交瘤细胞,有限稀释法克隆化。用ELISA测定单克隆抗体的效价,western blot和免疫荧光技术鉴定其特异性,用IsoStrip鉴定其抗体类型。结果筛选到1株稳定分泌抗Fba1单克隆抗体的杂交瘤细胞,抗体类型为IgG2a,轻链为κ型。鉴定结果表明,ELISA效价为1×106,能与念珠菌裂解物中天然Fba1和基因重组Fba1特异性结合。免疫荧光检测表明,该株单克隆抗体与白念珠菌、光滑念珠菌、近平滑念珠菌和热带念珠菌均发生反应。结论建立了1株持续分泌高效价抗念珠菌属特异性Fba1单克隆抗体的杂交瘤细胞株,为研究Fba1在IC中的致病作用提供了实验材料。     

ERG11基因突变与白念珠菌对氟康唑耐药性的初步研究

目的探讨白念珠菌氟康唑作用的靶酶编码基因（ERG11）突变与氟康唑耐药性的关系。方法采用聚合酶链反应（PCR）扩增临床分离的23株白念珠菌ERG11基因,包括2株耐氟康唑株、9株氟康唑剂量依赖敏感株和12株氟康唑敏感株,并进行双向测序。应用B last软件,将测序结果与网上已发表序列（GenBankAY856352）进行比对,以确定是否发生基因突变。结果23株白念珠菌的ERG11基因序列共检出16个同义突变位点和18个错义突变位点。错义突变中Y205E、I437V、A255V、E260V、K487N、G472R、N435V、D502E、K143Q为新发现的突变位点。结论Y205E、I437V、A255V位点突变发现于敏感株,可能与白念珠菌耐药无关;K487N、G472R、N435V、D502E、E260V发现于剂量依赖敏感株,K143Q发现于耐药株,可能与耐药的形成有关。     

The Candida albicans INT1 gene facilitates cecal colonization in endotoxin-treated mice

Increased intestinal colonization with Candida albicans is believed to be a major predisposing factor to systemic candidiasis. Previous evidence has implicated the C. albicans INT1 gene in hyphal development, epithelial adherence, and mouse virulence. The effect of INT1 on mouse cecal colonization was measured using a parent strain (CAF2, INT1/INT1), an int1 deletion homozygote (CAG3, int1/int1), and a heterozygous reintegrant (CAG5, int1/int1 + INT1). Forty-eight hours after oral inoculation of 10(7) C. albicans into normal mice, only low numbers of each strain were recovered from the cecal flora. In mice pretreated with oral bacitracin/streptomycin, cecal colonization of each C. albicans strain was increased compared to the corresponding strain inoculated into untreated mice, with the CAF2 parent strain greater (P < 0.01) than the two mutant strains, and with the heterozygous and homozygous mutants not different from each other. In mice pretreated with parenteral lipopolysaccharide (LPS), in addition to oral antibiotics, numbers of cecal CAF2, CAG5, and CAG3 were increased (P < 0.01) compared to the corresponding strain inoculated into mice treated with antibiotics alone. In LPS-treated mice, numbers of cecal C. albicans CAF2 (INT1/INT1) were greater (P < 0.05) than C. albicans CAG3 (int1/int1). Thus, parenteral LPS had an additive effect on C. albicans cecal colonization in antibiotic-treated mice, and the presence of two functional copies of the INT1 gene appeared to facilitate colonization in both antibiotic-treated mice and in mice treated with antibiotics plus parenteral endotoxin.     

牛奶培养基用于鉴定白色念珠菌芽管和厚膜孢子

１０ｇ／Ｌ脱脂奶粉或７．５％鲜牛奶培养基用于诱导白色念珠菌芽管和厚膜孢子的形成。在振荡条件下于３７℃培养３０ｍｉｎ或６０ｍｉｎ即可观察芽管，该培养基诱导芽管形成的百分率明显高于人血清，且特异性和人血清一样。白色念珠菌在牛奶培养基中于室温静置培养４８ｈ，可形成厚膜孢子。本法鉴定白色念珠菌简便、快速、特异、安全且培养基容易制备。     

Rapid identification of Pseudomonas aeruginosa from ocular isolates by PCR using exotoxin A-specific primers

The purpose of this research was to evaluate the use of PCR for the identification of ocular isolates of Pseudomonas aeruginosa by using primers specific to the exotoxin A gene of the bacteria. Genomic DNA was obtained from ocular microbial isolates of keratitis patients. Primers were designed based on the published sequence of the exotoxin A gene of P. aeruginosa. Using the primers designed, PCR reactions were performed on the DNA samples. The PCR was also examined for its specificity and sensitivity. In addition, a direct PCR using heating method was attempted on P. aeruginosa with no separate DNA extraction step. ATCC strains of P. aeruginosa were included as positive controls. The rest of the bacteria other than P. aeruginosa served as negative controls. A single band was obtained when analysed on agarose gel electrophoresis only from samples that contained genomic DNA of P. aeruginosa. The direct PCR method was also successful with the same band produced from the amplification. The whole process was completed within 4 h. The direct PCR amplification targeting at the exotoxin A gene of P. aeruginosa is potentially a rapid, specific, sensitive and relatively simple method for the identification of ocular isolates of P. aeruginosa.