Use of peptide nucleic acid-fluorescence in situ hybridization for definitive, rapid identification of five common Candida species

We investigated a 2.5-h peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) assay with five Candida species-specific probes to identify Candida colonies and compared it to standard 2-h to 5-day phenotypic identification methods. Suspensions were made and slides were prepared and read for fluorescence per the manufacturer's instructions. Sensitivity was 99% (109/110), and specificity was 99% (129/130). PNA-FISH can rapidly identify those Candida species isolated most frequently.     

Simple scheme for identification of common species of enterobacteriaceae

We propose a simple scheme for the identification of enterobacteriaceae species which routinely necessitates numerous biochemical tests and prolonged time span. In the scheme, family enterobacteriaceae is initially divided into four major groups depending on two important biochemical reactions viz. Lactose fermentation (L) and Methyl red test (MR). Each of the four groups, Group I (L + MR+), Group II (L + MR-), Group III (L- MR-), Group IV (L- MR+) can further be differentiated by using few tests. Eleven genera and 23 species can be identified by this scheme using limited biochemical tests. As many as 990 strains of enterobacteriaceae were subjected to standard biochemical tests and proposed simple scheme for identification. The discrepancy was observed only with 8 atypical strains of E. coli.     

Multiplex PCR-based detection and identification of the most common Salmonella second-phase flagellar antigens

Most Salmonella serotypes alternatively express phase 1 or phase 2 flagellar antigens encoded by fliC and fljB genes respectively. Flagellar phase reversal to identify both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fljB genes encoding H:1,w, H:e,n,x and H:e,n,z15 antigens have been sequenced and the specific sites for each antigen determined in selected Salmonella serotypes. These results, together with flagellar H1 complex variable internal sequences previously published, have been used to design a multiplex-PCR to identify H:1,2, H:1,5, H:1,6, H:1,7, H:1,w, H:e,n,x and H:e,n,z15 second-phase antigens. These antigens are part of the most common Salmonella serotypes possessing second-phase flagellar antigens. This multiplex-PCR includes 10 primers. A total of 140 Salmonella strains associated with 49 different serotypes were tested. Each strain generated one second-phase-specific antigen fragment, ranging between 50 and 400 bps. Twenty-five strains associated with 17 serotypes, with no second-phase antigen or with an antigen different from those tested in this work, did not generate any fragments. The method is quick, specific and reproducible and is independent of the phase expressed by the bacteria when tested.     

Evaluation of five commercial systems for identification of coagulase-negative staphylococci to species level

Five commercially available systems for identification of coagulase-negative staphylococci to the species level were evaluated using 163 clinical staphylococcal isolates. The conventional method of Kloos and Schleifer served as reference method. The API 20GP system showed the highest rate of agreement with the reference method, correctly identifying 98.8% of the isolates. The Microscan system had an overall rate of agreement of 96.3 %, and Staph-Trac, Sceptor and Staph-Ident rates of 88.8 %, 87.6 % and 84. % respectively.     

Comparative evaluation of five commercial systems for the rapid identification of pathogenic Neisseria species.

Prompt diagnosis and effective treatment of urogenital gonococcal infections require rapid isolation and identification of Neisseria gonorrhoeae from urogenital specimens. We evaluated a new, rapid (30-min) test called Gonochek II (E-Y Laboratories, San Mateo, Calif.) which utilizes chromogenic substrates for the identification of pathogenic Neisseria species. It was compared with the API NeIdent (Analytab Products, Inc., Plainview, N.Y.), Minitek (BBL Microbiology Systems, Cockeysville, Md.), and RapID NH (Innovative Diagnostics, Atlanta, Ga.), systems and the Phadebact GC (Pharmacia Diagnostics, Piscataway, N.J.) test for its performance in identifying known strains of N. gonorrhoeae (39 strains), Neisseria meningitidis (22 strains), Neisseria lactamica (12 strains), and Branhamella catarrhalis (17 strains). The Gonochek II system correctly identified 100% of N. gonorrhoeae, N. lactamica, and B. catarrhalis strains and 95.4% of N. meningitidis strains. The percent agreement for correct identification of all strains tested was 98.8%. In contrast, the Minitek, RapID NH, and API NeIdent systems correctly identified 86.6, 80.0, and 73.3% of the strains, respectively. The Phadebact GC test identified 94.9% of the N. gonorrhoeae isolates but also cross-reacted with 41.6% of the N. lactamica strains. The Gonochek II system is rapid, simple to perform, and easy to interpret, requires 1 to 2 min to set up, and more accurately identifies pathogenic Neisseria species when compared with other systems used in this study.     

Genome analysis of Cryphonectria hypovirus 4, the most common hypovirus species in North America

Many different viruses that reduce virulence and alter the phenotype to varying extents have been identified in the chestnut blight fungus Cryphonectria parasitica. Most viruses identified in this fungus fall within the Hypoviridae family of positive-sense RNA viruses, which contains one genus and four species. Different species predominate in different geographic locations in chestnut-growing areas around the world. In this paper, we describe the genome organization and some variants of Cryphonectria hypovirus 4 (CHV-4), the species most commonly found in eastern North America. CHV-4 is distinguished from other hypoviruses by having little effect on fungal virulence and colony morphology. The 9.1-kb genome of strain CHV-4/SR2 is the smallest of any member of the family characterized to date. Like the recently characterized species CHV-3, a single ORF was predicted from deduced translations of CHV-4/SR2. Sequence analysis revealed the presence of a putative glucosyltransferase domain in both CHV-4 and in CHV-3, but no such homolog was detected in the more thoroughly examined CHV-1 or in CHV-2. Alignments with 8 other CHV-4 isolates from different regions of eastern North America revealed sequence diversity within the species and the likelihood that RNA recombination has led to this diversity.     

An analysis of common isodisomic regions in five mUPD 16 probands

Intrauterine growth retardation (IUGR) with or without additional abnormalities is recognised as a common feature of maternal uniparental disomy for chromosome 16 (mUPD 16) and is usually associated with confined placental mosaicism (CPM). Although it is likely that the CPM largely contributes to the IUGR, postnatal growth retardation and other common abnormalities may also be attributed to the mUPD. Five cases with mUPD 16 and CPM were analysed for common regions of isodisomy using polymorphic markers distributed along the length of the chromosome. In each case the aberration was consistent with a maternal meiosis I error. Complete isodisomy was not detected in any of the patients although two patients were found to be mixed with both iso- and heterodisomy. Interestingly, the patient with the greater region of isodisomy was the most severely affected. The fact that there were no common regions of isodisomy in any of the patients supports the hypothesis that imprinted genes, rather than recessive mutations, may play a role in the shared phenotypes.     

An inexpensive and reliable method for routine identification of staphylococcal species

The aim of this study was to develop a simple, reliable, and inexpensive in-house system for routine species identification of staphylococci in clinical practice. The system combines 15 key tests (including carbohydrate fermentation) performed in micro-well strips and antimicrobial disk diffusion susceptibility tests performed on standardised paper disk method antibiotic sensitivity medium agar. Twenty-eight Staphylococcal reference strains belonging to 18 different species were correctly identified using this in-house system. A total of 291 clinical staphylococci isolates were evaluated with the in-house system and a conventional identification scheme. The in-house system identified 281 (96.6%) of these 291 isolates. Eleven different species were recognised. The five species most frequently identified wereStaphylococcus epidermidis (48.6%),Staphylococcus aureus (27.8%),Staphylococcus haemolyticus (8.2%),Staphylococcus hominis (5.7%), andStaphylococcus warneri (5.3%). There was an agreement of 86.3% between the species identification obtained with the in-house system and the conventional identification scheme. All coagulase-negative isolates initially identified as species other thanStaphylococcus epidermidis as well as indistinctly identified isolates were also evaluated with a commercial identification system. The agreement between species identification obtained with the inhouse system and the commercial system for 101 identified isolates was 73%. Several isolates that were difficult to distinguish with the conventional scheme and/or the commercial system were identified with the aid of the antimicrobial susceptibility test included in the in-house system. The described test scheme should be of value for identification of clinically significant staphylococci species.     

An optimized DNA extraction method for molecular identification of coccidian species

Parasitology Research - Molecular identification of Eimeria parasites infecting poultry and livestock has been commonly used for more than 20 years. An important step of the molecular...     

Identification of the five most common cystic fibrosis mutations in single cells using a rapid and specific differential amplification system

We describe a rapid and specific differential amplificationsystem which can detect five of the most common cystic fibrosismutations from a single cell. In the first round of the polymerasechain reaction (PCR), regions of exons 4, 10 and 11 of the cysticfibrosis transmembrane conductance regulator (CFTR) gene containingthe mutations     

Identification and phylogenetic relationship of the most common pathogenic Candida species inferred from mitochondrial cytochrome b gene sequences

We sequenced a 396-bp region of the mitochondrial cytochrome b gene of the most common clinically important Candida species: Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. lusitaniae. The recently described species of Candida, C. dubliniensis, associated with mucosal candidiasis in human immunodeficiency virus-infected individuals, was also included. Two to five strains of each species were examined. Some species represented intraspecies variation, which was not more than 1.8% (DNA). However, interspecies variations were more than 10 and 7%, respectively, for DNA and amino acid sequences. Multiple alignments of nucleotide and deduced amino acid sequences revealed species-specific nucleotides and amino acids. Nucleotide- and amino acid-based phylogenetic trees were constructed and are discussed. Using the database, it is possible to identify presumptive Candida species within a working day.     

Monoclonal antibody that recognizes an outer membrane antigen common to the pathogenic Neisseria species but not to most nonpathogenic Neisseria species.

A hybridoma derived from a mouse immunized with gonococcal outer membranes produced an antibody, designated H.8, that bound to all strains of Neisseria gonorrhoeae and N. meningitidis tested, and to N. lactamica and N. cinerea, but only rarely to other nonpathogenic Neisseria species. Studies with the gonococcal strain used in production of the antibody showed that the antibody bound to a surface-exposed, protease-sensitive, and heat-modifiable outer membrane antigen that we believe is distinct from previously described gonococcal outer membrane proteins.     

Sarcoglycanopathies: a multiplex molecular analysis for the most common mutations.

Sarcoglycanopathies (SGpathies) are highly frequent among severely affected limb-girdle muscular dystrophy patients. On the basis of the findings of 5 common mutations in the 4 sarcoglycan (SG) genes in the Brazilian population, we standardized a multiplex polymerase chain reaction-single-strand conformation polymorphism methodology for their concomitant analysis in DNA samples. The test was able to confirm the diagnosis in about 63% of new patients with a suspected SGpathy and was particularly important in patients in advanced stages of the disease, when obtaining a muscle biopsy for analysis may be very difficult. As common mutations have been described in several countries, this multiplex analysis could be useful for the diagnosis of SGpathies if established according to the most prevalent mutations in each population. Besides, even though the disorder studied is rare, the technique could be broadly applicable to other genes and disorders.     

Multiplex PCR for distinguishing the most common phase-1 flagellar antigens of Salmonella spp

Most Salmonella serotypes alternatively express either phase-1 or phase-2 flagellar antigens, encoded by the fliC and fljB genes, respectively. Flagellar phase reversal for the identification of both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fliC genes encoding the H:i, H:r, H:l,v, H:e,h, H:z(10), H:b, and H:d antigens have been sequenced; and the specific sites for each antigen in selected Salmonella serotypes have been determined. These results, together with flagellar G-complex variable internal sequences obtained by the Foodborne and Diarrheal Diseases Branch at the Centers for Disease Control and Prevention in Atlanta, GA, have been used to design a multiplex PCR to identify the G-complex antigens as well as the H:i, H:r, H:l,v, H:e,h, Hz(10), H:b, and H:d first-phase antigens. These antigens are part of the most common Salmonella serotypes possessing first-phase flagellar antigens. Salmonella enterica serotype Enteritidis is identified by adding a specific primer pair published previously. This multiplex PCR includes 13 primers. A total of 161 Salmonella strains associated with 72 different serotypes were tested. Each strain generated one first-phase-specific antigen fragment ranging from 100 to 500 bp; Salmonella serotype Enteritidis, however, generated two amplicons of 500 bp that corresponded to the G complex and a 333-bp serotype-specific amplicon, respectively. Twenty-three strains representing 19 serotypes with flagellar genes different from those targeted in this work did not generate any fragments. The method is quick, specific, and reproducible and is independent of the phase expressed by the bacteria when they are tested.     

基因释放剂结合异源双链分析快速检测β地贫最常见的一种突变

β地贫是一种分子病理具有高度异质性的、我国南方最为常见的遗传性血液病.在已发现的23种β地贫突变中,CD41-42(-TCTT)移码突变基因频率最高(达41.6%).根据PCR原理和该突变的特点,作者设计了一对引物进行PCR特异扩增,电泳分析即可检出此种突变.并引进基因释放剂DNA模板快速制备法,从而使此技术更为简便和实用.作者运用此法检测了广东珠海地区114份β地贫样品,CD41-42突变捡出率为40.35%,并成功地应用于产前诊断中.根据实验结果,作者建议在分析未知β地贫突变时,首先采用本法先对CD41-42突变进行初筛,而将RDB和ASO技术作为补充.