Hybridization analysis of the gene encoding a hemolysin (suilysin) of Streptococcus suis type 2: evidence for the absence of the gene in some isolates

A hemolysin gene was cloned from a virulent strain of Streptococcus suis type 2 strain 1933. Analysis of the gene and its product revealed that it is identical to a previously reported hemolysin (suilysin) of S. suis type 2. Southern hybridization analysis of the digested total genomic DNA from S. suis with the cloned hemolysin DNA sequences as probe indicated that the hemolysin gene is present as a single copy on the genome. Genomic DNA of 63 isolates of S. suis encompassing all known serotypes were examined by DNA hybridization and polymerase chain reaction (PCR) studies for the presence of the hemolysin gene homolog. The results of both techniques were identical and demonstrated the absence of the hemolysin gene in some isolates. In DNA hybridization studies, three DNA probes derived from the hemolysin encoding gene were used. Results showed that sequences encoding the C-terminal 257 amino acid residues (Probe 1) were the most conserved and hybridized to a 1.2 kb fragment in 32 (51%) strains and a 4.0 kb fragment in 23 (36%) strains respectively. Thus, Probe 2 hybridized to the DNA of 55 (87%) of the isolates tested. The first probe (Probe 1) comprising almost the entire hemolysin gene and the third probe (Probe 3) which consisted of the N-terminal sequences hybridized only to a 4.0 kb fragment in 23 (36%) of the strains tested. Eight (13%) of the strains tested were hybridization and PCR negative. The hybridization of the C-terminal end sequences (Probe 2) to the 1.2 kb fragment in 32 (51%) of the strains and the lack of hybridization of the probes to eight (13%) strains may suggest the presence of different types of hemolysin molecule in S. suis strains.     

Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification

AIMS: To examine whether Aeromonas bacteria isolated from municipally treated water had virulence factor genes. METHODS AND RESULTS: A polymerase chain reaction-based genetic characterization determined the presence of six virulence factors genes, elastase (ahyB), lipase (pla/lip/lipH3/alp-1) flagella A and B (flaA and flaB), the enterotoxins, act, alt and ast, in these isolates. New primer sets were designed for all the target genes, except for act. The genes were present in 88% (ahyB), 88% (lip), 59% (fla), 43% (alt), 70% (act) and 30% (ast) of the strains, respectively. Of the 205 isolates tested only one isolate had all the virulence genes. There was a variety of combinations of virulence factors within different strains of the same species. However, a dominant strain having the same set of virulence factors, was usually isolated from any given tap in different rounds of sampling from a single tap. CONCLUSIONS: These results show that Aeromonas bacteria found in drinking water possess a wide variety of virulence-related genes and suggest the importance of examining as many isolates as possible in order to better understand the health risk these bacteria may present. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a rapid method for characterizing the virulence factors of Aeromonas bacteria and suggests that municipally treated drinking water is a source of potentially pathogenic Aeromonas bacteria.     

A new set of PCR assays for the identification of multiple human adenovirus species in environmental samples

Aims:  To assess human adenovirus (HAdV) diversity in environmental samples based on sequence comparisons of hexon gene fragments amplified using newly designed HAdV-specific polymerase chain reaction (PCR) assays.
Methods and Results:  Six PCR primer sets were designed based on 56 aligned hexon sequences from NCBI GenBank to amplify different hexon gene sections (241–349 bp) of the six HAdV species. The amplified hexon genes from wastewater samples were cloned, sequenced, and compared with those in publicly accessible databases (i.e. NCBI GenBank) by using the B last program. A total of 46 analysed positive clones were affiliated to five HAdV serotypes, i.e. 1, 2, 12, 31 and 41. Similarities between the cloned and database hexon sequences ranged from 95·9 to 100% (with an average of 98·1 ± 1·0%).
Conclusion:  The designed primers showed higher amplification efficiencies when compared with the existing assays. Using the new assays, HAdV species A, C, and F (serotypes 1, 2, 12, 31 and 41 in particular) were identified in the studied municipal wastewater.
Significance and Impact of the Study:  The six PCR primer sets developed in this study can be used to efficiently amplify hexon gene fragments in HAdV. Multiple HAdV serotypes identified in the municipal wastewater provide new information about HAdV diversity in environmental samples.     

三种前处理在基质辅助激光解析电离飞行时间质谱鉴定假丝酵母菌属中的应用比较

目的 比较3种前处理方法在基质辅助激光解析电离飞行时间质谱(MALDI TOF MS)鉴定假丝酵母菌属中的结果可靠性。 方法 以ITS测序鉴定结果为金标准,对临床分离的66株假丝酵母分别采用传统直涂法、改良直涂法和甲酸-乙腈蛋白提取法进行前处理,MALDI TOF MS鉴定,比较3种方法的Biotyper Log值,分析质谱图的差异。 结果 传统直涂法、改良直涂法和甲酸-乙腈提取法对66株假丝酵母的属水平鉴定率分别为48.5%、50.0%和97.0%,Biotyper Log均值分别为1.628、1.674和2.010,其中甲酸-乙腈提取法对66株假丝酵母的种水平鉴定率为53.0%。甲酸-乙腈提取法得到的质谱图比另2种方法的质谱图离子峰更加密集,图像更复杂,鉴定结果可信度更高。 结论 甲酸-乙腈蛋白提取法对假丝酵母菌属的鉴定成功率和可靠性明显高于传统直涂法和改良直涂法,对临床假丝酵母菌病的准确诊断具有重要价值。     

Sequences from the aspergillopepsin PEP gene of Aspergillus fumigatus: evidence on their use in selective PCR identification of Aspergillus species in infected clinical samples

In immunodeficient patients, Aspergillus species emerge as circumstantial pathogens. Aspergillus fumigatus is a distant first among the pathogenic aspergilli, which cause deep-seated mycoses. Sequences of the pep gene of A. fumigatus as potential PCR primers, which have not been tested before, were used to identify this species and if possible, differentiate it from other, co-identified, clinically important species of the genus. We present results of the three most promising primer pairs, pep-1/pep-22, pep-15/pep-22 and pep-21/pep32. The second pair was of better specificity when tested with DNA extracted from pure cultures of a multitude of aspergilli, whereas the first co-amplified four clinically significant Aspergillus species. The compatibility of the PCR method with the CTAB DNA extraction protocol varied according to the biological fluid tested and the primer pair used. The first two pairs showed moderate adaptability to the different commercial DNA extraction kits, which were tested in whole blood, spiked with Aspergillus fumigatus hyphae and conidia - as were all the biological fluids used. Restriction of the amplification products with MspI produced distinct patterns for different Aspergillus spp. This approach, as a potential diagnostic tool, seems reliable and sensitive due to its flexibility, speed, low cost, ease of application and selectable breadth of detection.     

Evaluation of methods for molecular typing and identification of members of the genus Brevibacterium and other related species

The genus Brevibacterium includes pleomorphic Gram-positive bacteria with a high mol% G+C content. Species in the genus are difficult to identify by classical methods. The discriminatory power of DNA-based methods is assessed. Strains representing the four well established Brevibacterium species, and other related bacteria, were compared by amplified ribosomal DNA restriction analysis (ARDRA), repetitive-sequence-based PCR (rep-PCR) and ribotyping. Fingerprinting by rep-PCR and ribotyping provided complex genomic profiles with the highest discriminatory potential for molecular typing at the strain level, whereas ARDRA showed differentiation from the genus to the species levels. A high degree of heterogeneity within the genus Brevibacterium is apparent, thus indicating that the taxonomy of the genus should be further studied.     

Molecular identification of six species of scale insects (Quadraspidiotus sp.) by RAPD-PCR: assessing the field-specificity of pheromone traps

The San-José Scale Quadraspidiotus perniciosus (SJS) is a quarantine pest in Switzerland. Its occurrence is monitored by trapping males on glue traps treated with artificial female sex pheromone that is supposed to be species-specific. However, large numbers of males were caught in locations where, for ecological reasons, this species was not expected to occur, suggesting that the pheromone is attractive for one or several otrter scale species. Because no morphological species determination key is described for males of this genus, it was not possible to test this hypothesis until now. We used randomly amplified polymorphic DNA (RAPD-PCR) to establish a molecular identification key for six European Quadraspidiotus species. Because the glue used on the traps proved to be an excellent preservative, this key enabled us to identify males caught on pheromone traps in the field and to assess the species-specificity of the SJS-pheromone.     

Quantification of total mitochondrial DNA and the 4977-bp common deletion in Pearson's syndrome lymphoblasts using a fluorogenic 5'-nuclease (TaqMan) real-time polymerase chain reaction assay and plasmid external calibration standards

This study describes a multiplex real-time polymerase chain reaction (PCR) assay that quantifies total mitochondrial DNA (mtDNA(total)) and mtDNA bearing the 4977-base pair 'common deletion' (deltamtDNA4977) in lymphoblasts derived from an individual diagnosed with Pearson's syndrome. The method is unique in its use of plasmids as external quantification standards and its use of multiplex conditions. Standards are validated by comparison with purified mtDNA amplification curves and by the fact that curves are largely unaffected by nuclear DNA (nucDNA). Finally, slopes of standard curves and unknowns are shown to be similar to each other and to theoretical predictions. From these data, mtDNA(total) in these cells is calculated to be 3258 (+723/-592) copies per cell while deltamtDNA4977 averages 232 (+136/-86) copies per cell or 7% (+4.65/-2.81).     

Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA

Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.     

Species identification of Cecidophyopsis mites (Acari: Eriophyidae) from different Ribes species and countries using molecular genetics

Cecidophyopsis mites were studied by PCR amplification of parts of their ribosomal DNA, followed by restriction enzyme analysis. Mite specimens on Ribes nigrum (black currant) from six countries gave the same digestion pattern, which was distinct from the pattern for mites found on R. rubrum from Poland and Finland and for R. grossularia from the USA. This suggests that each Ribes species is host to a different mite species: C. ribis, C. selachodon and C. grossulariae, respectively. Two other mite samples from R. alpinum and R. aureum were identical but were distinct from each of the other species.     

Triacylglyceride composition and fatty acyl saturation profile of a psychrophilic and psychrotolerant fungal species grown at different temperatures

Pseudogymnoascus destructans is a psychrophilic fungus that infects cutaneous tissues in cave dwelling bats, and it is the causal agent for white nose syndrome (WNS) in North American (NA) bat populations. Geomyces pannorum is a related psychrotolerant keratinolytic species that is rarely a pathogen of mammals. In this study, we grew P. destructans and G. pannorum in static liquid cultures at favourable and suboptimal temperatures to: 1) determine if triacylglyceride profiles are species-specific, and 2) determine if there are differences in fatty acyl (FA) saturation levels with respect to temperature. Total lipids isolated from both fungal spp. were separated by thin-layer chromatography and determined to be primarily sterols (∼15 %), free fatty acids (FFAs) (∼45 %), and triacylglycerides (TAGs) (∼50 %), with minor amounts of mono-/diacylglycerides and sterol esters. TAG compositions were profiled by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI–TOF). Total fatty acid methyl esters (FAMEs) and acyl lipid unsaturation levels were determined by gas chromatography–mass spectrometry (GC–MS). Pseudogymnoascus destructans produced higher proportions of unsaturated 18C fatty acids and TAGs than G. pannorum. Pseudogymnoascus destructans and G. pannorum produced up to a two-fold increase in 18:3 fatty acids at 5 °C than at higher temperatures. TAG proportion for P. destructans at upper and lower temperature growth limits was greater than 50 % of total dried mycelia mass. These results indicate fungal spp. alter acyl lipid unsaturation as a strategy to adapt to cold temperatures. Differences between their glycerolipid profiles also provide evidence for a different metabolic strategy to support psychrophilic growth, which may influence P. destructans' pathogenicity to bats.     

Gene expression and immunolocalisation of a calcium-activated chloride channel during the stratification of cultivated and developing corneal epithelium

The spatial and temporal localisation of a calcium-activated chloride channel (CLCA) and its mRNA was investigated, during the in vivo and in vitro development of stratified epithelia, by fluorescence immunohistochemistry and quantitative polymerase chain reaction in embryonic chicken corneas and the expansion of excised human corneal stem cells on amniotic membrane. Single-layered human epithelial cultures on amniotic membrane and early day embryonic chicken corneas expressed relatively little human CLCA2 or its chicken homologue. However, as the epithelium in both models matured and the number of cell-layers increased, the gene expression level and protein staining intensity increased, primarily within the basal cells of both the cultured and embryonic tissues. These results demonstrate that human CLCA2 protein and mRNA expression are elevated during epithelial stratification, suggesting that this protein plays a role in the growth of multi-layered corneal epithelia during both natural development and tissue cultivation. This work was supported by the Japanese Society for the Promotion of Science (CJC) and The Royal Society (C.J.C.).     

PCR-based methods for identification of species of the Anopheles minimus group: allele-specific amplification and single-strand conformation polymorphism

We report two polymerase chain reaction (PCR)-based methods for distinguishing morphologically similar species based on amplification of a variable region of the 28S gene of ribosomal DNA. The four species we investigated are mosquitoes of the Anopheles minimus group: An. aconitus, An. varuna and An. minimus species A and C. The formally named species are vectors of human malaria parasites in south-east Asia but are difficult to distinguish with certainty on the basis of morphology. Allele-specific amplification was used to differentiate An. minimus A from An. minimus C. This technique has been widely used for the diagnosis of species. Single-strand conformation polymorphisms (SSCPs) were used to separate all four species. This technique, which has seldom been used for species identification, has many advantages: it does not require sequence information beyond that needed for amplification; it is ideally suited for the detection of heterozygotes; it utilizes more of the information in the PCR product than allele-specific amplification; it distinguishes all four species considered here and could easily be extended to other species; previously unknown intraspecific variation and additional species are likely to be detected. Thus, SSCPs provide valuable population genetic information which allele-specific amplification does not.     

Fine‐scale spatial variability in anatoxin‐a and homoanatoxin‐a concentrations in benthic cyanobacterial mats: implication for monitoring and management

Aims: The purpose of this study was to determine the variability in anatoxin‐a (ATX) and homoanatoxin‐a (HTX) concentrations in benthic cyanobacterial mats within sampling sites and to assess the applicability of using a PCR‐based approach to determine ATX‐ and HTX‐production potential. Methods and Results: ATX and HTX variability was investigated by collecting 15 samples from 10 × 10 m grids in seven rivers. ATX and HTX concentrations were determined using liquid chromatography–mass spectrometry (LC–MS). Samples from two sites contained no ATX or HTX and at one site ATX and HTX were detected in all samples. At four sites, both toxic and nontoxic samples co‐occurred and these samples were sometimes spaced less than 1 m apart. PCR amplification of a region of a polyketide synthase (ks2, putatively involved in the biosynthetic pathway of ATX and HTX) successfully distinguished ATX‐and‐HTX‐ and non‐ATX‐and‐HTX‐producing cultured Phormidium strains. Results from environmental samples were more variable, and the results were in congruence with the LC–MS data in only 58% of samples. Conclusions: Fine‐scale spatial variability in ATX and HTX concentrations occurs among benthic cyanobacterial mats. Significance and Impact of the Study: Multiple benthic cyanobacterial mat samples must be collected at a sampling site to provide an accurate assessment of ATX and HTX concentrations at that location. The PCR‐based technique offers the potential to be a useful early warning technique.     

Cytochrome b561 is not fatty acylated but acetylated at amino terminus in chromaffin vesicle membranes: an approach for the identification of posttranslational modification of transmembrane proteins

Summary.  We examined the nature of the posttranslational modification of bovine cytochrome b ₅₆₁, a membrane-spanning protein and an essential component of neuroendocrine secretory vesicles. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) showed two populations in the partially digested fragments of cytochrome b ₅₆₁, which were obtained by controlled treatment of cytochrome b ₅₆₁-proteoliposomes with trypsin. One population, containing the posttranslationally modified amino-terminal region, showed molecular masses which were by about 40 Da larger than the theoretical molecular masses. The other population, without the modified amino-terminal region, showed a reasonable matching with the theoretical masses. This result suggested that the posttranslational modification occurred only in the amino-terminal region. The amino-terminal peptide was isolated by tryptic peptide mapping followed by treatment with acylamino-acid-releasing enzyme. Amino acid sequence and MALDI-TOF-MS analyses of the amino-terminal peptide showed that the initial Met residue was acetylated. There was no other posttranslational modification in the amino-terminal region, such as covalent fatty acylation through an ester linkage to Ser or Thr residues. Received May 9, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Department of Molecular Science, Graduate School of Science and Technology, Kobe University, Rokkodai-cho 1-1, Nada-ku, Hyogo 657-8501, Japan.     

Single-molecule analysis of the hypermutable tetranucleotide repeat locus D21S1245 through sperm genotyping: a heterogeneous pattern of mutation but no clear male age effect

Single molecule genotyping of the hypermutable microsatellite locus D21S1245 was used for studying how the rate and pattern of mutation varied between alleles and different age groups. In total, 203 mutation events were scored from the genotyping of DNA corresponding to an estimated 8623 sperm cells from eight different men. Allele-specific mutation rates ranged from 0.007 to 0.052, a heterogeneity related in part to variation in the mutation rate among three allelic lineages identified after allele sequencing. Alleles from these lineages differed in the overall repeat structure of this complex microsatellite locus. Also, the pattern of mutation varied between lineages in that they differed in the relative proportions of expansion and contraction mutations. Surprisingly, a group of four men aged 18-23 years showed a higher mean mutation rate than a group of four men aged 48-56 years. To some extent this age difference can probably be explained by a bias in the distribution of alleles from the three allelic lineages among the age groups. However, the absence of a clear male age effect is at odds with the idea of an increasing male mutation rate with age, which is thought to arise from the continuous replication of germline cells throughout adulthood.     

Isolation and characterization of a cDNA encoding a serine proteinase from the root-knot nematode Meloidogyne incognita

This report describes the first serine proteinase gene isolated from the sedentary nematode Meloidogyne incognita. Using degenerate primers, a 1372bp cDNA encoding a chymotrypsin-like serine proteinase (Mi-ser1) was amplified from total RNA of adult females by RT-PCR and 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of Mi-ser1 encoded a putative signal peptide and a prodomain of 22 and 33 amino acids, respectively, and a mature proteinase of 341 amino acids with a predicted molecular mass of 37,680Da. Sequence identity with the top serine proteinases matches from the databases ranged from 23 to 27%, including sequences from insects, mammals, and other nematodes. Southern blot analysis suggested that Mi-ser1 is encoded by a single or few gene copies. The pattern of developmental expression analyzed by Northern blot and RT-PCR indicated that Mi-ser1 was transcribed mainly in females. The domain architecture composed of a single chymotrypsin-like catalytic domain and the detection of a putative signal peptide suggested a digestive role for Mi-ser1.     

Isolation and characterization of microsatellites in the columnar cactus: Polaskia chichipe and cross‐species amplification within the Tribe Pachycereeae (Cactaceae)

Microsatellite markers were developed for the columnar cactus Polaskia chichipe from central Mexico. After an enrichment procedure and three screening steps 87% of colonies contained microsatellites. A pair of primers for 10 loci (seven polymorphic) were developed, tested and used to estimate variation in samples of 18–45 individuals from the Tehuacan Valley, Mexico. Alleles per locus ranged from two to eight (mean 5.28; SD 2.5). Range of expected heterozygosity values was 0.188–0.797 (mean 0.502; SD 0.25). These loci are particularly useful for more precise evolutionary studies, such as gene flow and breeding systems, for this cactus species.