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PubMed1992

Development of a species-specific DNA probe for Mycoplasma capricolum.

A specific DNA probe for the detection and identification of Mycoplasma capricolum, one of the causative agents of contagious agalactia syndrome, was selected from a genomic library. It consists of a 900bp RsaI genomic fragment of M. capricolum (reference strain), cloned into the EcoRV site of the plasmid Bluescript. By using the appropriate stringency this radiolabelled probe reacts specifically with M. capricolum when tested by dot blot hybridization against various mycoplasmal DNAs. The current level of sensitivity of the 32P-labelled 900bp RsaI probe is 500 pg of homologous DNA, corresponding to 5 x 10(4) mycoplasmas. A non radioactive labelling method, using the digoxigenin-11-dUTP, was also tested. The specificity of the digoxigenin-labelled probe was equivalent to that obtained with the radioactive probe. However the sensitivity of detection decreased to 1 ng of homologous DNA detected, corresponding to 1 x 10(5) mycoplasmas. Tests performed with milk samples have demonstrated that the radioactive 900 bp RsaI probe indeed detected M. capricolum contained in milk. A positive signal was obtained when 10(5) M. capricolum were present in the spot.

Animals↗
PubMed1992

Sensitive and species-specific detection of Erwinia amylovora by polymerase chain reaction analysis.

Detection and identification of the fire blight pathogen, Erwinia amylovora, can be accurately done by polymerase chain reaction (PCR) analysis in less than 6 h. Two oligomers derived from a 29-kb plasmid which is common to all strains of E. amylovora were used to amplify a 0.9-kb fragment of the plasmid. By separation of the PCR products on agarose gel, this fragment wa specifically detected when E. amylovora DNA was present in the amplification assay. It was not found when DNA from other plant-pathogenic bacteria was used for the assay. A visible band specific to the 0.9-kb fragment was produced with DNA from fewer than 100 E. amylovora cells. A signal of similar strength was also obtained from E. amylovora cell lysates in the presence of the mild detergent Tween 20. Signals were weaker when bacteria were added to the PCR mixture without the detergent. As with results obtained from hybridization experiments using pEA29 DNA< the PCR signal was obtained with E. amylovora isolates from various geographic regions. This technique could also be used for detection of the fire blight pathogen in extracts of tissue obtained from infected plant material.

Base Sequence↗
PubMed1998

Species-specific and ubiquitous-DNA-based assays for rapid identification of Staphylococcus aureus.

Staphylococcus aureus is the cause of serious infections in humans, including endocarditis, deep-seated abscesses, and bacteremia, which lead to toxic and septic shock syndromes. Rapid and direct identification of this bacterium specifically and ubiquitously directly from clinical specimens would be useful in improving the diagnosis of S. aureus infections in the clinical microbiology laboratory. A wide variety of kits based on biochemical characteristics efficiently identify S. aureus, but the rapidity and the accuracy of each of these methods combined with testing of clinically relevant antibiotic resistance genes need to be improved. On the basis of hybridization assays with randomly selected clones from an S. aureus genomic library, we have identified a chromosomal DNA fragment which is specific for S. aureus and which detected all 82 S. aureus isolates tested. This 442-bp fragment was sequenced and was used to design a set of PCR amplification primers. The PCR assay was also specific and ubiquitous for the identification from bacterial cultures of 195 clinical strains of S. aureus isolated from a variety of anatomical sites and obtained from hospitals throughout the world. The PCR assay that we have developed is simple and can be performed in about 1 h. This DNA-based test provides a novel diagnostic tool for the diagnosis of S. aureus infections.

Cloning, Molecular↗
PubMed1992

Detection of Borrelia burgdorferi in ticks by species-specific amplification of the flagellin gene.

We developed a polymerase chain reaction (PCR) that specifically amplifies a fragment of the flagellin gene (fla) of Borrelia burgdorferi, the causative agent of Lyme disease. This fla target, amplified with nested primers, was conserved among all 80 strains of B. burgdorferi tested. Strains examined included cultures from ticks, humans, and rodents from major B. burgdorferi-endemic regions of the United States and parts of Europe and Asia. Templates from B. hermsii, B. parkeri, B. turicatae, and B. coriaceae were not amplified, nor were eukaryotic DNAs from three tick genera. Several host DNAs potentially present in a tick blood meal also were not amplified. Approximately six B. burgdorferi per PCR reaction could be detected by ethidium bromide staining of amplified DNA. Colony-raised Ixodes dammini were used to evaluate the method. One infected nymph in a pool of 40 ticks was routinely detected. The specificity of the assay for detecting B. burgdorferi-infected ticks in pools was 94% (29 of 31). This protocol should prove useful for assessing infection rates in other putative arthropod vectors.

Animals↗
PubMed2003

Demonstration of species-specific and cross-reactive components of hydatid cyst fluid antigens.

ELISA-hydatid cyst fluid antigen (HCF-Ag-ELISA) and conventional echinococcosis IHAT were assessed for cystic hydatid disease (CHD), confirmed by HCF-Ag immunoblotting assay. The sensitivity of the tests was 94.4% and 83.3% for HCF-Ag-ELISA and IHA respectively. HCF-Ag-ELISA showed some cross-reaction in tumor cases (5.5%), but no cross-reaction was observed in Rheumatoid arthritis patients. IHA showed 100% specificity. To confirm this data SDS-PAGE immunoblotting using HCF-Ag was applied. Echinococcus specific reactions were reported with 44Kda, 34Kda, 29Kda and 8Kda HCF-Ag resolved bands. Cross-reaction was found with 27Kda, 21Kda, 16Kda and 13Kda bands in tumor patients. Moreover, cross-reaction was found with 200Kda, 175Kda, 62Kda, 52Kda and 40Kda in serological anti-Schistosoma antibodies positive cases. So, using specific HCF-Ag components, immunoblotting provided 100% sensitivity and specificity in CHD.

Animals↗
PubMedDate not supplied

Polymerase chain reaction method to detect canis materials by amplification of species-specific DNA fragment.

Rapid identification of mammal materials in feeding stuffs and food is essential for effective control of a potential source of pathogens, such as those that cause bovine spongiform encephalopathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a canis-specific mitochondrial DNA sequence in foodstuffs and food. The amplified canis-specific PCR product was a 213 base pair band from the D-loop DNA fragment of mitochondria, a high copy gene which should improve the possibility of amplifying template molecules of adequate size among the degraded DNA fragments brought about by heat denaturation. The specificity of this method was confirmed by 8 canis blood DNA samples (from different breeds of dog) and 9 noncanis animal blood DNA samples (bovine, sheep, porcine, chicken, fish, donkey, rabbit, deer, horse). This method was able to detect the presence of canis material in foodstuffs and in food mixtures even when the concentration of canis-derived meat was reduced to 0.05%. Furthermore, it did not appear to be affected by prolonged heat treatment. This method was developed for detection of canis materials in feeding stuffs, and occasionally for medical jurisprudence detection of canis-derived materials.

Animals↗
PubMed1996

Species specific immunoassays to measure blood platelet and coagulation activation in the rat.

The purpose of this study was to develop specific and sensitive immunoassays to detect early indices of hypercoagulability in the rat. Rat platelet factor 4 (rPF4) and rat fibrinopeptide A (rFPA) assays, tools for the detection of activation of platelets and coagulation respectively, were designed using antibodies raised against purified rPF4 and against synthetic rFPA. The relevance of these new assays and of the commercially available ELISA kit for thrombin-antithrombin III (TAT) complexes was demonstrated in a rat model of a prethrombotic state induced by intravenous infusion of varying doses of thrombo-plastin (90 to 2400 microliters/kg/h). In this model, the immunoassays allowed simultaneous detection of low levels of rFPA and rPF4 which were correlated with fibrinogen and platelet consumption and TAT generation and further proved to be of higher sensitivity than the classical methods of platelet count or measurement of fibrinogen levels. Plasma concentrations of rFPA, rPF4 and TAT were dependent on infusion time and thromboplastin dose, while hirudin (1 mg/kg) prevented their appearance. Thus the new specific immunoassays for rPF4 and rFPA and the commercial human TAT assay represent useful tools for pathophysiological studies or the screening of antithrombotic drugs in rats.

Animals↗
PubMed1979

Response of nonhuman animals to speech and to species-specific sounds.

Three questions relate to how nonhuman species respond to speech and species-specific sounds: (1) Do nonhuman species perceive human speech in a human-like fashion? (2) How do nonhuman species perceive their own vocalizatios? and (3) How does human perception of animal sounds differ from the animals' perception of those sounds? Four methodologies are available for studying an animal's perception of sounds: (1) discriminative conditioning; (2) habituation-dishabituation; (3) playbacks in captivity; and (4) playbacks in situ. Each of these techniques has a different degree of ecological validity and has different intrinsic biases toward the conclusions one might draw. Experiments using these methodologies for each of the three questions are reviewed and the methodological problems of each are discussed. A two-stage model of perceiving species-specific sounds is presented which accounts for both categorical perception of sounds and within category discrimination of sounds.

Animal Communication↗
PubMed2005

Generation and characterization of SCARs by cloning and sequencing of RAPD products: a strategy for species-specific marker development in bamboo.

BACKGROUND AND AIMS: The aim of this study was to develop species-specific molecular markers for Bambusa balcooa and B. tulda to allow for their proper identification, in order to avoid unintentional adulteration that affects the quality and quantity of paper pulp production. METHODS: Two putative, species-specific RAPD markers, Bb836 for B. balcooa and Bt609 for B. tulda were generated using a PCR-based RAPD technique. Species-specificity of these two markers was confirmed through Southern hybridization in which RAPD gels were blotted and hybridized with radiolabelled cloned RAPD markers. Southern hybridization analyses were also performed to validate homology of the co-migrating Bb836 and Bt609 marker bands amplified from 16 different populations of B. balcooa and B. tulda, respectively. Sequence-characterized amplified region (SCAR) markers were developed from Bb836 and Bt609 sequences, using 20-mer oligonucleotide primers designed from both the flanking ends of the respective RAPD primers. KEY RESULTS: As anticipated, Bb836 hybridized with an amplified band from B. balcooa and Bt609 hybridized only with an amplified product from B. tulda; the two markers did not hybridize with the amplified products of any of the other 14 bamboo species studied. The two pairs of SCAR primers amplified the target sequences only in the respective species. The species-specific SCAR fragments were named as 'Balco836' for B. balcooa and 'Tuldo609' for B. tulda. The species-specific 'Balco836' was amplified from the genomic DNA of 80 individuals of 16 populations of B. balcooa studied. Similarly, the presence of 'Tuldo609' was noted in all the 80 individuals representing 16 populations of B. tulda assessed. These SCAR fragments contained no obvious repetitive sequence beyond the primers. CONCLUSION: These two molecular markers are potentially useful for regulatory agencies to establish sovereign rights of the germplasms of B. balcooa and B. tulda. In addition, this is the first report of species-specific SCAR marker development in bamboo.

Bambusa↗
PubMedDate not supplied

Development of a Chlamydophila psittaci species-specific and genotype-specific real-time PCR.

A Chlamydophila psittaci species-specific real-time PCR targeting the rDNA ribosomal spacer was developed as well as a genotype-specific real-time PCR targeting the Cp. psittaci outer membrane protein A (ompA) gene. The SYBR Green-based species-specific real-time PCR detected Cp. psittaci genotypes A to F, and the recently discovered E/B genotype. The genotype-specific real-time PCR could easily distinguish genotypes C, D, F by use of TaqMan probes. Genotypes A, B and E could not be distinguished from each other by simply using TaqMan probes. For this purpose, non-fluorescent competitor oligonucleotides, had to be used next to the TaqMan probes. Genotype E/B could only be detected by use of a minor groove binder (MGB) probe. Both real-time PCR assays allowed reproducible, sensitive (10 rDNA or ompA copies/microL DNA extract) and specific detection of Cp. psittaci DNA. The genotype-specific real-time PCR was compared to ompA sequencing and ompA restriction fragment length polymorphism (RFLP) analysis using five Cp. psittaci field isolates (99, 61/8, 7344/2, 8615/1 and 7778B15) each consisting of two different genotypes. The currently developed real-time PCR assays were used in a case study on a veterinary school and a turkey farm. In the veterinary school, Cp. psittaci genotypes D, E/B and F infection were detected in all five groups of turkeys, and one veterinarian who was taking care of all these turkeys. On the turkey farm, the presence of two Cp. psittaci genotype B infection waves was demonstrated in one randomly selected turkey, the first wave at the age of 6 weeks, and the second at the age of 12 weeks.

Animals↗
PubMed1991

Structure of the core promoter of human and mouse ribosomal RNA gene. Asymmetry of species-specific transcription.

In vitro transcription of the ribosomal RNA gene (rDNA) shows a remarkable species specificity such that human and mouse rDNA cannot use heterologous extracts of each other. The region that is responsible for this specificity has been studied using human-mouse chimeric genes and characteristic structures of both core promoters are presented. When the mouse sequence is substituted by the corresponding human sequence from upstream, the promoter activity in the mouse extract begins to decline at nucleotide -32 or -30, decreasing gradually and is lost completely at -19. A similar gradual decrease was noted for the 3' side substitution, which started at nucleotide -14 and was completed when up to the nucleotide -22 mouse position was replaced by the corresponding sequence from human. Thus, in the mouse rDNA core promoter, the sequence that is involved in species specificity resides only in a stretch encompassing the non-conserved region between the distal conserved sequence (DCS) and the proximal conserved sequence (PCS), plus two altered nucleotides in the PCS. When human rDNA is transcribed with human cell extract, the mouse sequence cannot substitute for the human sequence within the region from nucleotide -43 to +17 without affecting promoter activity significantly. This asymmetry of species specificity is due to the presence of nucleotides -43, +1 and +17, which are sensitive to change in only the human core promoter. The difference in the 5' border is ascribed to the species specificity of a transcription factor TFID, which recognizes this region. But the large difference of the 3' border is apparently due to another factor, possibly RNA polymerase I itself, because this region is not recognized by TFID in either human or mouse. Mammalian rDNA core promoter appears to consist of a tandem mosaic in which three evolutionarily conserved sequences alternate with non-conserved sequences having certain functionally important nucleotides. Not only non-conserved sequences and non-conserved nucleotides in conserved sequences, but also the spacings between the three conserved regions, play a crucial role in species specificity.

Animals↗
PubMed2000

Species-specific oligonucleotide probes for macroalgae: molecular discrimination of two marine fouling species of Enteromorpha (Ulvophyceae).

The green seaweeds Enteromorpha intestinalis and E. compressa are important fouling organisms commonly found in polluted and nutrient-enriched marine and brackish water habitats, where they are used in environmental monitoring. Discrimination of the two species is extremely difficult because of overlapping morphological characters. In this study a quick molecular method for species identification was developed based on the nuclear rDNA ITS2 sequence data of 54 E. intestinalis samples and 20 E. compressa samples from a wide geographical range. Oligonucleotide probes were designed for species-specific hybridization to dot-blots of the PCR-amplified ITS1, 5.8S gene and ITS2 fragment of both E. intestinalis and E. compressa. Specificity of the oligonucleotide probes was confirmed by tests with taxonomically diverse species that could morphologically be confused with E. intestinalis or E. compressa. This is the first use of species-specific probes for macroalgae. The restriction endonuclease NruI digested specifically the amplified PCR product from E. compressa into two fragments detectable on agarose gels, but no suitable restriction sites were identifiable in the PCR product of E. intestinalis.

Base Sequence↗