Improved methods for the detection of Bacillus anthracis spores by the polymerase chain reaction

Polymerase chain reactions (PCRs) for the capsule and oedema factor genes of Bacillus anthracis were used to assess methods for detecting B. anthracis spores. Untreated spore preparations were found to contain significant amounts of extracellular template DNA which probably accounted for observed amplification from these preparations without spore lysis. Germination of spores with suitable media allowed the detection of less than 10 spores in a PCR test. Mechanical disruption of spores with glass or zirconia beads yielded similar results to germination but in a much shorter time. The techniques described should improve the detection by PCR of B. anthracis and other sporulating bacteria.     

Detection of fungal spores from contaminated surfaces by the polymerase chain reaction

A method has been developed to detect fungal spores in dust samples collected from internal surfaces of air-conditioning ducts. The method is based on the utilization of the polymerase chain reaction (PCR) with specific primers for fungal species. PCR amplification is carried out directly in boiled samples avoiding time-consuming DNA preparation steps. The presence of bovine serum albumin in the reaction mixture overcame the inhibitory effect of the humic acids present in the dust.     

Germination of Bacillus anthracis spores

The influence of amino acids, nucleosides and inorganic components on the kinetics and effectiveness of the germination of B. anthracis spores was studied. The study revealed that the rapid germination of the spores took place after their activation at 65 degrees C in tris buffer with L-alanine in combination with inosine or adenosine added; less pronounced germinative action was caused by the addition of alanine only and the combination of phenylalanine, tyrosine and tryptophan. The rapidity of germination and the sets of effective germinants for spores of different strains were different. All B. anthracis strains under study had nucleotide sequences, of gene gerX in their genome.     

Genetic comparison of Bacillus anthracis and its close relatives using amplified fragment length polymorphism and polymerase chain reaction analysis

Amplified fragment length polymorphism (AFLP) analysis allows a rapid, relatively simple analysis of a large portion of a microbial genome, providing information about the species and its phylogenetic relationship to other microbes (Vos et al. 1995). The method simply surveys the genome for length and sequence polymorphisms. The AFLP pattern identified can be used for comparison to the genomes of other species. Unlike other methods, it does not rely on analysis of a single genetic locus that may bias the interpretation of results and does not require any prior knowledge of the targeted organism. Moreover, a standard set of reagents can be applied to any species without using species-specific information or molecular probes. We are using AFLP analysis to rapidly identify different bacterial species. A comparison of AFLP profiles generated from a large battery of Bacillus anthracis strains shows very little variability among different isolates (Keim et al. 1997). By contrast, there is a significant difference between AFLP profiles generated for any B. anthracis strain and even the most closely related Bacillus species. Sufficient variability is apparent among all known microbial species to allow phylogenetic analysis based on large numbers of genetically unlinked loci. These striking differences among AFLP profiles allow unambiguous identification of previously identified species and phylogenetic placement of newly characterized isolates relative to known species based on a large number of independent genetic loci. Data generated thus far show that the method provides phylogenetic analyses that are consistent with other widely accepted phylogenetic methods. However, AFLP analysis provides a more detailed analysis of the targets and samples a much larger portion of the genome. Consequently, it provides an inexpensive, rapid means of characterizing microbial isolates to further differentiate among strains and closely related microbial species. Such information cannot be rapidly generated by other means. AFLP sample analysis quickly generates a very large amount of molecular information about microbial genomes. However, this information cannot be analysed rapidly using manual methods. We are developing a large archive of electronic AFLP signatures that is being used to identify isolates collected from medical, veterinary, forensic and environmental samples. We are also developing the computational packages necessary to rapidly and unambiguously analyse the AFLP profiles and conduct a phylogenetic comparison of these data relative to information already in our database. We will use this archive and the associated algorithms to determine the species identity of previously uncharacterized isolates and place them phylogenetically relative to other microbes based on their AFLP signatures. This study provides significant new information about microbes with environmental, veterinary and medical significance. This information can be used in further studies to understand the relationships among these species and the factors that distinguish them from one another. It should also allow the identification of unique factors that contribute to important microbial traits, including pathogenicity and virulence. We are also using AFLP data to identify, isolate and sequence DNA fragments that are unique to particular microbial species and strains. The fragment patterns and sequence information provide insights into the complexity and organization of bacterial genomes relative to one another. They also provide the information necessary for the development of species-specific polymerase chain reaction primers that can be used to interrogate complex samples for the presence of B. anthracis, other microbial pathogens or their remnants.     

抗体-适配子混合夹心法检测炭疽芽孢杆菌芽孢的研究

通过SELEX(SystemEvolutionofLigandsbyEXponentialenrichment)体外筛选技术寻获的能与炭疽芽孢杆菌芽孢特异结合的DNA适配子(aptamer)考察其作为检测分子的能力。利用抗体-适配子混合夹心法,根据显色反应强度,评价适配子检测炭疽芽孢杆菌芽孢的能力。结果表明,适配子可以作为检测分子检测靶物质的存在。适配子检测范围在2-16μg,芽孢的检测范围在4×104-4×107,当适配子16μg,芽孢量为4×107显色的指示强度最强。说明适配子作为检测用分子,具有潜在的实用价值。     

Detection of dairy Leuconostoc strains using the polymerase chain reaction

This paper reports the design of a Leuconostoc -specific oligonucleotide based on 16S rRNA sequence data. When this oligonucleotide was used in a polymerase chain reaction (PCR) in conjunction with an oligonucleotide to a conserved region of the 16S rRNA sequence, a Leuconostoc -specific PCR product of approximately 470 bp was produced. The use of a second oligonucleotide to a conserved region allowed the production of an approximately 350 bp product in all PCRs, acting as a positive control. The PCR procedure described was particularly useful for detecting the presence of Leuconostoc in mixed mesophilic starter cultures. The Leuconostoc -specific oligonucleotide was used also as a specific hybridization probe.     

Detection of Bacillus anthracis from spores and cells by loop-mediated isothermal amplification without sample preparation

Loop-mediated isothermal amplification (LAMP) is a technique capable of rapidly amplifying specific nucleic acid sequences without specialized thermal cycling equipment. In addition, several detection methods that include dye fluorescence, gel electrophoresis, turbidity and colorimetric change, can be used to measure or otherwise detect target amplification. To date, publications have described the requirement for some form of sample nucleic acid extraction (boiling, lysis, DNA purification, etc.) prior to initiating a LAMP reaction. We demonstrate here, the first LAMP positive results obtained from vegetative cells and spores of Bacillus anthracis without nucleic acid extraction. Our data show that the simple addition of cells or spores to the reaction mixture, followed by heating at 63°C is all that is required to reproducibly amplify and detect target plasmid and chromosomal DNA via colorimetric change. The use of three primer sets targeting both plasmids and the chromosome of B. anthracis allows for the rapid discrimination of non-pathogenic bacteria from pathogenic bacteria within 30 min of sampling. Our results indicate that direct testing of B. anthracis spores and cells via LAMP assay will greatly simplify and shorten the detection process by eliminating nucleic acid purification. These results may allow more rapid detection of DNA from pathogenic organisms present in field and environmental samples.     

Gamma radiation resistance of Bacillus anthracis spores

The aim of the presented study was determined the effectiveness of action the gamma radiation on water suspension B. anthracis spores. The irradiation was performed using a Cobalt 60 (Co 60) source, by using single and fractionary irradiation doses. In the investigations was used B. anthracis stain "Sterne" 34F2. The obtained results show, that gamma radiation effectively inactivates B. anthracis spores. On the efficiency of sterilization process influence the irradiation's method and the number of spores in 1 ml suspension. In the suspension 1.5 x 10(9) spore in 1 ml, sporicidal doses gamma radiation amount to 25.0 kGy (single dose) or 41.5 kGy (fractionary dose). The volume suspension about definite inoculum of spores, subjected working the gamma rays has not influence on sporicidal effectiveness of radiation sterilization.     

Detection of Listeria monocytogenes by using the polymerase chain reaction.

A method was developed for detection of Listeria monocytogenes by polymerase chain reaction amplification followed by agarose gel electrophoresis or dot blot analysis with a 32P-labeled internal probe. The technique identified 95 of 95 L. monocytogenes strains, 0 of 12 Listeria strains of other species, and 0 of 12 non-Listeria strains.     

Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction

Five oligonucleotide sequences are described that were used as primers in the polymerase chain reaction (PCR) to amplify specific sequences from Listeria DNA. When all five primers were used in combination, three PCR products were possible; a Listeria specific product that occurs with DNA from any Listeria sp., a Listeria monocytogenes specific product that occurs only in the presence of DNA from this organism and a universal product that is found using DNA from any bacterial source. The occurrence of these PCR products was used as a diagnostic test on bacteria isolated from various food samples to detect Listeria sp. and L. monocytogenes.     

Sequential detection of Salmonella typhimurium and Bacillus anthracis spores using magnetoelastic biosensors

Multiple phage-based magnetoelastic (ME) biosensors were simultaneously monitored for the detection of different biological pathogens that were sequentially introduced to the measurement system. The biosensors were formed by immobilizing phage and 1mg/ml BSA (blocking agent) onto the magnetoelastic resonator's surface. The detection system included a reference sensor as a control, an E2 phage-coated sensor specific to S. typhimurium, and a JRB7 phage-coated sensor specific to B. anthracis spores. The sensors were free standing during the test, being held in place by a magnetic field. Upon sequential exposure to single pathogenic solutions, only the biosensor coated with the corresponding specific phage responded. As the cells/spores were captured by the specific phage-coated sensor, the mass of the sensor increased, resulting in a decrease in the sensor's resonance frequency. Additionally, non-specific binding was effectively eliminated by BSA blocking and was verified by the reference sensor, which showed no frequency shift. Scanning electron microscopy was used to visually verify the interaction of each biosensor with its target analyte. The results demonstrate that multiple magnetoelastic sensors may be simultaneously monitored to detect specifically targeted pathogenic species with good selectivity. This research is the first stage of an ongoing effort to simultaneously detect the presence of multiple pathogens in a complex analyte.     

UV resistance of Bacillus anthracis spores revisited: validation of Bacillus subtilis spores as UV surrogates for spores of B. anthracis Sterne

Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.     

Survival of Bacillus anthracis spores in baths using modern technologic tannery processes

The influence of skin tannery baths, according as wet-blue, wet-white and plant technology, on B. anthracis spores survival was investigated. As a result of this study there was explained that lime bath do not inactivated of all spores that are present in infected bath. As a result of these experiments the spores were inactivated completely not before pickle bath.     

Germination of Bacillus anthracis spores within alveolar macrophages

The fatal character of the infection caused by inhalation of Bacillus anthracis spores results from a complex pathogenic cycle involving the synthesis of toxins by the bacterium. We have shown using immunofluorescent staining, confocal scanning laser microscopy and image cytometry analysis that the alveolar macrophage was the primary site of B. anthracis germination in a murine inhalation infection model. Bacillus anthracis germinated inside murine macrophage-like RAW264.7 cells and murine alveolar macrophages. Germination occurred in vesicles derived from the phagosomal compartment. We have also demonstrated that the toxin genes and their trans -activator, AtxA, were expressed within the macrophages after germination.