Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real-time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S-layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex-PCR. Among them, heat treatment of samples suspended with sucrose plus non-ionic detergent was considered an effective spore disruption method because it detected up to 10 5 spores/g soil by multiplex-PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/l and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real-time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between CT value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 10 4 spores/g soil within 3 hr by real-time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real-time PCR, avoiding time and labor-consuming preparation steps such as enrichment culturing and DNA preparation.

The TaqMan real‐time PCR method for the quantitative detection of C. botulinum type A was developed based on sequence‐specific hybridization probes. The validity of this assay was verified by using 10 genera of 20 strains, including reference strains of C. botulinum types A, B, C, D, E and F. The detection limit of this assay was evaluated on C. botulinum type A, using a 10‐fold dilution series of DNA and spores. The DNA and spores were detected up to level of 0.1 ng/ml and 102 spores/ml, respectively. Spore spiked food sample preparation prior to the real‐time PCR was performed by two methods, heat treatment and GuSCN. The detection limits after heat treatment showed 102 spores/ml for spiked sausage slurry, and 103 spores/ml for spiked canned corn slurry, while detection limits after GuSCN precipitation showed 102 spores/ml in both sausage and canned corn. Therefore the real‐time PCR assay after GuSCN precipitation is useful for the quantification of C. botulinum type A because it showed identical CT values in both pure spore solutions and food slurries. We suggest that quantitative analysis of C. botulinum type A by TaqMan real‐time PCR can be a rapid and accurate assessment method for botulinal risk in food samples.

We analyzed the genetic relationships and molecular characteristics of 34 Bacillus anthracis isolates from soil and clinical samples in various regions of Korea and 17 related Bacillus species, using the amplified fragment length polymorphism (AFLP) and multilocus variable-number tandem repeat (MLVA) approaches. Triplicate AFLP profiles of these strains showed high reproducibility and identified 376 polymorphisms. AFLP phylogenetic analysis of B. anthracis isolates showed a high level of similarity, 0.93, and this monomorphic fragment profile proved to be useful to differentiate B. anthracis strains from other Bacillus species. The B. cereus group was separated from other Bacillus species at a level of similarity of 0.68. Among them, some B. cereus strains showed genetic interspersion with B. thuringiensis strains. The evolutionary pattern of nucleotide differences among B. anthracis strains with the eight MLVA markers showed nine MLVA types. Three MLVA types, M1 to M3, were pathogenic B. anthracis isolates and were assigned as new genotypes belonging to the A4 and B3 clusters, compared with 89 genotypes deduced from previous data. This indicates that differences in cluster prevalence and distribution may be influenced more by MLVA markers on two plasmids loci and human activity. Consequently, we suggest that the novel MLVA type may represent significant evidence for historic adaptation to environmental conditions of the Asian continent, particularly Korea. Therefore, MLVA techniques may be available for molecular monitoring on anthrax-release-related bioterrorism and further study is required for the continuous epidemiological study of variable anthrax collections.Bacillus anthracis is a causative agent of anthrax, a serious and often fatal infection of livestock and humans. Humans may contract the disease via three routes: cutaneous inoculation via a cut or abrasion, ingestion of contaminated meat, or inhalation of spores (2,3,7,39). The spores of this organism can remain stable for decades and can be used as a powerful bioterrorism agent (17,20). Recent bioterrorism-associated anthrax outbreaks demonstrated the need for the rapid molecular typing of B. anthracis isolates (11,14,17,18,28).B. anthracis is a member of the B. cereus group, which includes B. cereus and B. thuringiensis. This is one of the most taxonomically ambiguous groups of bacilli (16, 32). These species are indistinguishable by using DNA-DNA hybridization, 16S-23S rDNA sequences, and gyrB-gyrA intergenic spacer regions (1,5,12,25,38). It has been hypothesized that the members of this group evolved from a common ancestor (15,40).Molecular typing of pathogens has long been a part of pathogen identification and control. Pulsed-field gel electrophoresis data sets are not easily standardized for transfer throughout the public health community. Ribotyping is generally applicable to all bacteria but is limited by the number of ribosomal loci in the genome. These methods often may not distinguish between closely related species or may misidentify spe...

Conjugal transfer of plasmid pAM beta 1 from Lactococcus lactis to intestinal bacteria of BALB/c mice was studied. Plasmid transfer was observed to Enterococcus faecalis in vitro by a filter mating method with transfer frequencies of 2.3 x 10-3 and with lower frequencies to other species. In vivo, using gastric intubation with the pAM beta 1-bearing Lactococcus lactis as donor and Ent. faecalis as recipient, a few transconjugants were detected from faecal Ent. faecalis. However, when these mice were given erythromycin through drinking water, a large number of conjugated Ent. faecalis were detected in faeces. Plasmid transfer to Ent. faecalis occurred at high frequency, 1.2 x 10-3, in mice whose anus was artificially closed after gastric intubation with pAM beta 1-bearing Lactococcus lactis. These results demonstrate clearly that pAM beta 1 transfer occurs between Gram-positive bacteria in the gut of mice harbouring many species of bacteria.

Biomarkers enable early diagnosis, guide molecularly targeted therapy and monitor the activity and therapeutic responses across a variety of diseases. Despite intensified interest and research, however, the overall rate of development of novel biomarkers has been falling. Moreover, no solution is yet available that efficiently retrieves and processes biomarker information pertaining to infectious diseases. Infectious Disease Biomarker Database (IDBD) is one of the first efforts to build an easily accessible and comprehensive literature-derived database covering known infectious disease biomarkers. IDBD is a community annotation database, utilizing collaborative Web 2.0 features, providing a convenient user interface to input and revise data online. It allows users to link infectious diseases or pathogens to protein, gene or carbohydrate biomarkers through the use of search tools. It supports various types of data searches and application tools to analyze sequence and structure features of potential and validated biomarkers. Currently, IDBD integrates 611 biomarkers for 66 infectious diseases and 70 pathogens. It is publicly accessible at http://biomarker.cdc.go.kr and http://biomarker.korea.ac.kr.