Use of DNA diagnosis and molecular typing technique on the detection and discrimination of Bacillus cereus and Bacillus thuringiensis

Autor: Ming-Lun Chen, 陳名倫
Rok vydání: 2002
Druh dokumentu: 學位論文 ; thesis
Popis: 90
Bacillus cereus is one of the major foodborne pathogens. It is very closely related to B. anthracis, B. mycoides and B. thuringiensis. These species share many phenotypic properties and high levels of chromosomal sequence similarity. Hemolysin BL (HBL) is a major virulence factor for Bacillus cereus group strains. It is also a target enterotoxin for the most commonly used B. cereus detection kit, i.e., the BCET-RPLA kit. A survey of the hemolysin BL (HBL) activities and the cytotoxicities to the Chinese hamster ovary (CHO) cells for the B. cereus group strains, however, showed that although only part of the B. cereus group strains are HBL active, all strains show the cytotoxicity to the CHO cells. Thus, methods that allow the detection of not only the HBL, but also all of the B. cereus group strains are important. In this study, by comparison of the gene sequences of the 16S rRNA for B. cereus group and other bacteria strains, we designed primers B16S1 and B16S2 specific to all the B. cereus group strains. In addition, since HBL is a major enterotoxin, we also designed HBL gene specific PCR primers, i.e., Hm1 and Hm2, which generated the same results as those of the hemolysis and BCET-RPLA assays. Primers B16S1/B16S2 and Hm1/Hm2 could be combined into a multiplex PCR system for the simultaneous detection of B. cereus group cells and the possible presence of their HBL enterotoxins. Also, all these PCR systems allowed the detection of N x 100 CFU B. cereus cells per g of food sample if an 8 h enrichment step was performed prior to the PCR. B. thuringiensis is a widely used biopesticide. It can be distinguished from B. cereus by its ability to produce an insecticidal crystal inclusion inside the cell during sporulation. According to the differences in variable region V1 of 16S rRNA or gyrB gene between Bacillus cereus and B. thuringiensis, Giffel and Yamada et al. have suggested the use of DNA method to differentiate B. cereus from B. thuringiensis strains. However, other researchers have suggested that these two Bacillus species are indistinguished by molecular biological methods and should belong to the same species. In this study, we re-evaluated such DNA methods and tried to explain the results with DNA sequencing data. The results of PCR showed that six of eighty-two B. cereus strains were identified as B. thuringiensis and sixty-seven of seventy-three B. thuringiensis strains were identified as B. cereus. Sequence analysis of the primer annealing site showed that there is no clear-cut difference in the 16S rRNA gene or gyrB gene between B. cereus and B. thuringiensis strains. Therefore, we believe that of the methods to discriminate B. cereus and B. thuringiensis, parasporal crystal observation and cry gene detection may be the reliable methods. The comparison of the antibiograms, enterotoxin profiles, randomly amplified polymorphic DNA (RAPD), and pulsed-field gel electrophoresis (PFGE) patterns were also used to discriminate the strains of B. cereus and B. thuringiensis. 82 B. cereus and 73 B. thuringiensis strains were subtyped by antibiotic susceptibility testing, enterotoxin profile, RAPD and PFGE and results showed that for all those strains tested, 27 antibiogram and 8 enterotoxin profiles were found. Digestion with NotI generated 121 PFGE patterns. RAPD with primer 3F and 6F yielded 112 and 120 RAPD patterns, respectively. Thus, it is difficult to classify B. cereus ad B. thuringiensis strains by their RAPD or PFGE patterns. No specific RAPD or PFGE marker band could be found for the discrimination of these two Bacillus species. Due to the high frequency of genome rearrangements for these B. cereus ad B. thuringiensis strain, these strains display a higher level of heterogeneity within the chromosomal gene organization. This may also cause the diversified RAPD and PFGE patterns for B. cereus and B. thuringiensis strains.
Databáze: Networked Digital Library of Theses & Dissertations
Externí odkaz: