Species-identification dots: a potent tool for developing genome microbiology

Naimuddin, M.; Kurazono, Takayuki; Zhang, Yinghua; Watanabe, Takeshi; Yamaguchi, Masanori; Nishigaki, Koichi

doi:10.1016/s0378-1119(00)00502-3

Cited by 31 publications

(62 citation statements)

References 14 publications

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“…For successful strain differentiation by -TGGE analysis, the amplified region of the genome must be selected so that different strains have distinct GϩC contents, resulting in differences in the T m and the initial DNA melting point (P ini ) (Fig. 1D) (27). The genomic sequences of L. monocytogenes strains EGD-e (serovar 1/2a), F6854 (serovar 1/2a), F2365 (serovar 4b), and H7858 (serovar 4b) have been publicly released (10,28).…”

Section: Resultsmentioning

confidence: 99%

“…P ini , the position for DNA melting initiation; P min , the position for strand dissociation (27,30). Each experiment was repeated two times, and representative results are shown.…”

Section: Fig 1 -Tgge Analysis Of Mixed Pcr Products Of L Monocytogmentioning

confidence: 99%

“…These methods are based on variances in the electrophoretic mobilities of the partially melted DNA molecules (24,25,27,30). When a DNA fragment reaches its melting temperature (T m ) in TGGE or its melting point in a gel containing a linear gradient of DNA denaturants in DGGE, the fragment rapidly melts and its migration in the gel is slowed.…”

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confidence: 99%

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Rapid Discrimination of Listeria monocytogenes Strains by Microtemperature Gradient Gel Electrophoresis

Tominaga

2006

J Clin Microbiol

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Microtemperature gradient gel electrophoresis (-TGGE) was examined for use for the rapid subtyping of Listeria monocytogenes strains. Comparison of genomes between L. monocytogenes strains F2365 and H7858 identified a sequence encoding a portion of the PRT/PTS system IIA 2 protein domain as appropriate for -TGGE analysis. Thirty-one strains belonging to 10 different serovar types were tested by PCR, and sequence analysis of the amplified products revealed that the strains comprise 11 groups. All 55 possible pairs within the 11 groups were examined by -TGGE analysis. Of these, 47 pairs could be successfully discriminated, with a total electrophoresis time of only 7 min. Moreover, Cy3/Cy5 labeling allowed rapid identification of the sequence type in unknown strains of L. monocytogenes isolated from meat. These findings collectively indicate that -TGGE can be used for the rapid analysis of L. monocytogenes strains, facilitating determination of routes of contamination when these bacteria are found in food products.Listeria monocytogenes is considered one of the most dangerous food-borne pathogens because it causes approximately 2,500 cases of listeriosis per year in the United States, with a mortality rate of ϳ20% (22). Listeriosis outbreaks have been associated with L. monocytogenes contamination of foods such as coleslaw, cheese, and milk (8). Therefore, it is critical for food producers and administrators to be able to quickly trace the route of contamination when L. monocytogenes is detected in the final food product (42). Such field tests, however, can be complicated by the existence of L. monocytogenes in the natural environment.L. monocytogenes strains comprise 13 serovars grouped into three lineages (lineages I, II, and III) (14,31,43). Among these, serovars 1/2a, 1/2b, and, especially, 4b are implicated in many cases of human listeriosis (38). Various molecular typing methods beyond serovar typing have been investigated, including ribotyping (26), pulsed-field gel electrophoresis (PFGE) (34, 44), randomly amplified polymorphic DNA (RAPD) analysis (21), PCR-electrophoresis-based methods (15, 36), microarrays (4), esterase typing (9), amplified fragment length polymorphism analysis (1, 17), and multilocus enzyme electrophoresis (11). Among these, ribotyping and PFGE have better discriminatory powers and have been widely used (16). Recently, sequence typing (ST) and its extension, multilocus sequence typing (MLST), have been applied for analysis of L. monocytogenes (5,23,29,32,33,45). These methods have the benefit of delivering relatively standardized data, allowing easy comparisons between laboratories or against databases. In addition, MLST has been shown to be superior to ribotyping and PFGE in terms of discrimination power (32, 45). Unfortunately, ST and MLST are relatively laborious and time-consuming for analyses of large numbers of samples, limiting their usefulness in larger contexts.Temperature gradient gel electrophoresis (TGGE) and denaturing gradient gel electrophoresis (DGGE) are methods for the...

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Section: Resultsmentioning

confidence: 99%

“…P ini , the position for DNA melting initiation; P min , the position for strand dissociation (27,30). Each experiment was repeated two times, and representative results are shown.…”

Section: Fig 1 -Tgge Analysis Of Mixed Pcr Products Of L Monocytogmentioning

confidence: 99%

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Rapid Discrimination of Listeria monocytogenes Strains by Microtemperature Gradient Gel Electrophoresis

Tominaga

2006

J Clin Microbiol

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“…GP estimates the similarity of biological samples by comparing species identification dots (spiddos) extracted from profile images as the pattern similarity score (PaSS) Naimuddin et al 2000). In GP, fragments that are easily amplified in the genomic population are amplified and compared using an identical primer set, and the profile images of biological species with different genomic constitutions are different (Nishigaki et al 1991.…”

Section: Methodsmentioning

confidence: 99%

Genome profiling implies high genetic diversity in microsporidia isolated from the common cutworm, Spodoptera litura (Lepidoptera: Noctuidae), in Vietnam

et al. 2011

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In order to evaluate the potential application of microsporidia as a microbial control agent against lepidopteran insect pests, microsporidian infection in a field population of the common cutworm, Spodoptera litura (Fabricius), was surveyed in vegetable crop fields in Can Tho City, Vietnam, in March 2007. The infection rate of microsporidia was 46.7% (99/212 individuals) in adult S. litura, and 16 samples of infected adults were used to characterize the microsporidia at the molecular level. Analysis of the small subunit ribosomal RNA (SSU rRNA) sequences indicated that microsporidian strains isolated from S. litura were closely related to Nosema bombycis from the silkworm, Bombyx mori (Linnaeus); however, phylogenetic analysis based on genome profiling produced a different result from the SSU rRNA sequences. Temperature gradient gel electrophoresis profiles of 12 microsporidian strains from S. litura were closely related to N. bombycis strains, while the profiles of three microsporidian strains formed a different cluster. The Vietnamese strains did not form a single group, but were classified into at least three groups. These results suggested that the microsporidia isolated from S. litura in the Mekong Delta, Vietnam, are genetically diverse.

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“…It involves the analysis of random PCR products from biological samples by temperature gradient gel electrophoresis (TGGE) (Nishigaki et al 1991;Hamano et al 1996). The levels of similarity between samples are estimated by comparing species identification dots (Spiddos) extracted from the TGGE images, using pattern similarity scores (PaSS) Naimuddin et al 2000).…”

Section: Introductionmentioning

confidence: 99%

Phylogenetic relationships among Bacillus thuringiensis (Bacillaceae: Bacillales) strains based on a comparison of SSU rRNA sequences and genome profiling

2011

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Bacillus thuringiensis Berliner has previously been classified via the serological identification of flagellar antigens. However, the phylogenetic relationships among strains of B. thuringiensis cannot be investigated by serotyping. Furthermore, high levels of homology have been found in gene sequences among various strains, complicating the determination of their evolutionary relationships. In order to elucidate the phylogenetic relationships within B. thuringiensis, we analyzed 40 strains belonging to typical serotypes using two approaches: an analysis of small subunit (SSU) rRNA sequences and genome profiling (GP) based on temperature gradient gel electrophoresis of random PCR products. The SSU rRNA analysis resulted in all 40 strains forming a single cluster with Bacillus cereus Frankland & Frankland. The distances among the subclusters were too small to further classify the strains. On the other hand, the phylogenetic analysis based on GP resulted in three clusters of B. thuringiensis strains. These results suggest that GP is a better method for the determination of phylogenetic relationships within B. thuringiensis.

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Species-identification dots: a potent tool for developing genome microbiology

Cited by 31 publications

References 14 publications

Rapid Discrimination of Listeria monocytogenes Strains by Microtemperature Gradient Gel Electrophoresis

Rapid Discrimination of Listeria monocytogenes Strains by Microtemperature Gradient Gel Electrophoresis

Genome profiling implies high genetic diversity in microsporidia isolated from the common cutworm, Spodoptera litura (Lepidoptera: Noctuidae), in Vietnam

Phylogenetic relationships among Bacillus thuringiensis (Bacillaceae: Bacillales) strains based on a comparison of SSU rRNA sequences and genome profiling

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