RESUMO
Pulsed-field gel electrophoresis (PFGE) is considered the "gold standard" for molecular epidemiological study. Repetitive extragenic palindromic PCR (rep-PCR) is less time-consuming and more suitable for analyzing large numbers of bacterial strains in human populations. PFGE and rep-PCR provide comparable genotyping results for investigating Streptococcus mutans diversity and transmission.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Impressões Digitais de DNA/métodos , Reação em Cadeia da Polimerase/métodos , Streptococcus mutans/classificação , Streptococcus mutans/genética , Criança , Pré-Escolar , Análise por Conglomerados , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Humanos , Lactente , Masculino , Epidemiologia Molecular/métodos , Polimorfismo Genético , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/transmissão , Streptococcus mutans/isolamento & purificaçãoRESUMO
Cabbage and collard greens were inflicted with a previously undescribed virus-like disease during the fall 2000. Symptoms on leaves were yellow spots, vein clearing, mosaic, curling, and puckering. Symptomatic plants were widespread in Brooks, Colquitt, Grady, and Pierce counties in Georgia. Disease incidence ranged from 10 to 20% in the majority of the fields surveyed but some fields had 100% incidence. Fields were heavily infested by Bemisia argentifolii and the symptoms were suggestive of a whitefly-transmitted geminivirus infection. A polymerase chain reaction (PCR)-based diagnostic test for geminivirus was conducted. Total DNA was extracted from symptomatic cabbage and collard green plants collected from commercial fields. The two primers, 5'-GCCCACATYGTCTTYCCNGT-3' and 5'- GGCTTYCTRTACATRGG-3' (2,3), are "universal" for genus Begomovirus of family Geminiviridae. The primer pair could amplify a part of the replicase-associated protein and coat protein and the complete common region of DNA-A. The PCR gave a DNA band of expected size (1.1 kb) from both symptomatic cabbage and collard green samples, whereas no such product was obtained from healthy samples, suggesting that the causal agent could be a geminivirus. To establish the identity of the virus, the 1.1 kb PCR product was cloned into pGEM-T Easy (Promega) and sequenced. GenBank search showed that the geminivirus isolated in Georgia was most closely related (98% sequence identity) to Cabbage leaf curl virus (accession number U65529) reported from Florida (1). The virus was mechanically transmitted to healthy cabbage and collard green plants under experimental conditions. To our knowledge, this is the first report of Cabbage leaf curl virus from Georgia. References: (1) A. M. Abouzid et al. Phytopathology 82:1070, 1992. (2) S. S. Pappu et al. Plant Dis. 84:370, 2000. (3) M. R. Rojas et al. Plant Dis. 77:340-347, 1993.
RESUMO
The genetic diversity of Streptococcus mutans has been extensively studied using a variety of genotyping methods. Repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) is a genotyping approach used for screening large numbers of bacterial isolates. This two-part study used multilocus sequence typing (MLST) analysis to evaluate genotypes previously identified as unique using rep-PCR. In part one, an isolate was selected from each of the 22 S. mutans rep-PCR genotype groups representing 8000 clinical isolates. For part two, four additional isolates were selected from the six most commonly occurring genotype groups (GG) for further analysis. Real-time PCR was performed using eight housekeeping S. mutans gene loci and the amplicons were sequenced. Sequence data analysis was performed using CLC DNA Workbench and alleles were compared with the PubMLST database for Oral Streptococcus using the Nakano scheme. Concatenated sequences were evaluated with MEGA using a minimum evolution method with bootstrap. All 22 rep-PCR genotypes were unique by MLST analysis. Within rep-PCR GGs, MLST matched rep-PCR in three groups demonstrating clonality; three groups exhibited more diversity with MLST. The discovery of three clonal groups is unique to this study and suggests that S. mutans genotypes are shared between unrelated subjects. Furthermore, MLST defined 19 new alleles and 26 new sequence types that have been confirmed and registered with PubMLST. Methods for processing were streamlined and a process for using MLST with rep-PCR is suggested. In conclusion, MLST verified that rep-PCR is a reliable and cost-effective method for screening large numbers of S. mutans strains for epidemiological study.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Sequências Repetidas Invertidas/genética , Tipagem de Sequências Multilocus/métodos , Streptococcus mutans/genética , Oxirredutases do Álcool/genética , Alelos , Isomerases de Aminoácido/genética , Proteínas de Bactérias/genética , Criança , Pré-Escolar , Mapeamento Cromossômico , Células Clonais , DNA Girase/genética , DNA Concatenado/genética , Variação Genética/genética , Genótipo , Glutamato Sintase/genética , Glutamato-Amônia Ligase/genética , Guanina , Humanos , Proteínas de Membrana/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Análise de Sequência de DNA/métodos , Serina Endopeptidases/genética , Streptococcus mutans/classificação , Transcetolase/genéticaRESUMO
Mutans streptococci (MS) are key organisms associated with the etiology of dental caries. Using probabilities that were tested by oversampling, we designed this study to determine the minimal number of MS isolates from an individual required to evaluate diversity of genotypes. MS isolates were genotyped by repetitive extragenic palindromic-polymerase chain-reaction (rep-PCR). Analysis of 20 isolates from individuals resulted in a mean of 1.6 and 2.4 genotypes in children (N = 12) and adults (N = 10), respectively. In a follow-up study, reducing the number of isolates to 7-10 resulted in a theoretical probability of up to 78% for detecting up to 4 genotypes. A mean of 1.5 genotypes was found in 35 children and 10 adults. These findings provide evidence for the design of studies of MS genotyping that can serve as a model for the analysis of genotypes within individuals.