RESUMEN
Criteria for sub-typing of microbial organisms by DNA sequencing proposed by Olive and Bean were applied to several genes in Escherichia coli to identify targets for the development of microbial source tracking assays. Based on the aforementioned criteria, the icd (isocitrate dehydrogenase), and putP (proline permease) genes were excluded as potential targets due to their high rates of horizontal gene transfer; the rrs (16S rRNA) gene was excluded as a target due to the presence of multiple gene copies, with different sequences in a single genome. Based on the above criteria, the mdh (malate dehydrogenase) gene was selected as a target for development of a microbial source tracking assay. The mdh assay was optimized to analyze a 150 bp fragment corresponding to residues G191 to R240 (helices H10 and H11) of the Mdh catalytic domain. 295 fecal isolates (52 horse, 50 deer, 72 dog, 52 seagull and 69 human isolates) were sequenced and analyzed. Target DNA sequences for isolates from horse, dog plus deer, and seagull formed identifiable groupings. Sequences from human isolates, aside from a low level (ca. 15%) human specific sequence, did not group; nevertheless, other hosts could be distinguished from human. Positive and negative predictive values for two- and three-way host comparisons ranged from 60% to 90% depending on the focus host. False positive rates were below 10%. Multiple E. coli isolates from individual fecal samples exhibited high levels of sequence homogeneity, i.e. typically only one to two mdh sequences were observed per up to five E. coli isolates from a single fecal sample. Among all isolates sequenced from fecal samples from each host, sequence homogeneity decreased in the following order: horse>dog>deer>human and gull. For in-library isolates, blind analysis of fecal isolates (n=12) from four hosts known to contain host specific target sequences was 100% accurate and 100% reproducible for both DNA sequence and host identification. For blind analysis of non-library isolates, 18/19 isolates (94.7%) matched one or more library sequences for the corresponding host. Ten of eleven geographical outlier fecal isolates from Florida had mdh sequences that were identical to in-library sequences for the corresponding host from California. The mdh assay was successfully applied to environmental isolates from an underground telephone vault in California, with 4 of 5 isolates matching sequences in the mdh library. 146 sequences of the 645bp mdh fragment from five host sources were translated into protein sequence and aligned. Seven unique Mdh protein sequences, which contained eight polymorphic sites, were identified. Six of the polymorphic sites were in the NAD+ binding domain and two were in the catalytic domain. All of the polymorphic sites were located in surface exposed regions of the protein. None of the non-silent mutations of the Mdh protein were in the 150bp mdh target. The advantages and disadvantages of the assay compared to established source tracking methods are discussed.
Asunto(s)
Técnicas de Tipificación Bacteriana , Infecciones por Escherichia coli/microbiología , Escherichia coli/clasificación , Escherichia coli/genética , Malato Deshidrogenasa/genética , Epidemiología Molecular/métodos , Análisis de Secuencia de ADN , Animales , Secuencia de Bases , Dominio Catalítico/genética , Charadriiformes/microbiología , Ciervos/microbiología , Perros , Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Heces/microbiología , Genes Bacterianos , Caballos/microbiología , Humanos , Malato Deshidrogenasa/química , Datos de Secuencia Molecular , Mutación , Polimorfismo Genético , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Homología de Secuencia de Ácido NucleicoRESUMEN
The quantification of plasmid DNA by the PicoGreen dye binding assay has been automated, and the effect of quantification of user-submitted templates on DNA sequence quality in a core laboratory has been assessed. The protocol pipets, mixes and reads standards, blanks and up to 88 unknowns, generates a standard curve, and calculates template concentrations. For pUC19 replicates at five concentrations, coefficients of variance were 0.1, and percent errors were from 1% to 7% (n=198). Standard curves with pUC19 DNA were nonlinear over the 1 to 1733 ng/microL concentration range required to assay the majority (98.7%) of user-submitted templates. Over 35,000 templates have been quantified using the protocol. For 1350 user-submitted plasmids, 87% deviated by >or=20% from the requested concentration (500 ng/microL). Based on data from 418 sequencing reactions, quantification of user-submitted templates was shown to significantly improve DNA sequence quality. The protocol is applicable to all types of double-stranded DNA, is unaffected by primer (1 pmol/microL), and is user modifiable. The protocol takes 30 min, saves 1 h of technical time, and costs approximately $0.20 per unknown.
Asunto(s)
Análisis de Secuencia de ADN/métodos , Plásmidos , Moldes GenéticosRESUMEN
We assessed the quality of nine standard primers for automated fluorescent dye terminator DNA sequencing by whether their melting temperatures (Tms) were in the optimal range for DNA sequencing, and the degree to which their sequences matched the sequences of 36 common vectors. The M13F (-21/-20), M13F (-41/-40), M13R, and T7R primers showed optimal physicochemical characteristics and were not redesigned. The M13R (-41/-40), T3, and SP6 primers showed mismatches and/or Tm values outside of the optimal range and were redesigned by these two criteria. With few exceptions, the redesigned primers did not significantly improve DNA sequencing quality as assessed by Phred scores compared to the corresponding original primer. However, both redesigned T7 primers, which were also redesigned to function as pET primers, strikingly improved sequencing on pET vectors. The original and redesigned T7 primers also improved the sequence quality for the Bluescript family of vectors compared to the original and redesigned M13R (-41/-40) primers. The mismatch-redesigned T7, SP6, and M13R (-41/-40) primers marginally improved the sequence quality over the corresponding original primers for specific vector types. Within limits, decreasing the %GC and Tm below reported optimal levels did not affect the sequence quality. Commercially available standard vector primers were surveyed.