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Diarrhea is a world epidemic diseases, health conditions and backward areas have a higher prevalence, and has obvious seasonal. In China, the incidence of infectious diarrhea ranks first of all infectious diseases. Our monitoring results indicate, the the diarrhea bacteria Escherichia coli cause diarrhea proportion upward trend. Separation of the application of conventional microbiological induced diarrhea pathogen detection clinical and biochemical identification, time-consuming, and the lack of reagents, different standards, it is difficult to adapt to the rapid detection and identification of need. Although molecular biology techniques have been applied to the detection of the infectious pathogen, but complicated to operate, the reagent is incomplete and unstable, limiting molecular biology techniques in clinical practice. Escherichia coli cause diarrhea, not yet mature, both molecular biology methods to identify different types of strains. This study aims to establish the the five diarrhea caused by Escherichia coli, a simultaneous detection of multiple PCR, to explore factors affect multiplex PCR; explore analog stool specimen pretreatment method, will establish a multiplex PCR method is applied to detect clinically isolates as well as analog stool specimens, evaluation of the multiplex PCR method, the multiplex PCR directly from clinical specimens to detect and identify the corresponding cause diarrhea Escherichia coli. In this study, DNAStar software, designed for the five diarrhea caused by Escherichia coli EAEC, EHEC, EIEC,, EPEC, ETEC virulence gene and verify that the appropriate primers to detect five kinds of diarrhea caused by Escherichia coli single PCR method. The lowest singlet PCR capable of rapid, sensitive, specific, identified five diarrhea caused by Escherichia coli, each reaction can be detected by varying the amount of bacteria, the sensitivity from 51 CFU / reaction to 1.31 × 10 ~ 5 CFU / reaction ranging. On the basis of the single-plex PCR method, by adjusting the main factors affecting the multiplex PCR system exploration and optimization of the reaction conditions, so step PCR simultaneously identified five Escherichia coli, better amplification results established multiplex PCR method can be simultaneously detected. Its sensitivity is slightly lower than the corresponding single-plex PCR. Through the different strains were amplified, to verify the specificity of this method, compared with the single-plex PCR and identified 16 diarrhea caused by E. coli isolates, 15 of the detection results coincide with each other, only one EIEC strains multiplet PCR amplification was negative 93.8%, in line with the single-plex PCR. Simulated fecal specimens to the EAEC standard strain representative strains were prepared containing a single strain of the EAEC, select different processing methods, pre-treatment of the simulated fecal specimens to obtain bacterial DNA. Singlet PCR extracting corresponding analog stool specimens template for amplification, to evaluate and determine the analog pre-processing method in the stool specimens. Seven simulated fecal specimens pre-treatment methods, the final screening method of processing analog stool specimens suitable for PCR amplification. Which set of bacteria after boiling purification method, to extract genomic collecting bacteria, directly after boiling purification method, kit prepared by the template can be used for single-plex PCR amplification, the first two methods in simulated specimens after centrifugation, precipitation the boundaries of the supernatant enough easy to form the stool suspension, experimental results are closely related to the operation of the laboratory personnel; kit extracted according to the operation manual, good repeatability, but it takes a long time, complicated operation, higher costs ; directly after boiling purification method is simple, rapid, and reproducible. The comprehensive analysis of the singlet PCR amplified directly after boiling purification method is the preferred method of specimen processing. The the preparation laboratory simulation specimens containing five kinds of diarrhea caused by E. coli, total nucleic acid extracted from simulated samples were used directly after boiling method of purification method and kit, multiplex PCR amplification results show that, kit method is superior to direct after boiling method of purification of nucleic acids, the preferred method for multiplex PCR processing analog stool specimens. Equivalent to the amount of simulation specimens were inoculated in LB medium enrichment 4 ~ 6h later boiled the legal system to take the template, the template can be detected in each reaction vaccination 2CFU. This study to establish a stable, sensitive, specific and rapid multiplex PCR reaction method, the method can simultaneously detect five kinds of diarrhea caused by Escherichia coli, than conventional bacteriological diagnosis fast, simple and practical. Application of the multiplex PCR method to detect the analog stool specimens directly typing identified specimens of diarrhea caused by Escherichia coli.
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