| Pseudomonas aeruginosa is an opportunistic Gram-negative pathogens. P. aeruginosa is easy to prevail in immunocompromised individuals, so many hospital and community acquired infection cases were caused by P. aeruginosa infection. Now P. aeruginosa is one of the most serious problems widely. Since study showed that 10 to 100 cells of P. aeruginosa can lead to gut colonization in patients, early detection p. aeruginosa is crucial. Nanotechnology is one of the most important technologies in the past decades. The magnetic nanoparticles (MNPs) have been widely applied in the detection of various biological signals because of that they can be easily collected under the external magnetic field and, furthermore, they have the characteristics of high surface area. This study intends to develop highly sensitive detection and genotyping method using magnetic nanoparticles which is easy to realize automation. Concrete research contents were as follows.1. Preparation and surface modification of high dispersion MNPsBecause of the wide applications of MNPs in biotechnology, biomedical, material science, engineering, and environmental areas, much attention has been paid to the synthesis of different kinds of MNPs. In this paper, MNPs were prepared by solvothermal method with PEG-4000 as a surfactant, and then were modified. The result showed that the prepared MNPs under optimum conditions present a uniform morphology and good dispersion, and the size is from 350 to 450 nm. The precipitation time of MNPs is greatly increased in comparison with that of commercially available MNPs, and keeps good dispersion after 12 hours. In order to obtain more stable functional ized particles, the MNPs were modified. The obtained functionalized particles were characterized with scanning electron microscope (SEM), transmission electronic microscopy (TEM) and fourier transform infrared (FTIR), and the images showed that they have good dispersion and uniform morphology. The elemental analysis indicated 0.33% of the content of the surface of MNPs@Probe are P element, also showing that the modification is successful.2. Development of method for highly sensitive detection of P. aeruginosa based on magnetic enrichment PCR and magnetic enrichment nested PCRPseudomonas aeruginosa is a common opportunistic pathogen in clinics and one of major threats to human health. In recent years, genomic DNA separation and detection based on MNPs has been rapidly developed. In this study, the method of magnetic enrichment PCR was built using p. aeruginosa specificity gene gyrB as target. Compared with traditional PCR, the magnetic enrichment PCR has higher utilization rate of the template, so its amplification efficiency is higher and the limit of detection (LOD) is 100 cfu/mL. The species-specific ecfX gene of Pseudomonas aeruginosa has high specificity. In this study, we also have combined magnetic enrichment and nested PCR technology to develop a high specific and sensitive approach for P. aeruginosa detection, and the specific ecfX gene of Pseudomonas aeruginosa was used as the detection object. This method displays high sensitivity and specificity, the LOD is 10 cfu/mL. The method provides a reliable, timely and accurate platform for early detection of Pseudomonas aeruginosa. Furthermore, the method can shorten the procedure and time from DNA extraction to detection, making the assay procedure more convenient.3. Highly sensitive and rapid detection of P. aeruginosa based on magnetic enrichment, magnetic separation and chemiluminiscenceAlthough magnetic enrichment PCR method can realize highly sensitive detection of P. aeruginosa, it still difficult to find wide clinical application owing to it is difficult to achieve automation and quantitative detection. A method for highly sensitive and rapid detection of P. aeruginosa, based on magnetic enrichment, magnetic separation and chemiluminiscence, is described in this paper. The factors which could influence the chemiluminiscence were studied in detail, and the P. aeruginosa samples were successfully and quantitatively detected. Three of commercially available P. aeruginosa standard strains (ATCC27853, ATCC9027, CMCC10104) were detected by this method, and the results showed that the chemiluminescent intensity of the samples has significant difference in comparison with those of the controls and the Q values were greater than 2.1. The LOD of this method was 10 cfu/mL or 30 fM PCR products, while the detection of a single Pseudomonas aeruginosa can also be achieved. Experiment results proved that the primer and probe had high specificity to P. aeruginosa compared with Escherichia coli, Salmonella enteritidis, and Shigella dysenteriae.4. Genotyping of P. aeruginosa type III secretion system using magnetic enrichment Multi-PCR and chemiluminescenceThe pathology characteristic and the toxicity mechanism of P. aeruginosa are different for those who contain different Type III secretion system (T3SS) gene, so the genotyping of p. aeruginosa T3SS is significant for understanding its virulence and predict clinical results. A rapid method for T3SS genotyping was developed using enrichment Multi-PCR and chemiluminescence. Three of P. aeruginosa standard strains (ATCC27853, ATCC9027, CMCC10104) were analysed by this method, and the results showed the chemiluminescent intensity of gene exoT, exoY and exoS of these strains were 10 times greater than that of the control, and the Q value were greater than 2.1. These results were consistent with the results of electrophoresis, indicating the method was reliable.22 of clinical isolates were tested using the method. In these isolates sample,100% of isolates contained exoT, 72.7% contained exoY,95.5% contained exoS, and 4.5% contained exoU gene. All of isolates contained either the exoS or the exoU genes but not both. All genotyping results of isolates were consistent with PCR results.5. Single nucleotide polymorphisms classification method was developed using magnetic enrichment PCR and dual-color hybridizationAll of T3SS toxin gene of P. aeruginosa contains single nucleotide polymorphisms (SNP). The previous studies have shown that 95.5% of 22 clinical isolates contain exoS gene. Therefore, in this study, we developed a SNP classification method of exoS gene (G162A and G434C) using magnetic enrichment PCR, magnetic separate and dual-color fluorescence which will provide technical basis for understanding T3SS genotype of variation. Three of P. aeruginosa standard strains (ATCC27853, ATCC9027, CMCC10104) were analysed by this method for the two SNP loci, and the results indicated the two SNP loci of all these strains were wild subtype. All of G values were greater than 0.8 and I value were greater than 3, so the classification had statistical significance. In addition,21 of clinical isolates were analysed by this method for G162A and G434C SNP loci for monitoring clinical mutation. The results showed, in G162A SNP locus.57.1% of isolates were wild subtype, 23.8% were mutation subtype, and 9.5% were heterozygous subtype. However two isolates can not be decided since the G value was meanless. In G434C SNP locus,80.9% of isolates were wild subtype,14.3% were mutation subtype, and 4.8% were hybrid subtype. |
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