Establishment And Validation Of The Gene Chip Technique In Detection Of Vancomycin-resistant Enterococcus

Enterococcus is a kind of gram positive coccus. It is widely distributed in the natural environment and human and animal in the digestive tract. It has weak pathogenicity and can cause infection only in the conditions of breeding in the host organization parasitic, Bearing the body immune system and defense mechanism and causing pathological changes. But due to the heavy use of broad spectrum antibiotics especially vancomycin in clinical in recently years and unreasonable action in the application, vancomycin-resistant enterococcus(VRE) has appeared. This caused wide attention around the world. For the past few years a large number of studies have shown that the detection,identification and diagnosis of VRE has become an important issue of doctors and medical researchers because of its causing nosocomial infection and spread easier. The common method to detect VRE is bacteriology method. It is time consuming and can not accurately identify types of enterococcus strains. There are many differences in antibacterial drug resistance in enterococcus genera. Also the method can not identify genotype of VRE. Relative to the shortage of the medicine sensitive experiment, two common enterococcus were chosen. Then we aimed at application of gene chip detection technology can distinguish betweenVRE type and resistant genotypes. As in-depth development of gene chip detection VRE been more widely used in clinical basis lay the foundation.The 16 SrDNA genes were chosen simultaneously as targets for screening of specific probes and designing universal primers to detect and identify specific vancomycin resistant genes and enterococcus specific genes.According to two kinds of common enterococcus specific gene sequences(ddl) and vancomycin resistance gene(VanA, VanB) in GeneBank,oligonucleotide probe were designed and compounded which could detect and distinguish special genes and drug resistance genes of enterococcus by the program of BLAST and so on. Gene chip of detection of vancomycin resistant enterococcus was prepared. The target DNA fragments of vancomycin-resistant enterococcus were labeled with biotin by multiple PCR amplification, and then hybridized with oligonucleotied probes on slide. The results were analyzed by portable imager after cleaning and chemiluminescence method. The specificity, sensitivity and reproducibility of the chip were evaluated. Then it was applied in the clinical test samples under the conditions of optimized multiple PCR system, hybridization reaction and chemiluminescence method. The genes was finally determined were vancomycin resistance gene(VanA,VanB) and enterococcus specific genes(ddl). The 16 SrDNA genes were reference genes. We selected a couple of universal primers, four couples of specific primers, a general probe and four specific probes of bacteria detection. The multiple PCR system and cutoff of probes were determined. The sensitivity could achieve 103cfu/ml. Coefficient of variation is under 15%. 40 cases of clinical samples were tested. The results of test were consistent with the regular training results. At last a gene chip technique has been set up for the detection of vancomycin-resistant enterococcus. It is possible to detect and distinguish type and phenotype of vancomycin-resistant enterococcus by gene chip technique rapidly, sensitively and specifically. This invention provided anewer method in the clinical for detection of VRE, Individualized drug treatment, monitoring clinical outcomes and nosocomial infection. It is true and reliable, simple and low cost. This method was of great significance for reducing waste of resources and avoiding the more risks of antibiotic.