The Construction And Clinical Application Of DNA Microarray For Gram-negative Bacteria Drug Resistance Detection

Background:Mixed infection has become a common phenomenon in clinical drug resistance, increased the difficulty of clinical and laboratory diagnosis, drug susceptibility testing and PCR and molecular hybridization technology to detect bacterial resistance genes have some limitations, so there is a need to establish a multi-detection methods, In pathogen detection, DNA microarray has unique advantages, which in high-throughput parallel testing approach can make a variety of drug-resistant strains in the resistance gene was detected at the same time, with fast, efficient, sensitive features, especially for large-scale samples. In this study, gene chip technology, to use PCR and DNA markers to construct genetic testing chip resistant Gram-negative bacteriaObjective:According to Gram-negative bacteria resistant gene cassette in the gene sequence, designed and synthesized 63 kinds of resistance genes for specific probes, a gram-negative bacteria developed resistance gene cassette test chip, Lattice on the same chip can be rapid and accurate identification of gram-negative bacteria resistant gene cassette of the 63 subtypes. The establishment of Gram-negative bacteria resistant gene microarray for clinical detection of bacterial resistance, for the bulk sample testing and large-scale epidemiological survey to provide fast, parallel, sensitive method. Resistant to disease surveillance and monitoring of the test provides a reliable means of comprehensive and technical reserves, has important practical significance.Method:1, Aminoglycoside-resistant gene aadA1 chip production will be for aminoglycoside-resistant gene aadAl gene probe designed for 82, plus three positive control and three negative control, a total of 88 serial cross the United States Agilent Technologies Inc., the chip is divided into three sub-array printing, production of aminoglycoside-resistant gene aadAl chip. And collected clinical samples containing aadAl resistance gene by sequencing of eight gram-negative bacteria and resistance genes in non-aadAl 8 gram-negative bacteria tested to assess the probe and the chip's sensitivity, specificity, sex.2, Gram-negative bacteria resistance gene chip detection of drug resistance genes in Gram-negative bacteria produce chips were to detect 63 kinds of detection of drug resistance genes and the positive control group for a sub-array probe; then this Parallel Construction of the 8 sub-arrays for a large array. Between the 8 sub-arrays, respectively, the blank line as the interval. Meanwhile, a small amount of clinical samples to evaluate the chip's specificity, sensitivity and reproducibility were evaluated.Results:1, With the aminoglycoside resistance gene aadAl microarray Gram-negative bacteria samples, test results can be positive probe rate (PPR), as one of the most direct and simple parameter index, the report or show test samples were ownership of the actual test results consistent with expectations. Therefore, the subject developed aminoglycoside resistance gene aadAl array chip can be clearly marked to distinguish different samples2, We developed Gram-negative bacteria resistance gene chip analysis of clinical samples, microarray results and sequencing results are generally consistent, but there are still differences. To blaTEM gene, for example, chip to detect 11 of them found to contain the gene, five without the resistance gene, and sequencing results, and higher specificity; to detect the catB8, aadAl, aacA4, dfrA17, dfrAl resistance drug gene, the chip result were completely consistent with sequencing. None of the two detection methods to detect blaVIM resistance gene; chip test results show blaSHV, blaOXA, blaPSE, sull resistance gene, respectively, in which a clinical resistant isolates detected, blaIMP-4 resistance gene in which the two detected in clinical resistant strains, but PCR sequencing of the gene was not found; blaSHV, blaVEB, blaCTX, aadB resistance gene, respectively, in which a clinical resistant isolates detected by PCR sequencing, but negative for the gene microarray. Description blaSHV, blaVEB, blaCTX, aadB, blaOXA, blaPSE, sull, blaIMP-4 sub-array probe specific parts of the poor performance of the chip to improve the next round of optimization, the need to increase testing of samples specific probes.Conclusion:1, Build a gram-negative bacteria resistant gene pool and gram-negative bacteria resistance gene probe library 2, Successful design of the Gram-negative bacteria resistant gene detection chip, Gram-negative bacteria with resistance genes in microarray detection of clinical samples, confirmed that the chip has good repeatability, sensitivity and specificity.