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Objective: to establish a multiplex PCR system, simultaneous detection of seven kinds of bacterial aminoglycoside antibiotic resistant gene aac (3) - Ⅱ, aac (6 ') - Ⅰ b, aac (6') - Ⅱ, ant (3 \) - I, aph (3 ') - Ⅳ armA and rmtB evaluation of 56 clinical isolates of Klebsiella pneumoniae strains resistant to genetic testing, the clinical application of the multiplex PCR system. 2, the establishment of a multi- reverse transcription - polymerase chain reaction (RT-PCR) method, the human hand, foot and mouth disease caused by genotyping nine common intestinal virus - human enterovirus 71 (HEV71), Coxsackie virus group A ( CVA) 16,4,5,9,10 and Coxsackie virus B group (CVB) 1,3,5 type of 180 clinical stool specimens of hand, foot and mouth pathogen detection, evaluation of the multiplex RT-PCR system clinical application methods: 1, the establishment of multi-drug resistant gene detection system and clinical application (1) queries each target gene sequences after ClustalX than, conservative area design multiple primer; (2) to collect the aminoglycoside antibiotic resistance strains of bacterial genomic DNA was extracted; (3) singlet PCR to verify the specificity of each pair of primers, establishment of GeXP multiple detection system to verify the specificity of the multiple detection system known positive mixed template; (4) optimize the reaction system to positive gene cloning plasmid analysis of the sensitivity of the detection system; (5) detection system for screening of of 56 aminoglycoside antibiotic-resistant clinically isolated Klebsiella pneumoniae, and to observe the distribution of various genes, and compared with the traditional single-plex PCR and agarose gel electrophoresis analysis to observe the consistency of the two methods to evaluate the clinical application of the multiple system, hand, foot and mouth disease pathogen spectrum the parting detection system and its clinical application Evaluation (1) select a design for enterovirus 5'UTR region universal primers PE, and 10 pairs of specific primers designed nine serotypes of enterovirus VP1 region, establish GeXP multiplex RT-PCR detection system; (2) Using the known serotypes of enteroviruses cell cultures to analyze the specificity of the RT-PCR detection system GeXP multiplet; (3 serial dilutions of) TCID50 quantitative cell culture and cloning plasmid in vitro transcribed RNA analysis multiple detection the sensitivity of the system; blind sieve (4) 180 parts clinical stool specimens, and the strain separation method, serum antibodies and the aggregated results of the trial and single re-sequencing of the RT-PCR analysis of the control, evaluation GeXP multiplex RT-PCR intestinal clinical application of virus detection system. Results: 1, successfully established a multiplex PCR detection system based on GeXP system, simultaneous detection of seven kinds of aminoglycoside antibiotic resistance genes, the detection sensitivity of 10 copies. GeXP multiplex PCR screening 56 strains of Klebsiella pneumoniae clinical isolates, aac (3) - Ⅱ, aac (6 ') - Ⅰ b, ac (6') - Ⅱ, ant (3 \and rmtB positive rate of 71.43%, 25.00%, 14.29%, 75.00%, 17.86%, 71.43% and 30.36%, respectively. Traditional single-plex PCR detected 56 Klebsiella pneumoniae clinical specimens GeXP multiplex PCR detection sensitivity of 83.33% to 100%, a specificity of 85.71% to 100%, consistent with the higher rate compared with traditional methods. 2, the successful establishment of a detection system of multiplex RT-PCR based on GeXP system simultaneously conservative fragment common enterovirus and type-specific fragment amplified. Optimized multi-detection system detects HEV71 and CVA16 cell culture, sensitivity 100.05 TCID50, and can simultaneously detect the remaining seven kinds of enterovirus RNA, a sensitivity of 100 copies. Detection and analysis of 180 clinical specimens, multiplex RT-PCR, GeXP system detected PE sensitivity HEV71 and CVA16 were 98.79%, 91.67% and 91.67%, specificity 80.00%, 98.48%, and 100%, other 7 serotypes test results summary of the test results consistent with traditional typing methods was 92.59% (25/27). Conclusion: 1, successfully established multiplex PCR based on GeXP system detection system, rapid, sensitive screening aminoglycoside antibiotic resistance genes, the multiplex PCR detection system with higher consistency with the traditional single-plex PCR can be used to aminoglycoside antibiotic resistance gene screening. 2, successfully established based on multiple RT-PCR system of GeXP system, rapid, sensitive and specific detection of common enteroviruses cause HFMD 9, higher consistency of the method with the traditional virus typing methods, can be used to HFMD pathogen spectrum of molecular epidemiological investigation.
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