Genotyping Of P. Aeruginosa Type Ⅲ Secretion System Using Magnetic Separation And Chemiluminescence

Pseudomonas aeruginosa(P.aeruginosa)is a highly viable conditioned pathogenic bacterium which causes many severe clinical infections such as cystic fibrosis and pneumonia.Nowdays,the P.aeruginosa has become one of the most serious problems in hospital infection.Type Ⅲ secretion system(T3SS)is a vital virulence secretion system of pseudomonas aeruginosa.T3SS has four virulence proteins:ExoU,ExoT,ExoS and ExoY.The differences of T3SS genotypes among P.aeruginosa strains can cause its infection of the pathological characteristics and make toxicity mechanism differ.Therefore,the testing of the T3SS genotypes is of great significance to acquaint the toxicity of bacteria and predict clinical efficacy.This study is intended to use magnetic separation and chemiluminescence technology to establish a test method which is easy to be automated for the detection of the pseudomonas aeruginosa T3SS genotypes.1.The development of genome DNA extraction kits based on magnetic separationThe extraction of nucleic acids is usually the first step in all experiments with nucleic acids.This research developed rapid and high-quality genome DNA extraction kits using magnetic nanoparticles as the carrier of nucleic acids.The results showed that the best lysis time is 5 min.The whole extraction process can be completed in half an hour.The nucleic acid extraction rate of the kit for DNA samples is more than 90%.Compared with the two commercial type kits,the kit has higher nucleic acid extraction rate,and the extracted nucleic acids have higher amplification efficiency.2.The development of magnetic enrichment multiplex PCR amplification systemWith the T3SS genes of P.aeruginosa as the amplification target,magnetic enrichment multiplex PCR amplification system was developed and optimized.The optimum annealing temperature of multiple PCR amplification is 50 ℃.The optimum proportion of multiple PCR primers is exoU:exoS:exoT:exo Y=0.8:0.8:0.9:1.3.Genotyping of P.aeruginosa type Ⅲ secretion system using magnetic separation and chemiluminescenceMagnetic separation nucleic acid extraction,magnetic enrichment multiplex PCR and chemiluminescence were successfully applied to T3SS genotyping of P.aeruginosa.The T3SS genotypes of P.aeruginosa standard strains ATCC9027,ATCC27853 and CMCC10104 were tested respectively.The results showed that all of the three strains contained exoS,exoT and exoY without exoU.T3SS genotyping was performed on 22 clinical samples by the method.It was found that all of the samples contained the exoT gene,72.7%of the samples contained the exoY gene,95.5%of the samples contained the exoS gene,and only 4.5%of the samples contained the exoU gene.