The Establishment Of IntI-ISCR1-qnrB Multiplex PCR And Its Clinical Application

At present, quinolones is a broad-spectrum antibioticsits which is commonly used, and its drug-resistance is attracting extensive attention. Gene mutation of chromosome and plasmid mediated quinolone resistance are the main resistance mechanisms. Among them, qnr gene is a research foucs in recent years, qnr genotypes such as qnr A、qnr B、qnr C、qnr S has been found one after the other and qnr B is the main genotype in this area. qnr B gene could locate in the variable region of ISCR1(Insertion Sequence Common Regions 1),connecting with ISCR1 and thus spread drug resistance. ISCR1 is another gene transfer element which always connect with class 1 integron and they compose complex class I integron. Since complex class I integron has both class I integron and ISCR1, so it has strong gene spreading capacity. Previous methods for integron and qnr detecting including PCR-RFLP, PFGE, Southern Blot and so on. As these methods are too tedious, time-consuming, labor-consuming, so it is necessary to establish a rapid and simple method to detect Int I-ISCR1-qnr B.Given this, this study investigated 199 clinical isolated strains. Firstly, a Int I-ISCR1-qnr B multiplex PCR method which could quickly detect class I integron, ISCR1 and qnr B was established. Then the method’s specificity and sensitivity were evaluated. On this basis, the established Int I-ISCR1-qnr B multiplex PCR method and existing drug resistance test were used to detect clinical strains, hereby to analyze the clinical application value of the established Int I-ISCR1-qnr B multiplex PCR method, and provide quinolone resistance alarm information for clinical treatment. Methods1.Bacteria strains: 199 strains of gram-negative bacteria were collected from clinical departments from Nov.9,2014 to May 15, 2014.2.Antibiotics susceptibility: All isolated strains were identified and antibiotic susceptibility were determined by bacteria identification system.3.Specificity primers of int I1, conserved region of ISCR1-orf513,and qnr B gene were designed respectively by Microsoft Primer Premier5.0 for Int I-ISCR1-qnr B multiplex PCR system establishment.4. The most optimal annealing temperature for Int I-ISCR1-qnr B mutiplex PCR was determined by gradient PCR.5.Int I,ISCR1 and qnr B were detected by multiplex PCR, and the amplification products were analysised by agarose gel electrophoresis. Microsof Image J was used to scanned Agarose gel electrophoresis bands’ OD value, then the target gene’s concentration could be determined.6. 80 isolated strains weredetected by Int I-ISCR1-qnr B multiplex PCR and amplification products were all sequenced to evaluate the established method’s specificity.7.Mc Far Land method was used to detect bacteria concentration and original concentration was diluted to 108 cfu/ml, then 107、106、105、104、103、102、101cfu/ml was diluted by multiple proportion dilution. Then they were detected by Int I-ISCR1-qnr B mutiplex PCR.8.119 isolated strains were detected by Int I-ISCR1-qnr B mutiplex PCR,and the results were compared with drug sensitive test.9.SPSS 17.0 was adopted for statistical analysis,and square test was used to analyze two methods’ difference, α=0.05.1.The most optimal annealing temperature for Int I-ISCR1-qnr B mutiplex PCR was 57.5℃.2.The Int I-ISCR1-qnr B multiplex PCR method can amplify the Int I,ISCR1 and qnr B effectively, 280bp、475bp、607bp were the products, which were in accord with target sequences.3.80 isolated strains were detected by Int I-ISCR1-qnr B multiplex PCR and 19 strains were positive, the average OD value was Int I:217.9±33.3,ISCR1:243.2±60.6,qnr B:203.7±59.4. 61 strains were Int I-ISCR1-qnr B negative. After sequencing, there were 19 Int I-ISCR1-qnr B positive. Blast verifyed that all of the sequencing results were confirmed to the public sequences in Gene Bank, and the Gene Bank Accession numbers were HQ018645.1,KM111273.1,NG036203.1.4.The sensitivity of Int I-ISCR1-qnr B multiplex PCR was 101cfu/ml.5.119 isolated strains were analyzed by Int I-ISCR1-qnr B multiplex PCR to detect Int I-ISCR1-qnr B. 35 strains were Int I-ISCR1-qnr B positive and 84 strains were negative. Drug sensitive test showed 92 strains were quinolone resistant,27 strains are unresistant. c2 =3.36<c2(0.05,1)=3.84, P>0.05,which state that there was no statistical difference between the established Int I-ISCR1-qnr B multiplex PCR method and existing drug sensitive test. Conclusions1.The Int I-ISCR1-qnr B multiplex PCR method which can simultaneously detect class I integron,ISCR1 and qnr B was established successfully, and the method was quickly,exactly and specifically.2.The established Int I-ISCR1-qnr B multiplex PCR for qnr B detecting was compliant with drug sensitive test.3.Int I-ISCR1-qnr B multiplex PCR method detecting Int I-ISCR1-qnr B could provide quinolone resistance alarm information for clinical. Results...