Rapid detections of Opr D2 gene in Pseudomoas aeruginosa using loop-mediated isothermal amplification

Abstract

Objective To establish a rapid detection approach by visual interpretation directly for OprD2 resistance gene of Pseudomonas aeruginosa based on the Loop-mediated isothermal amplification (LAMP),and provide a quick and effective method for clinical monitoring of Pseudomonas aeruginosa strains. Methods Totally 47 strains of Pseudomonas aeruginosa collected from December 2011 to June 2012 in Southwest Hospital of microorganisms were prospectively studied.Four LAMP primers (two inner,two outer) were designed according to the six zones of the OprD2 gene of Pseudomonas aeruginosa.A positive reaction is indicated by the color change after adding an intercalating dye (hydroxy naphthol blue) to the reaction solution.This method was used to detect and analyze the distribution of OprD2 resistance gene in 47 strains of Pseudomonas aeruginosa and its correlation with antibiotic resistance. Results The LAMP assays showed 100% specificity for the OprD2 gene,and the sensitivity (with the lowest detection limits of 17.414μg/L) was 10-fold higher than that of conventional PCR assays.The OprD2 gene was negative in 23 strains by both conventional PCR and LAMP.In OprD2 negative strains,the resistance rate of cefotaxime,levofloxacin,aztreonam,piperacillin,imipenem and meropenem was 100% (23/23),57% (13/23), 48% (11/23),48% (11/23),48% (11/23) and 43% (10/23).Compared with the OprD2 positive strains,statistical analysis showed that the resistance rate of imipenem,levofloxacin and meropenem in OprD2 negative strains increased significantly (chisquare value is 9.155,4.846,4.037,P value was 0.002,0.028,0.045, and so there was significant difference). Conclusions The established LAMP approach in this study enables rapid,sensitive and specific detection of OprD2 gene in Pseudomonas aeruginosa by visual interpretation.Deficiency of OprD2 gene confers Pseudomonas aeruginosa a basal level of resistance to carbapenems especially to imipenem.The identification of OprD2 gene distribution in Pseudomonas aeruginosa is helpful to the selection of antimicrobial agents in the infection treatment.(Chin J Lab Med,2013,36:543-547) Key words: Pseudomonas aeruginosa; Drug resistance; bacterial