Multiplex TaqMan real-time PCR platform for detection of Neisseria gonorrhoeae with decreased susceptibility to ceftriaxone

Abstract

A multiplex TaqMan real-time PCR platform was developed in this study for combined detection of opa and/or porA genes (identification of N. gonorrhoeae) and the key mutations (Ala501Val/Thr/Pro, and/or Gly545Ser) in penicillin-binding protein 2 (PBP2) associated with decreased susceptibility to extended-spectrum cephalosporins (ESCs). Firstly, the specificities of the TaqMan probes/primers for the multiplex TaqMan real time PCR platform were confirmed by Basic Local Alignment Search Tool (BLAST) analysis. Then the multiplex PCR platform was performed on 77 isolates with decreased susceptibility to ceftriaxone (CRO) and 100 isolates with full susceptibility to CRO under universal optimized reaction conditions. As a result, based on cultivation-based matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and antimicrobial susceptibility testing in vitro, the multiplex platform had a sensitivity of 100% and a specificity of 95.0% for identifying cultured isolates of Neisseria gonorrhoeae (N. gonorrhoeae, NG, GC) with decreased susceptibility to CRO. When directly screening N. gonorrhoeae with decreased susceptibility to CRO from clinical urogenital swabs, the multiplex platform offered a sensitivity of 96.1% and a specificity of 95.0%. Therefore, on the basis of sample culture and antimicrobial susceptibility testing in vitro, the multiplex TaqMan real time PCR platform has been proven to be a sensitivity of 100% and a specificity of 95.0% useful tool for screening cultured isolates of N. gonorrhoeae with decreased susceptibility to CRO, which can be finished within 2 days.