Multiplex Real-Time PCR Assay for Simultaneous Identification of Neisseria gonorrhoeae and Its Ciprofloxacin Susceptibility Status

Abstract

A real-time PCR (RT-PCR) assay was designed for the simultaneous identification of Neisseria gonorrhoeae and its ciprofloxacin susceptibility status. A SYBR green-based multiplex RT-PCR format was used; it comprised two different forward primers and a common reverse primer to detect single nucleotide polymorphisms (SNPs) in gyrA of N. gonorrhoeae The primer pairs were evaluated for their sensitivity and specificity using genomic DNA from 254 N. gonorrhoeae isolates (82 were ciprofloxacin susceptible and 172 were ciprofloxacin resistant) and 23 non-N. gonorrhoeae species isolates. The performance of the primers was validated using genomic DNA from 100 different N. gonorrhoeae isolates (46 were ciprofloxacin susceptible and 54 were ciprofloxacin resistant) and 52 non-N. gonorrhoeae isolates. The latter panel was revalidated by testing 99 (46 isolates were ciprofloxacin susceptible and 53 isolates were ciprofloxacin resistant) of the N. gonorrhoeae isolates and 23 non-N. gonorrhoeae isolates. These primers detected N. gonorrhoeae and its ciprofloxacin susceptibility status with over 99% sensitivity and specificity for all panels tested. This assay has the potential to be an inexpensive and rapid test for the simultaneous identification of N. gonorrhoeae and its ciprofloxacin susceptibility status.

Keywords: Neisseria gonorrhoeae; antimicrobial susceptibility (AMS); ciprofloxacin; gyrA; multiplex RT-PCR assay.

FIG 1

Multiplex RT-PCR melt curve representation of ciprofloxacin-susceptible and intermediate/resistant N. gonorrhoeae isolates. (A) Ciprofloxacin-susceptible (n = 47) and intermediate/resistant (n = 47) N. gonorrhoeae isolates (subset of panel 1). Blue lines represent susceptible isolates, orange lines represent intermediate/resistant isolates, and black lines represent no template negative controls. (B) WHO N. gonorrhoeae reference strains (n = 8). Strains B, C, F, O, and P are susceptible (melt curve ∼78°C), and strains K, L, and M are intermediate/resistant (melt curve 80.0-83.0°C).