Simple, Rapid, and Inexpensive Detection of Neisseria gonorrhoeae Resistance Mechanisms Using Heat-Denatured Isolates and SYBR Green-Based Real-Time PCR

Abstract

Neisseria gonorrhoeae has developed resistance to multiple classes of antimicrobials. There is now growing concern that without the availability of appropriate public health strategies to combat this problem, gonorrhea could become untreatable. For this reason, surveillance for gonococcal antimicrobial resistance must be optimal both in terms of obtaining a representative sample of gonococcal isolates and in terms of having the appropriate tools to identify resistance. To aid with this surveillance, molecular tools are increasingly being used. In the present study, we investigated the use of a simple heat denaturation protocol for isolate DNA preparation combined with SYBR green-based real-time PCR for the identification of mutations associated with N. gonorrhoeae antimicrobial resistance. A total of 109 clinical gonococcal isolates were tested by highresolution melting (HRM) curve analysis for chromosomal mutations associated with gonococcal resistance to beta-lactam antibiotics: a penA 345A insertion, ponA L421P, mtrR G45D, substitutions at positions 120 and 121 in porB1b, and an adenine deletion in the mtrR promoter. An allele-specific PCR assay was also investigated for its ability to detect the adenine deletion in the mtrR promoter. The results were compared to those obtained by DNA sequencing. Our HRM assays provided the accurate discrimination of heat-treated isolates in which the sequence types differed in GC content, including isolates with the penA 345A insertion and the ponA L421P and mtrR G45D mutations. The allele-specific PCR assay accurately identified isolates with the adenine deletion in the mtrR promoter. Heat-denatured DNA combined with SYBR green-based real-time PCR offers a simple, rapid, and inexpensive means of detecting gonococcal resistance mechanisms. These methods may have broader application in the detection of polymorphisms associated with phenotypes of interest.

Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis

“…A limitation was that several SNPs could not be identified. This was not investigated further, but probably resulted from sequence variations within assay targets [20]. Notably, these variations did not alter AES‐1, AES‐2 and P42 detection by either the HRM20SNP or HRM10SNP profiles, given that these strains differed by at least two SNPs from every other PFGE pulsotype.…”

“…The 40 oligonucleotides designed and the alleles targeted are presented in Table 1. HRM curve reactions were performed with a standard reaction mix and previously described cycling conditions [20]. Each reaction mix contained 10.0 μL of 2 × SYBR green qPCR SuperMix‐UDG (Invitrogen Australia, Mulgrave, NSW, Australia), 0.5 μM forward and reverse primers, 1.0 μL of heat‐denatured isolate and DNase‐free water to a total volume of 20.0 μL.…”

“…Bacterial DNA was released by a basic heat-treatment method and stored at )20°C until testing [20]. Primers were designed to flank the 20 SNPs of interest.…”

Real-time PCR genotyping of Neisseria gonorrhoeae isolates using 14 informative single nucleotide polymorphisms on gonococcal housekeeping genes

“…17 A total of 17 reactions were used to characterize the 14 SNPs. Briefly, high-resolution melting (HRM) curve analysis was used for 11 SNPs, and for 3 SNPs for which HRM was found to be unsuitable (data not shown), an allelespecific PCR (ASP) approach was used.…”

“…17 Briefly, each reaction mix contained 10.0 mL of 2× SYBR Green qPCR SuperMix-UDG (Invitrogen, Australia), 0.5 mM of forward and reverse primers and 2.0 mL of heat-denatured isolate, and were made up to a total volume of 20.0 mL with DNase-free water. Cycling was performed on a Rotor-Gene Q instrument (Qiagen, Australia) with the following cycling conditions: an initial enzyme activation step at 958C for 2 min, followed by 40 cycles of denaturation at 958C for 15 s and primer annealing and elongation at 608C for 30 s, with fluorescence signal read on green at 608C following extension.…”