Article,

Simple, rapid and inexpensive detection of Neisseria gonorrhoeae resistance mechanisms using heat-denatured isolates and SYBR-green based real-time PCR

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Antimicrobial Agents and Chemotherapy, (June 2009)PMID: 19528267.
DOI: 10.1128/AAC.00385-09

Abstract

Neisseria gonorrhoeae has developed resistance to multiple classes of antimicrobials. There is now growing concern that without appropriate public health strategies to combat this problem, gonorrhoea could become untreatable. For this reason, surveillance of gonococcal antimicrobial resistance must be optimal, both in terms of obtaining a representative sample of gonococcal isolates as well as having appropriate tools to identify resistance. To aid in this surveillance, molecular tools are being increasingly used. In this study, we investigated the use of a simple heat-denaturing protocol for isolate DNA preparation combined with SYBR-green based real-time PCR for identifying mutations associated with N. gonorrhoeae antimicrobial resistance. A total of 109 clinical gonococcal isolates were tested using high resolution melting (HRM) curve analysis for chromosomal mutations associated with gonococcal resistance to beta-lactam antibiotics; penA 345A insertion, ponA L421P, mtrR G45D, porB1b 120 and 121 substitutions and an adenine deletion in the mtrR promoter. An allele-specific PCR assay was also investigated for detection of the adenine deletion in the mtrR promoter. The results were compared to DNA sequencing. Our HRM assays provided accurate discrimination using heat-treated isolates where sequence types differed in GC content, including the penA 345A insertion, ponA L421P, and mtrR G45D mutations. The allele-specific PCR assay accurately identified isolates with the mtrR promoter adenine deletion. 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.

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