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

Kugelman, Gayle, Tapsall, John W., Goire, Namraj, Syrmis, Melanie, Limnios, Athena, Lambert, Stephen B., Nissen, Michael D., Sloots, Theo P. and Whiley, David M. (2009) Simple, rapid, and inexpensive detection of Neisseria gonorrhoeae resistance mechanisms using heat-denatured isolates and SYBR green-based real-time PCR.. Antimicrobial Agents & Chemotherapy, 53 10: 4211-4216. doi:10.1128/AAC.00385-09

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Author Kugelman, Gayle
Tapsall, John W.
Goire, Namraj
Syrmis, Melanie
Limnios, Athena
Lambert, Stephen B.
Nissen, Michael D.
Sloots, Theo P.
Whiley, David M.
Title Simple, rapid, and inexpensive detection of Neisseria gonorrhoeae resistance mechanisms using heat-denatured isolates and SYBR green-based real-time PCR.
Formatted title
Simple, rapid, and inexpensive detection of Neisseria gonorrhoeae resistance mechanisms using heat-denatured isolates and SYBR green-based real-time PCR.
Journal name Antimicrobial Agents & Chemotherapy   Check publisher's open access policy
ISSN 0066-4804
Publication date 2009-10
Year available 2009
Sub-type Article (original research)
DOI 10.1128/AAC.00385-09
Open Access Status File (Publisher version)
Volume 53
Issue 10
Start page 4211
End page 4216
Total pages 6
Editor George M. Eliopoulos
Place of publication Washington, DC, United States
Publisher American Society for Microbiology
Collection year 2010
Language eng
Subject 060502 Infectious Agents
060506 Virology
920109 Infectious Diseases
C1
920203 Diagnostic Methods
920117 Skin and Related Disorders
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-Iactam antibiotics: apenA 345A insertion,ponA L421P, mtrR G45D, substitutions at positions 120 and 121 in porBlb, 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 resistancemechanisms. These methods may have broader application in the detection of polymorphisms associated with phenotypes of interest. Copyright © 2009 American Society for Microbiology. All rights reserved.
Formatted 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-Iactam antibiotics: apenA 345A insertion,ponA L421P, mtrR G45D, substitutions at positions 120 and 121 in porBlb, 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 resistancemechanisms. These methods may have broader application in the detection of polymorphisms associated with phenotypes of interest.

Copyright © 2009 American Society for Microbiology. All rights reserved.
Keyword Gonorrhoea
Resistance
Mechanisms
PCR
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

 
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Created: Wed, 10 Mar 2010, 09:55:32 EST by Lesley Arnicar on behalf of Clinical Medical Virology Centre