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

被引:24
作者
Kugelman, Gayle [1 ,2 ,3 ]
Tapsall, John W. [2 ,4 ]
Goire, Namraj [1 ,2 ,3 ]
Syrmis, Melanie W. [1 ,3 ]
Limnios, Athena [2 ,4 ]
Lambert, Stephen B. [1 ,2 ,3 ]
Nissen, Michael D. [1 ,2 ,3 ,5 ,6 ]
Sloots, Theo P. [1 ,2 ,3 ,5 ,6 ]
Whiley, David M. [1 ,2 ,3 ]
机构
[1] Royal Childrens Hosp & Hlth Serv Dist, Sir Albert Sakzewski Virus Res Ctr, Queensland Paediat Infect Dis Lab, Herston, Qld 4029, Australia
[2] Royal Childrens Hosp, Infect Dis Lab, Queensland Childrens Med Res Inst, Herston, Qld 4029, Australia
[3] Univ Queensland, Clin Med Virol Ctr, Brisbane, Qld 4072, Australia
[4] Prince Wales Hosp, SE Area Lab Serv, Dept Microbiol, WHO Collaborating Ctr STD & HIV, Sydney, NSW, Australia
[5] Royal Brisbane Hosp Campus, Div Microbiol, Brisbane, Qld, Australia
[6] Univ Queensland, Dept Paediat & Child Hlth, Brisbane, Qld, Australia
关键词
MEDIATED PENICILLIN RESISTANCE; ANTIMICROBIAL RESISTANCE; REDUCED SUSCEPTIBILITY; ANTIBIOTIC-RESISTANCE; TAQMAN PROBES; MUTATIONS; PENA; SURVEILLANCE; STRAINS; ASSAY;
D O I
10.1128/AAC.00385-09
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 high-resolution 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.
引用
收藏
页码:4211 / 4216
页数:6
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