Molecular typing of Neisseria gonorrhoeae isolates by pyrosequencing of highly polymorphic segments of the porB gene

Journal of Clinical Microbiology
M UnemoH Fredlund

Abstract

For prevention and control of gonorrhea, an objective, highly discriminating, and reproducible molecular epidemiological characterization of Neisseria gonorrhoeae is essential. In the present study, in pursuance of providing such qualities, pyrosequencing technology, a fast real-time DNA sequence analysis, was applied to six short, highly polymorphic porB gene segments, with subsequent genetic variant (genovar) determination of the bacterial isolates. The sequencing templates were obtained by real-time PCR amplification, which also included fluorescence melting curve analysis of the entire porB gene in order to determine the genogroup (porB1a or porB1b allele) prior to pyrosequencing analysis. The PSQ 96 MA system used allowed rapid (in approximately 1.5 h) determination of 96 sequences of 20 to 65 correct nucleotides each. The results were reproducible and mostly in concordance with the results of conventional Sanger dideoxy sequencing, with the exception of shorter read lengths and some uncertainty in determining the correct number of identical nucleotides in homopolymeric segments. The number of sequence variants identified in each of the six highly polymorphic segments of the porB1a and porB1b alleles (encoding surface-expo...Continue Reading

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Citations

Dec 18, 2009·International Journal of Microbiology·Pejvak KhakiPawan Sharma
Feb 3, 2007·Experimental Parasitology·C R StensvoldR C A Thompson
Jul 6, 2005·APMIS : Acta Pathologica, Microbiologica, Et Immunologica Scandinavica·Magnus UnemoHans Fredlund
Jul 29, 2006·International Journal of Antimicrobial Agents·Emma LindbäckBengt Wretlind
Sep 6, 2005·Clinica Chimica Acta; International Journal of Clinical Chemistry·Elizabeth A Mothershed, Anne M Whitney
Sep 7, 2005·International Journal for Parasitology·R J TraubI D Robertson
May 21, 2009·FEMS Microbiology Reviews·Wenjun LiPierre-Edouard Fournier
Oct 5, 2011·Clinical Microbiology and Infection : the Official Publication of the European Society of Clinical Microbiology and Infectious Diseases·A CarannanteP Stefanelli
Apr 6, 2007·Clinical Microbiology and Infection : the Official Publication of the European Society of Clinical Microbiology and Infectious Diseases·B EdvinssonUNKNOWN ESCMID Study Group on Toxoplasmosis
Jan 30, 2008·The Journal of Antimicrobial Chemotherapy·Mingmin LiaoJo-Anne R Dillon
Mar 21, 2007·APMIS : Acta Pathologica, Microbiologica, Et Immunologica Scandinavica·Magnus UnemoHans Fredlund
Nov 1, 2005·Expert Review of Molecular Diagnostics·Stuart C Clarke

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