Genotyping Neisseria gonorrhoeae using fluorescent amplified fragment length polymorphism analysis

Abstract

An evaluation of fluorescent amplified fragment length polymorphism (FAFLP) for typing Neisseria gonorrhoeae using 52 isolates revealed that its discriminatory power approached that of Opa-typing in identifying epidemiologically linked isolates. Automated, accurate sizing of FAFLP amplified fragments permits objective data analysis and storage, making it an attractive method for large surveillance projects.

FIG. 1

Genotyper outputs of strain 14890 after serial subculture for the size range 135 to 163 bp. The boxed numbers under the peaks of the traces are the fragment sizes in base pairs assigned by comparison with the standard curve generated with the internal size standard. The panels show fragment sizes in base pairs before subculture (top two panels) and after 11 subcultures (bottom two panels).

FIG. 2

Genotyper output of FAFLP analysis of unrelated strains 13072 (top panel), 16973 (middle panel), and 7575 (bottom panel) in the size range of 197- to 237-bp fragments. It is clear that the fragment at 201 bp (boxed) is present for strains 13072 and 16973 but not for strain 7575. Also, the fragment present in the dashed box at 236 bp is present for strain 7575 but absent for the other two strains and the fragment present at 237 bp for strain 13072 is absent for the other two strains.

FIG. 3

FAFLP-generated distance tree for 52 strains of N. gonorrhoeae. FAFLP was carried out in duplicate for all strains. These gave identical profiles except for strain 12589, which differed by one fragment (12589a and 12589b). Epidemiologically related strains (solid circle) were indistinguishable by FAFLP. Epidemiologically unrelated strains of the auxotype AHU (underlined), and to some extent PA°U (boxed), clustered together by FAFLP. Strains of other auxotypes did not group together. Three strains of the auxotype PA°U (7103, 7249, and 8652) were indistinguishable by FAFLP but differed by two to three fragments when analyzed by Opa-typing. Three pairs of strains not known to be epidemiologically related (dashed circle) were indistinguishable by FAFLP or Opa-typing. The phenotype of strains is given as auxotype/serovar. Auxotypes are as follows: A, arginine requiring; AHU, arginine, hypoxanthine, and uracil requiring; NR, nonrequiring; P, proline requiring; and PA°U, proline requiring, arginine requiring (not satisfied by ornithine), and uracil requiring. Serovars are as follows: IA, Porin IA, IB, and Porin IB, with numbers designated according to the established scheme (12).