[Establishment of fluorescent amplified fragment length polymorphism in Vibrio cholerae and evaluation in molecular typing].
OBJECTIVE
To develop fluorescent amplified fragment length polymorphism (AFLP) method and to evaluate the its typing capability with pulsed-field gel electrophoresis (PFGE) in molecular typing of Vibrio cholerae.


METHODS
Forty-seven strains of V. cholerae, with different PFGE patterns, were selected as the reference group to optimize the selective primers of AFLP analysis. Eighty-three strains including 20 strains from one epidemic episode, isolated from different provinces during 1961 and 2005, were used to compare the typing abilities of AFLP and PFGE. LI-COR4300 DNA sequencing system was used for AFLP electrophoresis. The images were recorded by Saga(MX) software and transferred to BioNumerics for clustering analysis. A standard protocol for V. cholerae from PulseNet was used in PFGE.


RESULTS
When comparison was made with different selective primers on AFLP based on the 47 strains, results showed that the optimized selective primer pair was EcoR I-G/Mse I-T, and the reproducibility of the tests was 99.2%. Eighty-three isolates showed 52 AFLP patterns and 44 PFGE patterns, with D values as 0.9545 (AFLP) and 0.9251 (PFGE) respectively.


CONCLUSIONS
The protocol of fluorescent AFLP on V. cholerae typing was established. AFLP was higher than PFGE in discrimination of V. cholerae which could be used for molecular typing. When combined with PFGE, AFLP became a more insightful tool to identify genome difference of different isolates.
Lou
Jing
J
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Diao
Bao-wei
BW
Wang
Hong-xia
HX
Cui
Zhi-gang
ZG
Qi
Guo-ming
GM
Kan
Biao
B
D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods*; D016521:Electrophoresis, Gel, Pulsed-Field; D005838:Genotype; D010802:Phylogeny; D014734:Vibrio cholerae / Q000145:classification / Q000235:genetics*