[Simultaneous detection and differentiation of O1 classical E1 Tor, O139 and nonO1 nonO139 strains of Vibrio cholerae by using multiplex PCR].
OBJECTIVE
To explore the rapid and sensitive procedures for detection and differentiation of O1 classical(CVC), El Tor(EVC), O139 and nonO1 nonO139 strains of Vibrio cholerae.


METHODS
Four pairs of primers were designed from toxin sub-unit A(ctxA) gene, O139 special gene, Haemolytic subunit A gene and Toxin coregulated pilus subunit A gene. Multiplex PCR was established to simultaneously detect those four genes. CVC, EVC, O139 and nonO1 nonO139 strains can be detected and differentiated by the size and numbers of Multiplex PCR bands via electrophoresis. Two pairs of inside primers were designed from ctx A and O139 special gene, and a nested PCR was established.


RESULTS
60 EVC, 2 CVC, 15 O139 and 30 nonO1 nonO139 strains isolated from patients were detected with multiplex PCR and nested PCR, and the results were correct. 10 EVC and 15 O139 strains isolated from environment samples were detected, and the results meant that EVC were toxic and O139 were non-toxic. The sensitivity of multiplex PCR reached 100 cfu, and that of nested PCR reached 1-10 cfu.


CONCLUSIONS
This method is rapid, specific, and sensitive, and possess great value for practical application.
Rui
Y
Y
Department of Epidemiology, First Military Medical University, Guangzhou 510515, China.
Cai
C
C
Xiao
B
B
D004269:DNA, Bacterial / Q000032:analysis*; D005838:Genotype; D006801:Humans; D008565:Membrane Proteins; D016133:Polymerase Chain Reaction; D014734:Vibrio cholerae / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification
D004269:DNA, Bacterial; D008565:Membrane Proteins