[Polymerase chain reaction (PCR) for the identification of toxigenic Vibrio cholerae O1 in oysters].
PCR was made with ctx2 (CGG GCA GAT TCT AGA CCT CCT G) y ctx3 (CGA TGA TCT TGG AGC ATT CCC AC) primers for subunit A of cholera toxin, 30 cycles of temperature on samples of 50 g of oysters added in 450 ml of peptone alcaline water that were inoculated with 15 x 10(6), 0.75 x 10(6) and 0.15 x 10(6) CFU/ml of toxigenic 6707 V. cholerae O1 reference strain. The samples were tested by three microbiological methods: INDRE's method uses 1 x 10(-1) dilution of sample, two fold pass to peptone alcaline water pH 9 incubated 18 h and 6 h at 37 degrees C, the Food and Drugs Administration (FDA) method uses 10(-1) to 10(-6) dilutions of sample, 6 h incubation and reincubation for 18 h at 37 and 42 degrees C and the Mexican laboratories (LMD) with 10(-4) to 10(-3) dilutions, the samples were incubated for 6 h and then reincubated for 18 h at two temperatures 37 and 42 degrees C. The PCR by INDRE's method was positive with 3 x 10(2) CFU/ml/g oyster. In the FDA's method the PCR detected DNA in 10(-4) dilution with 3 x 10(1) CFU/ml/g oyster and in LMD's method the PCR was positive in 10(-3) with 3 CFU/ml/g oyster. The results of the PCR were obtained between 5-6 h, and later V. cholerae O1 was isolated by three microbiological methods. The PCR reproducibility was better on DNA sample diluted 1:4 and 10 microliters of sample increased from 1:1000 to 1:10000 the sensitivity of PCR.
Rodríguez-Angeles
M G
MG
Instituto Nacional de Diagnóstico y Referencia Epidemiológicos, México, D.F., Mexico.
Giono-Cerezo
S
S
Moreno-Escobar
A
A
Valdespino-Gómez
J L
JL
D000818:Animals; D001431:Bacteriological Techniques; D001483:Base Sequence; D002772:Cholera Toxin / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010125:Ostreidae / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D014734:Vibrio cholerae / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*
D004269:DNA, Bacterial; D002772:Cholera Toxin