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Detection of Vibrio cholerae and V. mimicus heat-stable toxin gene sequence by PCR
A C Vicente1, A M Coelho, C A Salles
1Department of Genetics, Institute Oswaldo Cruz, Federal University of Rio de Janeiro, Brazil.
Abstract:
Previously the heat-stable enterotoxin in Vibrio cholerae and V. mimicus has been detected by suckling mouse assay, a non-specific approach, and by DNA probes, a time-consuming method. This report describes a polymerase chain reaction (PCR) procedure for the detection of the stn (NAG-ST) and sto (O1-ST) gene sequences that is rapid and specific, allowing toxin gene molecular characterisation. A total of 34 V. cholerae and V. mimicus isolates was examined for ST and CT genes. The NAG-ST gene sequence was amplified in 13 of 22 non-O1/non-O139 V. cholerae and three of five V. mimicus strains. A new enterotoxin gene sequence pattern was found with MseI and TaqI restriction endonuclease PCR fragment digestion of two V. cholerae isolates, in addition to the pattern anticipated from the Genbank sequence, and found with the other ST+. These results show that ST-PCR detection is useful for the characterisation of V. cholerae and V. mimicus.
Insights
A new, rapid polymerase chain reaction (PCR) method accurately detects heat-stable enterotoxin genes in Vibrio cholerae and V. mimicus. This molecular characterization offers a significant improvement over older, less specific, and time-consuming techniques.
Area of Science:
- Microbiology
- Molecular Biology
- Bacteriology
Background:
- Traditional methods for detecting heat-stable enterotoxin (ST) in Vibrio cholerae and V. mimicus, such as the suckling mouse assay and DNA probes, are either non-specific or time-consuming.
- Accurate and rapid detection of enterotoxin genes is crucial for the molecular characterization of Vibrio species.
Purpose of the Study:
- To develop and evaluate a rapid and specific polymerase chain reaction (PCR) procedure for detecting heat-stable enterotoxin (ST) gene sequences (stn and sto) in Vibrio cholerae and V. mimicus.
- To enable molecular characterization of toxin genes in these bacterial species.
Main Methods:
- Development of a polymerase chain reaction (PCR) assay targeting the stn (NAG-ST) and sto (O1-ST) gene sequences.
- Analysis of 34 isolates of Vibrio cholerae and V. mimicus for the presence of ST and CT genes using the developed PCR method.
- Restriction endonuclease digestion (MseI and TaqI) of PCR products for further molecular characterization of enterotoxin gene sequences.
Main Results:
- The ST-PCR assay successfully amplified the NAG-ST gene sequence in 13 of 22 non-O1/non-O139 V. cholerae isolates and in three of five V. mimicus strains.
- Restriction fragment digestion revealed a novel enterotoxin gene sequence pattern in two V. cholerae isolates, distinct from the expected Genbank sequence.
- The ST-PCR method demonstrated high specificity and speed for detecting and characterizing enterotoxin genes in Vibrio species.
Conclusions:
- The developed ST-PCR detection method is a rapid, specific, and valuable tool for the molecular characterization of enterotoxin genes in Vibrio cholerae and V. mimicus.
- This molecular approach significantly improves upon existing methods for identifying toxin-producing Vibrio strains.