Detection of the Vibrio cholerae heat-stable enterotoxin gene by polymerase chain reaction.
A polymerase chain reaction assay was developed for detection of the Vibrio cholerae heat-stable enterotoxin gene (sto). The assay is based on two oligonucleotide primers suitable for amplification of the entire sto open reading frame. Reaction conditions were defined to obtain optimal results in terms of specificity and sensitivity. Under these conditions the assay was highly sensitive and enabled good results to be obtained using, as a template, crude DNA preparations from single bacterial colonies. A rapid protocol for direct sequence analysis of the amplification product, which may be used in combination with the PCR assay to confirm the product identity and analyse sequence polymorphisms within the sto gene, was also developed. Twenty-two V. cholerae non-O1 isolates from sporadic cases of V. cholerae non-O1-associated gastroenteritis from Cuba were analysed by this PCR assay. Four strains (18.2%) were found to carry the sto gene. The prevalence of V. cholerae non-O1 strains carrying the sto gene among clinical isolates is higher than that reported for other geographical areas, except in the case of epidemics.
Istituto di Clinica delle Malattie Infettive, Università di Siena, Italy.
P
P
Guglielmetti
L
L
Bravo
A
A
Zanchi
R
R
Montè
G
G
Lombardi
G M
GM
Rossolini
D000595:Amino Acid Sequence; D001483:Base Sequence; D002771:Cholera / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D004768:Enterotoxins / Q000235:genetics*; D005784:Gene Amplification; D005798:Genes, Bacterial; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014734:Vibrio cholerae / Q000235:genetics* / Q000302:isolation & purification
D004269:DNA, Bacterial; D004768:Enterotoxins; D009841:Oligonucleotides; C037420:stN protein, Vibrio cholerae