Specific detection of toxigenic vibrio cholerae based on in situ PCR in combination with flow cytometry.
- Author:
Li ZHU
1
,
2
;
Jun-Peng CAI
;
Qing CHEN
;
Shou-Yi YU
Author Information
- Publication Type:Journal Article
- MeSH: Cholera Toxin; genetics; Colony Count, Microbial; DNA, Bacterial; analysis; Flow Cytometry; Genes, Bacterial; genetics; Microscopy, Fluorescence; Polymerase Chain Reaction; Reproducibility of Results; Vibrio cholerae; genetics; isolation & purification
- From: Biomedical and Environmental Sciences 2007;20(1):64-69
- CountryChina
- Language:English
-
Abstract:
OBJECTIVETo develop an in situ PCR in combination with flow cytometry (ISPCR-FCM) for monitoring cholera toxin positive Vibrio cholerae.
METHODSIn running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells.
RESULTSA length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene (ctxAB gene). Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low (10(5) cells/mL), while the specificity was high.
CONCLUSIONWe have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.
