Specific detection of toxigenic vibrio cholerae based on in situ PCR in combination with flow cytometry.
OBJECTIVE
To develop an in situ PCR in combination with flow cytometry (ISPCR-FCM) for monitoring cholera toxin positive Vibrio cholerae.


METHODS
In 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.


RESULTS
A 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.


CONCLUSIONS
We 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.
Zhu
Li
L
School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong, China. zhuli89@126.com
Cai
Jun-Peng
JP
Chen
Qing
Q
Yu
Shou-Yi
SY
D002772:Cholera Toxin / Q000235:genetics; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis; D005434:Flow Cytometry; D005798:Genes, Bacterial / Q000235:genetics; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D014734:Vibrio cholerae / Q000235:genetics / Q000302:isolation & purification*
D004269:DNA, Bacterial; D002772:Cholera Toxin