[Development and application of real-time polymerase chain reaction to detect Vibrio cholerae O1 and O139 in river water].
OBJECTIVE
To develop a real-time SYBR Green polymerase chain reaction (PCR) for detection of Vibrio cholerae serogroups O1 and O139, and to evaluate its reliability through detection of estuary water samples.


METHODS
O antigen rfb genes specific for O1 and O139 were used for the design of PCR primers. The real-time SYBR Green PCR system in detecting O1 and O139 specific rfb genes in one tube was developed, and its sensitivity, specificity and reproducibility were evaluated. The ability of the real-time PCR in detection of estuary water samples was compared with the routine PCR and bacteria isolation.


RESULTS
The amplification of O1 or O139 specific target gene could be detected according to the melt curve temperature of amplicons. No amplification was observed in the templates of other 10 non-cholerae vibrios. When comparing to the real-time PCR to bacteria isolation in detection of 524 estuary water samples, it showed high sensitivity, plus also positive in real-time PCR detection among all the samples in which bacteria of O1 or O139 were isolated.


CONCLUSIONS
The real-time SYBR Green PCR could be used as the first step of rapid environment screen of V. cholerae in water samples thus might enhance the efficiency of isolation in screening of large amount of water samples.
Wang
Xiao-Mei
XM
State Key Laboratory for Infectious Disease Prevention and Control, National Institute of Communicable Disease Control & Prevention, Chinese Center of Disease Control and Prevention, Beijing 102206, China.
Wang
Duo-Chun
DC
Tan
Hai-Ling
HL
Zhong
Hao-Jie
HJ
Chen
Jing-Diao
JD
Li
Bai-Sheng
BS
Ke
Chang-Wen
CW
Yan
Mei-Ying
MY
Zhang
Jing
J
Kan
Biao
B
D004784:Environmental Monitoring / Q000379:methods; D005798:Genes, Bacterial; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D045483:Rivers / Q000382:microbiology*; D012680:Sensitivity and Specificity; D041241:Vibrio cholerae O1 / Q000302:isolation & purification*; D041242:Vibrio cholerae O139 / Q000302:isolation & purification*