Establishment of a triplex real-time PCR for the detection of cholera toxin gene ctx and heat labile enterotoxin gene elt.

Jie LI; Biao Kan; Jingyun Zhang

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Establishment of a triplex real-time PCR for the detection of cholera toxin gene ctx and heat labile enterotoxin gene elt.

Author: Jie LI ¹ ; Biao KAN ¹ ; Jingyun ZHANG ²
Author Information

1. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
2. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China. Email: zhangjingyun@icdc.cn.
Publication Type:Journal Article
MeSH: Cholera Toxin; genetics; Enterotoxigenic Escherichia coli; genetics; Enterotoxins; genetics; Real-Time Polymerase Chain Reaction; methods; Sensitivity and Specificity
From: Chinese Journal of Epidemiology 2014;35(6):720-723
CountryChina
Language:Chinese
Abstract:
OBJECTIVETo establish a triplex TaqMan real-time PCR system containing internal amplification control (IAC) to detect cholera toxin gene ctxA and enterotoxigenic Escherichia coli (ETEC)heat-labile enterotoxin gene elt.
METHODSPrimers and probes were designed based on the sequences of ctxA, elt and IAC. Both sensitivity and specificity were analyzed and interactions between different reactions were evaluated.
RESULTSThis system showed that the sensitivity of ctxA was 94 copies/reaction while the elt 79 copies/reaction and the amplification efficiency were 94.7% and 98.1%, respectively. Under the ratio of copy numbers on gene ctxA to elt as between 1 : 1-1 : 10, when both targets were detected, with impact was less on each other. However, when the amount of elt or ctxA was 100 times of IAC, the amplification of IAC was significantly inhibited.
CONCLUSIONThis system showed both satisfactory sensitivity and specificity, thus could be used to detect pathogenic bacteria in diarrhea stools. The detection of IAC could prompt the presence of PCR inhibitors in samples being tested.