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基于sfmD基因，通过动力学5'-核酸酶聚合酶链反应（实时PCR）对大肠杆菌进行定量分析。

Quantification of Escherichia coli by kinetic 5'-nuclease polymerase chain reaction (real-time PCR) oriented to sfmD gene.

作者信息

Kaclíková E, Pangallo D, Oravcová K, Drahovská H, Kuchta T

机构信息

Department of Microbiology and Molecular Biology, Food Research Institute, Bratislava, Slovak Republic.

出版信息

Lett Appl Microbiol. 2005;41(2):132-5. doi: 10.1111/j.1472-765X.2005.01736.x.

DOI:10.1111/j.1472-765X.2005.01736.x

PMID:16033509

Abstract

AIMS

A kinetic 5'-nuclease polymerase chain reaction (real-time PCR) for the quantification of Escherichia coli was developed.

METHODS AND RESULTS

Specific primers and a fluorogenic probe oriented to sfmD gene, encoding a putative outer membrane export usher protein, were designed. The PCR system was highly specific and sensitive for E. coli, as determined with 37 non-E. coli strains (exclusivity, 100%) and 24 E. coli strains (inclusivity, 100%). When used in real-time PCR, linear calibration lines were obtained in the range from 10(2) to 10(8) CFU ml(-1) for three E. coli strains. Salmonella Enteritidis (10(6) CFU ml(-1)) or Citrobacter freundii (10(6) CFU ml(1)) had no effect on quantification of E. coli by the method.

CONCLUSIONS

The developed real-time PCR is suitable for rapid quantification of E. coli.

SIGNIFICANCE AND IMPACT OF THE STUDY

In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications.

摘要

目的

开发一种用于定量检测大肠杆菌的动力学5'-核酸酶聚合酶链反应（实时荧光定量PCR）。

方法与结果

设计了针对编码假定外膜输出引导蛋白的sfmD基因的特异性引物和荧光探针。用37株非大肠杆菌菌株（特异性为100%）和24株大肠杆菌菌株（包容性为100%）检测，该PCR系统对大肠杆菌具有高度的特异性和敏感性。在实时荧光定量PCR中，对三株大肠杆菌菌株在10²至10⁸CFU ml⁻¹范围内获得了线性校准曲线。肠炎沙门氏菌（10⁶CFU ml⁻¹）或弗氏柠檬酸杆菌（10⁶CFU ml⁻¹）对该方法定量检测大肠杆菌没有影响。