An improved template preparation technique for PCR assay for detection of Enterobacter sakazakii in infant formula

摘要

In the present study, an assay using polymerase chain reaction (PCR) was developed for detecting Enterobacter sakazakii in infant formula. Based on solvent extraction technology, FTA filter was used to extract E.sakazakii DNA from artificially contaminated infant formula. The FTA paper coat was able to efficiently remove inhibitors that could affect the PCR reaction. Primers targeting the 16S rRNA gene were used to amplify a 149 bp DNA fragment, which was confirmed by DNA sequencing. Experiments to determine the sensitivity of the PCR indicated that it could detect as few as 7×102 CFU/ml of E. sakazakii bacteria in infant formula directly and 7×100 CFU/ml after a 4-h enrichment step. This novel FTA-PCR assay allows for detection of E.sakazakii in infant formula in < 6 h, this is a substantial improvement over the conventional PCR with enrichment method which requires 7days. Thus, PCR amplification using FTA filters provides a faster and more sensitive method of E.sakazakii detection than the standard cultivation method.