Rapid detection of Enterobacter sakazakii using TaqMan real-time PCR assay.
Enterobacter sakazakii is an emerging food pathogen, which induces severe meningitis and sepsis in neonates and infants, with a high fatality rate. The disease is generally associated with the ingestion of contaminated infant formula. In this study, we describe the development of a real-time PCR protocol to identify E. sakazakii using a TaqMan probe, predicated on the nucleotide sequence data of the 16S rRNA gene obtained from a variety of pathogens. To detect E. sakazakii, four primer sets and one probe were designed. Five strains of E. sakazakii and 28 non-E. sakazakii bacterial strains were used in order to ensure the accuracy of detection. The PCR protocol successfully identified all of the E. sakazakii strains, whereas the 28 non-E. sakazakii strains were not detected by this method. The detection limits of this method for E. sakazakii cells and purified genomic DNA were 2.3 CFU/assay and 100 fg/assay, respectively. These findings suggest that our newly developed TaqMan real-time PCR method should prove to be a rapid, sensitive, and quantitative method for the detection of E. sakazakii.
Department of Food Science and Technology, Dongguk University, Seoul 100-715, Korea.
Sil Eun
SE
Kang
Yong Suk
YS
Nam
Kwang Won
KW
Hong
D015373:Bacterial Typing Techniques; D015169:Colony Count, Microbial; D044083:Cronobacter sakazakii / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis; D005516:Food Microbiology; D006801:Humans; D007225:Infant Food / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D019914:Taq Polymerase / Q000378:metabolism*; D013997:Time Factors
D015342:DNA Probes; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D019914:Taq Polymerase