Characterization of the gene encoding the 16S rRNA of Enterobacter sakazakii and development of a species-specific PCR method

Hassan, Abdulwahed Ahmed; Akineden, Ömer; Kress, Claudia; Estuningsih, Sri; Schneider, Elisabeth; Usleber, Ewald

doi:10.1016/j.ijfoodmicro.2006.12.011

Cited by 53 publications

(30 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Several targets have been described for PCR detection of Cronobacter genus, including 16S ribosomal (r)DNA (Iversen et al, 2004), 16S-23S rDNA internal transcribed spacer (Liu et al, 2006), 23S rDNA (Derzelle et al, 2007), transfer (t)RNAGlu (Hassan et al, 2007), dnaG (Seo and Brackett, 2005), ompA (Mohan Nair and Venkitanarayanan, 2006), gluA (Iversen et al, 2007), wehC, wehI , wzx (Jarvis et al, 2011), zpx (Jaradat et al, 2009), and others.…”

Section: Introductionmentioning

confidence: 99%

Mining for sensitive and reliable species-specific primers for PCR for detection of Cronobacter sakazakii by a bioinformatics approach

Chen

Tao

Bie

et al. 2015

Journal of Dairy Science

View full text Add to dashboard Cite

Although several studies have reported PCR assays for distinguishing Cronobacter sakazakii from other species in the genus, reports regarding assay sensitivity and specificity, as well as applications for food testing, are lacking. Hence, the objective of this study was to develop a sensitive and reliable PCR-based method for detection of C. sakazakii by screening for specific target genes. The genome sequence of C. sakazakii in the GenBank database was compared with that of other organisms using BLAST. Thirty-eight DNA fragments unique to C. sakazakii were identified, and primers targeting these sequences were designed. Finally, 3 primer sets (CS14, CS21, and CS38) were found to be specific for C. sakazakii by PCR verification. The detection limit of PCR assays using the 3 pairs of primers was 1.35 pg/μL, 135 fg/μL, and 135 fg/μL, respectively, for genomic DNA, and 5.5×10(5), 5.5×10(3), 5.5×10(3) cfu/mL, respectively, using pure cultures of the bacteria, compared with 13.5 pg/μLand 5.5×10(5) cfu/mLfor primer set SpeCronsaka, which has been previously described. Cronobacter sakazakii were detected in artificially contaminated powdered infant formula (PIF) by PCR using primer sets CS21 and CS38 after 8h of enrichment. The detection limit was 5.5×10(-1) cfu/10g of PIF. Thus, the PCR assay can be used for rapid and sensitive detection of C. sakazakii in PIF.

show abstract

Section: Introductionmentioning

confidence: 99%

Mining for sensitive and reliable species-specific primers for PCR for detection of Cronobacter sakazakii by a bioinformatics approach

Chen

Tao

Bie

et al. 2015

Journal of Dairy Science

View full text Add to dashboard Cite

show abstract

“…Recently, a number of molecular-based techniques for the rapid detection of Cronobacter spp. have been reported, including PCR assays (12)(13)(14), the DuPont Qualicon BAX system (15), real-time PCR (5,8,11), and oligonucleotide arrays (14). Real-time PCR assays using the TaqMan probe have been increasingly exploited, and these assays have replaced ordinary PCR methods in many cases.…”

Section: Resultsmentioning

confidence: 99%

Real-time PCR targeting OmpA gene for detection of Cronobacter spp. in powdered infant formula

Dong¹,

Wu²,

Wu³

et al. 2013

Food Sci Biotechnol

View full text Add to dashboard Cite

Cronobacter spp. (formerly Enterobacter sakazakii), a pathogen commonly found in powdered infant formula (PIF), is a rare cause of invasive infection with a high mortality rate in neonates. In the present study, a realtime PCR assay was conducted to identify the pathogens in PIF using a TaqMan probe targeting the outer membrane protein A gene (ompA) of Cronobacter spp. The specificity of the PCR assay was tested against 25 strains of Cronobacter spp. and 38 non-Cronobacter bacterial species. The detection limits of this method are 1.0×10 2 copy/µL in standard plasmid, 1.1 CFU/100 g in PIF through 38 h of enrichment, and 2.8×102 CFU/mL in phosphate-buffered saline (pH 7.0). Based on the detection limits, real-time PCR is more sensitive than simplex and duplex PCR. These methods were successfully applied to actual samples, indicating that this real-time PCR assay can be used for the detection of Cronobacter spp. in PIF.

show abstract

“…(Iversen et al, 2007;Jaradat et al, 2009). However, these studies have not given consistent results highlighting the need for other methods of confi rmation such as PCR analysis and 16S rDNA sequencing (Lehner et al, 2006;Hassan et al, 2007).…”

Section: Resultsmentioning

confidence: 99%

Isolation and characterization of Cronobacter spp. from environmental and food resources

Putthana¹,

Marounek²,

Břeňová³

et al. 2012

Agricultura Tropica Et Subtropica

View full text Add to dashboard Cite

Cronobacter spp. (formerly Enterobacter sakazakii) has been isolated from a wide range of environmental and several food sources. Cronobacter spp. is an opportunistic pathogen causing serious infection in infants, particularly neonates. The aim of this study was to isolate and characterize Cronobacter spp. from food sources (infant food, herbs and spices and vegetables) and from environmental sources as dust from vacuum cleaners. Isolation of Cronobacter spp. was performed on selective chromogenic agars, fi rstly using commercial ESIA agar and thereafter on Kim and Rhee-KR agar described in the literature. Phenotypic characteristics were obtained by commercial miniaturized biochemical ENTEROTEST 24 kits and the fi nal confi rmation of isolated strains was performed by molecular techniques (PCR, PCR -DGGE analysis, and 16S rDNA sequencing). Altogether, 99 samples were analyzed (47 samples of foods and 52 samples of dust). In total, 43 isolates of presumptive Cronobacter spp. were initially identifi ed, however, only 22 isolates (51%) were identifi ed as Cronobacter spp. with high identity scores (75-99%). The occurrence of presumptive cronobacters in environmental samples was signifi cantly higher than in samples of food (18 out of 52 vs. 4 out of 47; P = 0.003). No cronobacters were found in 17 samples of infant food, 3 isolates originated from herbs and spices, 1 isolate from spinach and 18 isolates from samples of dust (households, restaurants, dormitory rooms). It can be concluded that Cronobacter spp. is a ubiquitous pathogen contaminating food and environment. Cronobacter spp. could be well identifi ed by means of ENTERO24 test kits with high probability. Both phenotypic and genotypic methodology could be used for identifi cation of Cronobacter spp. and they can be combined for reliable identifi cation.

show abstract

Characterization of the gene encoding the 16S rRNA of Enterobacter sakazakii and development of a species-specific PCR method

Cited by 53 publications

References 38 publications

Mining for sensitive and reliable species-specific primers for PCR for detection of Cronobacter sakazakii by a bioinformatics approach

Mining for sensitive and reliable species-specific primers for PCR for detection of Cronobacter sakazakii by a bioinformatics approach

Real-time PCR targeting OmpA gene for detection of Cronobacter spp. in powdered infant formula

Isolation and characterization of Cronobacter spp. from environmental and food resources

Contact Info

Product

Resources

About