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Stoop B,
Lehner A,
Iversen C,
Fanning S,
Stephan R,
( 2009 ) Development and evaluation of rpoB based PCR systems to differentiate the six proposed species within the genus Cronobacter. PMID : 19467725 : DOI : 10.1016/j.ijfoodmicro.2009.04.023 Abstract >>
Although there are various PCR based methods described in the literature to detect the genus Cronobacter, none of these methods is able to differentiate the six proposed species within this genus. A species differentiation is important for epidemiological studies. Moreover, the different species show differences in sensitivity to chemical agents and antibiotics. Here we report for the first time different rpoB based PCR systems which enable the identification to species level of strains previously confirmed to belong to the genus Cronobacter. Different primer pairs based on the rpoB sequences of the six Cronobacter species type strains were designed. Thereafter, 57 target and non-target strains, previously described in the Cronobacter taxonomy paper, were included for the specificity evaluation. C. turicensis, C. muytjensii, C. dublinensis and C. genomospecies 1 can be reliably identified by the proposed single primer pairs. Only target strains showed a correctly sized amplification product, whereas no amplification product was obtained for all non-target strains used in this study (100% specificity). However, as the rpoB gene sequences of C. sakazakii and C. malonaticus are closely related, a two step procedure is necessary. We therefore recommend a two-step procedure in which the primer pairs Cmalf/Cmalr are used in a follow up PCR on strains that are found to be positive in the amplification with the C. sakazakii specific primers Csakf/Csakr.
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Chen W,
Ai L,
Yang J,
Ren J,
Li Y,
Guo B,
( 2013 ) Development of a PCR assay for rapid detection of Cronobacter spp. from food. PMID : 24102218 : DOI : 10.1139/cjm-2013-0243 Abstract >>
The occurrence of outbreaks of necrotizing meningitis caused by Cronobacter spp. in neonates highlights the need for rapid detection and accurate identification of this pathogenic species. The gold standard for isolation and identification of Cronobacter spp. from powdered infant formula is time consuming and labor intensive. The gyrB gene that encodes the B subunit of DNA gyrase (topoisomerase type II) was found to be suitable for the identification of Cronobacter spp. A region of the gyrB gene of 38 Cronobacter spp. strains and 5 Enterobacter spp. strains was amplified and sequenced, and a pair of primers was designed and synthesized based on the sequence of the gyrB gene. A polymerase chain reaction (PCR) system was developed and optimized to detect Cronobacter spp. The PCR assay amplified a 438 bp DNA product from all 38 Cronobacter spp. strains tested but not from 34 other bacteria. The detection limit was 1.41 pg/PCR (equivalent 282 genomic copies) when the genomic DNA of Cronobacter sakazakii ATCC 29544 was 10-fold diluted. Infant formula powders from 3 different commercial brands were inoculated with strains ATCC 29544 at a level of 56 colony-forming units, and the target fragment were produced after samples were enriched for 6 h at 37 �XC. Twenty-five food samples were evaluated by the PCR assay and the conventional method. A PCR product of the expected size was obtained from 3 samples; however, Cronobacter spp. strains were isolated from only 2 samples by the conventional method. This method is a useful tool for rapid identification of Cronobacter spp. in food and potentially environmental samples.
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( 2013 ) Use of novel species-specific PCR primers targeted to DNA gyrase subunit B (gyrB) gene for species identification of the Cronobacter sakazakii and Cronobacter dublinensis. PMID : 22963906 : DOI : 10.1016/j.mcp.2012.08.004 Abstract >>
Cronobacter sakazakii and its phylogenetically closest species are considered to be an opportunistic pathogens associated with food-borne disease in neonates and infants. Neither phenotypic nor genotypic (16S ribosomal DNA sequence analysis) techniques can provide sufficient resolutions for accurately and rapidly identification of these species. The objective of this study was to develop species-specific PCR based on the gyrB gene sequence for direct species identification of the C. sakazakii and Cronobacter dublinensis within the C. sakazakii group. Two pair of species-specific primers were designed and used to specifically identify C. sakazakii and C. dublinensis, but none of the other C. sakazakii group strains. Our data indicate that the novel species-specific primers could be used to rapidly and accurately identify the species of C. sakazakii and C. dublinensis from C. sakazakii group by the PCR based assays.
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