Aptamer-based detection of Salmonella enteritidis using double signal amplification by Klenow fragment and dual fluorescence
- 602 Downloads
This article describes a sensitive and selective fluorometric method for the determination of Salmonella enteritidis by exploiting the polymerase activity of the Klenow fragment and dual fluorescence. First, one end of a target-selective aptamer was labeled with the fluorophore 6-carboxyfluorescein (FAM). Once the labeled aptamer binds to graphene oxide (GO) via π-stacking interaction, the fluorescence of FAM is quenched. However, the addition of target (16S rRNA) leads to the restoration of fluorescence due to the binding of probe and target which shifts the FAM fluorophore away from the quenching GO. By using the Klenow fragment and by exploiting the synergistic effect of FAM and the DNA probe SYBR Green I (which is strongly fluorescent in presence of dsDNA only), fluorescence is strongly amplified and sensitivity improved. The analyte 16SrRNA can be determined by this method in the 60 pM to 100 nM concentration range, and the detection limit is 60 pM. It is also shown that Salmonella enteritidis can be determined in milk samples by this method in concentrations between 102 to 105 cfu⋅mL‾1, with a detection limit of 300 cfu⋅mL‾1. This assay displays high sensitivity and selectivity and may possess wide applications in pathogen detection.
KeywordsFAM SYBR green I Graphene oxide Foodborne pathogen 16S rRNA
This work was financially supported by the National Natural Science Foundation (81271660), Doctoral Program of Higher Education from the Ministry of Education (20114306110006).
- 2.Pui CF, Wong WC, Chai LC, Tunung R, Jeyaletchumi P, Noor Hidayah MS, Ubong A, Farinazleen MG, Cheah YK, Son R (2011) Salmonella: a foodborne pathogen. International Food Research Journal 18:465–473Google Scholar
- 3.Herikstad H, Motarjemi Y, Tauxe RV (2002) Salmonella surveillance: a global survey of public health serotyping. Epidemiol Infect 129:1–8Google Scholar
- 9.Lungu B, Waltman WD, Berghaus RD, Hofacre CL (2012) Comparison of a real-time PCR method with a culture method for the detection of Salmonella enterica serotype enteritidis in naturally contaminated environmental samples from integrated poultry houses. J Food Prot Apr. 75(4):743–747. doi: 10.4315/0362-028X.JFP-11-297
- 10.Sokolov DM, Sokolov MS (2013) Rapid methods for the genus salmonella bacteria detection in food and raw materials. Vopr Pitan 82:33–40Google Scholar
- 11.Zhi Y, Sasai D, Okubo Y, Shinozaki M, Nakayama H, Yamagata Murayama S, Wakayama M, Ide T, Zhang Z, Shibuya K (2013) Comparison between the effectiveness of polymerase chain reaction and in situ hybridization in detecting the presence of pathogenic fungi by using the preserved DNA in formalin-fixed and paraffin-embedded tissues. Jpn J Infect Dis 66:173–179CrossRefGoogle Scholar
- 15.Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925Google Scholar
- 23.Cordeiro M, Giestas L, Lima JC, Baptista PJ (2013) Coupling an universal primer to SBE combined spectral codification strategy for single nucleotide polymorphism analysis. Biotechnol. 168(1):90–94Google Scholar