An evaluation of immunomagnetic separation-real-time PCR (IMS-RTiPCR) combined assay for rapid and specific detection of Escherichia coli O157:H7 in raw milk and ground beef

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Abstract

Abstract The aim of this study is to optimize a rapid and specific method for detection of E. coli O157:H7 (EHEC) from food samples with high background flora using an immunomagnetic separation-real-time polymerase chain reaction assay (IMS-RTiPCR). For this, EHEC cells were recovered from raw milk and ground meat samples that were artificially contaminated to the final concentrations of 101, 103 and 105 cfu EHEC/mL or g, after a non-selective pre-enrichment at 35 °C for 8 h following either with or without capturing by micro-sized beads. Then, EHEC cells were identified by RTiPCR. The study was also carried out without any enrichment to evaluate the enrichment efficiency of the assay. By comparing the assay with and without pre-enrichment, we showed that even the usage of specific micro-sized beads did not improve EHEC detection in raw milk and ground beef samples and so gave false negative results unless an 8-h pre-enrichment was applied. Besides, the assay can be completed in less than 9 h with a minimum detection limit of 103 cfu EHEC/mL or g in foods and hence was found to be simple routine-based method that exhibits great potential for implementing as a rapid screening of EHEC in microbial tests as compared to existing techniques.

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Most cited references 35

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Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters.

Jessica Nordstrom, Michael Vickery, George M Blackstone … (2007)

Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected 10(4) CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh(+) and trh(+) strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.

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Rapid detection of Listeria monocytogenes by nanoparticle-based immunomagnetic separation and real-time PCR.

Hua Yang, Liangwei Qu, Adrienne N Wimbrow … (2007)

The objective of this study was to develop a method combining nanoparticle-based immunomagnetic separation (IMS) with real-time PCR for a rapid and quantitative detection of Listeria monocytogenes. Carboxyl modified magnetic nanoparticles were covalently bound with rabbit anti-L. monocytogenes via the amine groups. Several factors, such as the amount of immunomagnetic nanoparticles (IMNPs), reaction and collection times, and washing step, were optimized, and the nanoparticle-based IMS in combination with real-time PCR was further evaluated for detecting L. monocytogenes from artificially contaminated milk. The cell numbers calculated from the means of threshold cycles (CT) of PCR amplification curves were compared to those from plate counts in order to determine the correspondence degree of quantitative data. The capture efficiency (CE) by plating from IMNP-based IMS was 1.4 to 26 times higher than those of Dynabeads-based IMS depending on the initial cell concentrations inoculated into milk samples. When combined with real-time PCR, L. monocytogenes DNA was detected in milk samples with L. monocytogenes >or=10(2) CFU/0.5 ml. In the range of 10(3) to 10(7)L. monocytogenes CFU/0.5 ml, cell numbers calculated from CT values were 1.5 to 7 times higher than those derived from plate counts. Our results demonstrated that both the use of nanoparticles and the choice of anti-L. monocytogenes in our IMNP-based IMS in combination with real-time PCR has improved the sensitivity of L. monocytogenes detection from both nutrient broth and milk samples.

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Author and article information

Contributors

Birce MERCANOGLU TABAN: Role: ND

Sait Aykut AYTAC: Role: ND

Journal

Journal ID (publisher-id): cta

Title: Food Science and Technology

Abbreviated Title: Food Sci. Technol

Publisher: Sociedade Brasileira de Ciência e Tecnologia de Alimentos (Campinas, SP, Brazil )

ISSN (Print): 0101-2061

ISSN (Electronic): 1678-457X

Publication date (Print and electronic): December 2019

Volume: 39

Issue: 4

Pages: 955-961

Affiliations

[02] Beytepe orgnameHacettepe University orgdiv1Faculty of Engineering orgdiv2Food Engineering Department Turkey

[01] Diskapi orgnameAnkara University orgdiv1Faculty of Agriculture orgdiv2Dairy Technology Department Turkey

Article

Publisher ID: S0101-20612019000400955

DOI: 10.1590/fst.15818

SO-VID: 8241fa65-524b-4012-a65f-bc33767194bd

License:

This work is licensed under a Creative Commons Attribution 4.0 International License.

History

Date accepted : 04 March 2019

Date received : 08 May 2018

Page count

Figures: 0, Tables: 0, Equations: 0, References: 40, Pages: 7

Product

SciELO Brazil