Systematic loop-mediated isothermal amplification assays for rapid detection and characterization of Salmonella spp., Enteritidis and Typhimurium in food samples

Garrido‐Maestu, Alejandro; Fuciños, Pablo; Azinheiro, Sarah; Carvalho, Joana; Prado, Marta

doi:10.1016/j.foodcont.2017.05.011

Cited by 41 publications

(20 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Actually, the plate counts indicated that 2 cfu/25 g could be reliably detected. This result compares favorably with other molecular biology‐based methods previously published for this and/or other bacterial pathogens (Fachmann et al., ; Garrido‐Maestu et al., ; Garrido‐Maestu, Fuciños, Azinheiro, Carvalho, & Prado, ). The RLOD was determined to be 1.6, with a z‐Test of 0.975 (<2).…”

Section: Discussionsupporting

confidence: 81%

Combination of Immunomagnetic Separation and Real‐Time Recombinase Polymerase Amplification (IMS‐qRPA) for Specific Detection of Listeria monocytogenes in Smoked Salmon Samples

Garrido‐Maestu

Azinheiro

Carvalho

et al. 2019

Journal of Food Science

Self Cite

View full text Add to dashboard Cite

Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real‐time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real‐time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre‐enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as “almost complete concordance” between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real‐time PCR. Practical Application The methodology described in this study significantly reduces the detection time of L. monocytogenes, when compared with culture‐based methods, and it requires fewer steps than other molecular methods, making it a reliable and more convenient method for routine testing. Finally, the evaluation of the methodology in spiked food samples, confirms its reliability.

show abstract

Section: Discussionsupporting

confidence: 81%

Combination of Immunomagnetic Separation and Real‐Time Recombinase Polymerase Amplification (IMS‐qRPA) for Specific Detection of Listeria monocytogenes in Smoked Salmon Samples

Garrido‐Maestu

Azinheiro

Carvalho

et al. 2019

Journal of Food Science

Self Cite

View full text Add to dashboard Cite

show abstract

“…Salmonella DNA in turbid food samples was successfully detected with a detection limit of 10 copies/mL using the electrochemical platform; however, the optical method could not be used. This demonstrates the reliability of the methods described in this study, which may compete with the previous Salmonella in food detection method (SE, SP, and AC were higher than 97 % ). Additional advantages include a faster, direct identification without DNA isolation (total run time <40 min).…”

Section: Resultsmentioning

confidence: 99%

An Integrated Real‐time Electrochemical LAMP Device for Pathogenic Bacteria Detection in Food

2018

View full text Add to dashboard Cite

A novel, integrated, real‐time electrochemical loop‐mediated isothermal amplification (LAMP) device for pathogen detection in food was developed. The electrochemical LAMP device for detection of the vt gene in E. coli O157 : H7 and invA gene in Salmonella enterica was implemented with DNA intercalating probes‐methylene blue. To improve assay efficiency, the optimal concentration of MB was determined to be 30 μM. The real‐time electrochemical LAMP results of gene serial dilutions were compared to the fluorescence‐based detection method. For E. coli O157 and Salmonella, the assay detection limits were 10 DNA copies and 1 DNA copy, respectively, in a reaction time of less than 30 min. The electrochemical and optical LAMP method analyses were realized with 10 % milk to simulate the turbid environment of real food samples. In real‐time LAMP of Salmonella in a turbid environment, the detection limit of 10 copies without pretreatment was possible using the electrochemical method only. Our electrochemical device was used for bacterial detection in 150 turbid food samples, including juice, milk, and soy milk. This electrochemical device has many advantages, including size, portability, high specificity, and high sensitivity, while also being a user‐friendly device that provides stable signals. Thus, to the best of our knowledge, this new electrochemical device demonstrates the best performance for bacterial detection in the rapid contaminated food detection category.

show abstract

“…typhimurium that had been artificially inoculated into chicken samples at concentrations of 4.5–4.5 × 10 5 CFU/ml with a high specificity. Garrido‐Maestu et al (2017) evaluated a set of LAMP methods for the detection of S . typhimurium using STM4497 as the target gene.…”

Section: Discussionmentioning

confidence: 99%

“…The species‐specific gene STM4497 , which has been used in previous real‐time PCR and Loop‐mediated isothermal amplification (LAMP) detection assays (Garrido‐Maestu et al, 2017; Park et al, 2008) was utilized for the detection of S . typhimurium , whereas sdfI was targeted for the specific detection of S .…”

Section: Methodsmentioning

confidence: 99%

“…Among them, real‐time polymerase chain reaction (PCR) is widely used for the detection of pathogenic bacteria on foods, including poultry products, eggs, fruits, and vegetables (Hyeon & Deng, 2017; Mafu, Pitre, & Sirois, 2009; Rodriguez‐Lazaro et al, 2014), due to the fact that the risk of cross contamination is reduced and the accuracy of detection is improved. Recently, nucleic acid‐based new isothermal molecular technologies are being developed due to its simpler thermoblock at a fixed temperature and with shorter time compared with PCR‐based method (Du, Zhou, Li, & Wang, 2017; Garrido‐Maestu, Fuciños, Azinheiro, Carvalho, & Prado, 2017; Jeyaprakash & Hoy, 2004). Recombinase polymerase amplification (RPA) technique is a DNA amplification technique developed by Piepenburg, Williams, Stemple, and Armes (2006), which employs a recombinase–primer complexes to scan double‐stranded DNA and to facilitate strand exchange at cognate sites.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Detection of Salmonella enteritidis and Salmonella typhimurium in foods using a rapid, multiplex real‐time recombinase polymerase amplification assay

Ren

Man

et al. 2020

Journal of Food Safety

View full text Add to dashboard Cite

Salmonella has been recognized as a major foodborne pathogen for humans and animals. In this study, a multiplex real‐time recombinase polymerase amplification (RPA) was developed for simultaneous detection of Salmonella enterica serovars, Salmonella enteritidis and Salmonella typhimurium, from chicken, eggs, lettuce, and papaya. The reaction was performed for 20 min at 35°C, and the detection limit of the assay was 102 CFU/ml for pure culture. In food application, the limit of detection (LOD) of S. enteritidis and S. typhimurium using multiplex real‐time RPA without enrichment procedure was 102 CFU/25 g, respectively. After enrichment, the LOD of S. enteritidis and S. typhimurium was 10 CFU/25 g. Moreover, the result for Salmonella spp. was not significantly different from those obtained using a culture‐based method. Additionally, the assay has a lower cross‐reactivity with other pathogenic microorganisms and a good stability performance. Thus, the developed multiplex RPA assay could be used as a rapid tool for the detection of S. enteritidis and S. typhimurium in food.

show abstract

Systematic loop-mediated isothermal amplification assays for rapid detection and characterization of Salmonella spp., Enteritidis and Typhimurium in food samples

Cited by 41 publications

References 53 publications

Combination of Immunomagnetic Separation and Real‐Time Recombinase Polymerase Amplification (IMS‐qRPA) for Specific Detection of Listeria monocytogenes in Smoked Salmon Samples

Combination of Immunomagnetic Separation and Real‐Time Recombinase Polymerase Amplification (IMS‐qRPA) for Specific Detection of Listeria monocytogenes in Smoked Salmon Samples

An Integrated Real‐time Electrochemical LAMP Device for Pathogenic Bacteria Detection in Food

Detection of Salmonella enteritidis and Salmonella typhimurium in foods using a rapid, multiplex real‐time recombinase polymerase amplification assay

Contact Info

Product

Resources

About