Monitoring Pathogenic Viable E. coli O157:H7 in Food Matrices Based on the Detection of RNA Using Isothermal Amplification and a Paper-Based Platform

Author(s):  
Sabrina Petrucci ◽  
Connor Costa ◽  
David Broyles ◽  
Avinash Kaur ◽  
Emre Dikici ◽  
...  
TECHNOLOGY ◽  
2016 ◽  
Vol 04 (03) ◽  
pp. 194-200 ◽  
Author(s):  
Evangelyn C. Alocilja ◽  
Parul Jain ◽  
Kasey Pryg

An antibody-based sensor (immunosensor) has been developed that features an all-in-one extraction and detection of the enteric pathogen E. coli O57 : H7 using the same redox-active polyaniline-coated iron oxide nanoparticles (PIONs). Capture efficiency for E. coli O57 : H7 is shown to be 81–99% in various food matrices with varying properties. The immunosensor's detection range is 100 to 105 CFU/mL with a detection limit of 5 CFU. Furthermore, magnetic separation is showing great promise as an alternative to existing conventional sample processing systems. Given the bacteria's low contamination level in food and water, this biosensor technology has great potential in monitoring enteric microbial contaminants in the food supply chain where simplicity, sensitivity, and ease of use are important.


2014 ◽  
Vol 618 ◽  
pp. 293-297 ◽  
Author(s):  
Yang Deng ◽  
Li Li Ji ◽  
Lin Li ◽  
Bing Li ◽  
Jian Yu Su

Escherichia coliO157,Psuedomonas aeruginosa,Salmonella,Vibrio parahaemolyticusandListeriaare important pathogens for human. With increased awareness in public health, development of a rapid, sensitive, cost-effective and easy-operating bacteriological detection is of the utmost importance and urgent necessity. In this study, we developed and applied a simple amplification kit based on loop-mediated isothermal amplification (LAMP) methods for rapid detection of various pathogens includingEscherichia coliO157,Psuedomonas aeruginosa,Salmonella,Vibrio parahaemolyticusandListeria, as well as related virulence. Nine targets, includingrfbE(E. coli-specific),stx1 (coding for Shiga toxin 1),stx2 (coding for Shiga toxin 2),oprI(P. aeruginosa–specific),invA(Salmonella-specific),hlyA(Listeria-specific),tlh(coding for thermolable haemolysin),tdh(coding for thermostable direct haemolysin) andtrh(coding for TDH-related haemolysin), were selected for identification forEscherichia coliO157,Psuedomonas aeruginosa,Salmonella,Vibrio parahaemolyticusandListeria, as well as related virulence.. Six primers, including outer primers, inner primers and loop primers, were specially designed for recognizing eight distinct sequences on the targets. Three solutions labeled A, B and C was included in the kit. The experiment were carried out in a total of 25 μl reaction mixture: solution A containing 1.6 μM (each) of the primers FIP and BIP, 0.2 μM (each) of the primers F3 and B3, 0.8 μM (each) of primers LF and LB; solution B containing 1.6 mM of deoxynucleoside triphosphates, 6 mM MgSO4, 1 M betain (Sigma, St. Louis, MO, USA), 1 X thermopol buffer (New England Biolabs, Ipswich, MA, USA); solution C containing BST polymerase. Twenty-two reference strains, including various species of gram-negative and-positive isolates, were included in this study to evaluate and optimize LAMP assays. Application of the optimized LAMP assays was performed on a total of 200 strains (with 40 strains for each species of the pahtogens). The optimal reaction condition was found to be 65°C for 45 min. Application of the kit assays were performed on various types of pathogens, the sensitivity for the 9 targets was found to be 100%; with a 100% specificity and positive predictive value (PPV) for all the 9 targets targets. In conclusion, the isothermal amplification kits were demonstrated to be useful and powerful tools for rapid differentiation of various pathogens (includingEscherichia coliO157,Psuedomonas aeruginosa,Salmonella,Vibrio parahaemolyticusandListeria), and undoubtedly, the rapidness, easiness and cost-effectiveness of LAMP assay will aid in the broad application of bacteriological detection of common pathogens.


Sensors ◽  
2021 ◽  
Vol 21 (5) ◽  
pp. 1749
Author(s):  
Alejandra Ben Aissa ◽  
Narayanan Madaboosi ◽  
Mats Nilsson ◽  
Maria Isabel Pividori

Isothermal amplification techniques are emerging nowadays for the rapid and accurate detection of pathogenic bacteria in low resource settings, where many infectious diseases are endemic, and the lack of reliable power supply, trained personnel and specialized facilities pose critical barriers for timely diagnosis. This work addresses the detection of E. coli based on DNA isothermal amplification performed on magnetic particles (MPs) followed by electrochemical genosensing on disposable electrodes by square-wave voltammetry. In this approach, the bacterial DNA is preconcentrated using a target-specific magnetic probe and then amplified on the MPs by rolling circle amplification (RCA). Two different electrochemical readout methods for the RCA amplicons are tested. The first one relied on the labelling of the magnetic RCA product with a digoxigenin probe followed by the incubation with antiDIG-HRP antibody as electrochemical reporter. In the second case, the direct detection with an HRP-probe was performed. This latter strategy showed an improved analytical performance, while simultaneously avoiding the use of thermocyclers or bulky bench top equipment.


2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Cindy Shuan Ju Teh ◽  
Kek Heng Chua ◽  
Yvonne Ai Lian Lim ◽  
Soo Ching Lee ◽  
Kwai Lin Thong

We have successfully developed a Loop-mediated isothermal amplification(LAMP) assay that could specifically detect genericEscherichia coli(E. coli). This assay was tested on 85 bacterial strains and successfully identified 54E. colistrains (average threshold time, Tt = 21.26). The sensitivity of this assay was evaluated on serial dilutions of bacterial cultures and spiked faeces. The assay could detect 102 CFU/mL for bacterial culture with Tt = 33.30 while the detection limit for spiked faeces was 103 CFU/mL (Tt = 31.12). We have also detected 46 genericE. colifrom 50 faecal samples obtained from indigenous individuals with 16% of the positive samples being verocytotoxin-producingE. coli(VTEC) positive. VT1/VT2 allele was present in one faecal sample while the ratio of VT1 to VT2 was 6 : 1. Overall, our study had demonstrated high risk of VTEC infection among the indigenous community and most of the asymptomatic infection occurred among those aged below 15 years. The role of asymptomatic human carriers as a source of dissemination should not be underestimated. Large scale screening of the VTEC infection among indigenous populations and the potential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay.


Author(s):  
Tanis McMahon ◽  
Jillian Bastian ◽  
Inas Alshawa ◽  
Alexander Gill

Verotoxin-producing Escherichia coli (VTEC; also known as Shiga toxin-producing E. coli ) are a significant cause of foodborne illnesses around the world. Due to the serological and genomic diversity of VTEC, methods of detection for VTEC in food samples require detection of verotoxin or its gene vt (also known as stx ). The current taxonomy of vt identifies three vt1 (a,c,d) and seven vt2 (a to g) subtypes. PCR detection of vt is convenient and rapid, but protocols may not detect all currently identified variants or subtypes of vt . The Health Canada Compendium of Analytical Methods for the analysis of food for VTEC is MFLP-52. MFLP-52 includes a VT Screening PCR that is used to determine the presumptive presence of VTEC by the detection of vt in food enrichments, and to differentiate VTEC from other isolates. The VT Screening PCR was developed prior to the establishment of the current vt taxonomy. An evaluation of VT Screening PCR for detection of the ten established vt -subtypes was performed and it was discovered that the method could not detect subtypes vt1d and vt2f . Additional primers and a modified protocol were developed and the modified VT Screening PCR was tested against an inclusivity panel of 50 VTEC strains, including representatives of ten vt -subtypes, and an exclusivity panel of 30 vt negative E. coli from various sources, to ensure specificity. The reliability of MFLP-52 with the modified VT Screening PCR was assessed by analysis of four priority food matrices (ground beef, lettuce, cheese and apple cider) inoculated with a VTEC strain at 2 to 5 CFU per 25 g. The modified VT Screening PCR was determined to be able to detect all ten vt -subtypes and reliably detect the presence of VTEC in all tested food enrichments.


2018 ◽  
Vol 8 (4) ◽  
pp. 490-496
Author(s):  
P. Baraily ◽  
R. J. Zende ◽  
D. P. Kshirsagar ◽  
V. M. Vaidya ◽  
R. N. Waghamare ◽  
...  

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Lu Han ◽  
Kaidi Wang ◽  
Lina Ma ◽  
Pascal Delaquis ◽  
Susan Bach ◽  
...  

ABSTRACT Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the “viable but nonculturable” (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting the wzy gene of E. coli O157:H7 and the agfA gene of S. enterica and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture and were 5.13 × 103 or 5.13 × 104 CFU/g for VBNC E. coli O157:H7 and 1.05 × 104 or 1.05 × 105 CFU/g for VBNC S. enterica in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stress conditions and to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive, and specific way to determine levels of VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the preharvest and postharvest stages of fresh produce.


Foods ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1741
Author(s):  
Stefanie M. Allgöwer ◽  
Chris A. Hartmann ◽  
Clarissa Lipinski ◽  
Vera Mahler ◽  
Stefanie Randow ◽  
...  

Soybean (Glycine max) allergy can be life threatening. A lack of causative immunotherapy of soybean allergy makes soybean avoidance indispensable. Detection methods are essential to verify allergen labeling and unintentional allergen cross contact during food manufacture. Here, we aimed at evaluating our previously described primers for loop-mediated isothermal amplification (LAMP) of multicopy gene ORF160b, combined with a lateral flow dipstick (LFD)-like detection, for their performance of soybean detection in complex food matrices. The results were compared with those obtained using quantitative real-time Polymerase Chain Reaction (qPCR) as the current standard of DNA-based allergen detection, and antibody-based commercial lateral flow device (LFD) as the current reference of protein-based rapid allergen detection. LAMP-LFD allowed unequivocal and reproducible detection of 10 mg/kg soybean incurred in three representative matrices (boiled sausage, chocolate, instant tomato soup), while clear visibility of positive test lines of two commercial LFD tests was between 10 and 102 mg/kg and depending on the matrix. Sensitivity of soybean detection in incurred food matrices, commercial retail samples, as well as various processed soybean products was comparable between LAMP-LFD and qPCR. The DNA-based LAMP-LFD proved to be a simple and low-technology soybean detection tool, showing sensitivity and specificity that is comparable or superior to the investigated commercial protein-based LFD.


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