Name: foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7
Uploaded: 2017-09-17T14:00:00
Description: Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

This work is supported by K-INBRE P20GM103418, Kansas Soybean Commission (KSC/PSU 1663), ACS PRF 56629-UNI7 and PSU polymer chemistry startup fund, all to SS. We thank the university videographer, Mr. Jacob Anselmi, for his outstanding work with the video. We also thank Mr. Roger Heckert and Mrs. Katha Heckert for their generous support for research.

1.Synthesis and Functionalization of Multi-Parametric Magneto-Fluorescent Nanosensors (MFnS).
<ol>
	<li>Synthesis of superparamagnetic iron oxide nanoparticles (IONPs)
	<ol>
		<li>To prepare for IONP synthesis, prepare the following 3 solutions: Solution 1: FeCl3 (0.70 g) and FeCl2 in H2O (2 mL), Solution 2: NH4OH (2.0 mL, 13.4 M) in H2O (15 mL), and Solution 3: polyacrylic acid (0.855 g) in H2O (5 mL).</li>
		<li>Add 90 &#181;L of 2 M Hydrochloric acid ...

<ol>
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J., Dostalek, J., Sessitsch, A., Knoll, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+surface+plasmon-enhanced+fluorescence+spectroscopy+biosensor+for+ultrasensitive+detection+of+E.+coli+O157:H7.">Long-range surface plasmon-enhanced fluorescence spectroscopy biosensor for ultrasensitive detection of E. coli O157:H7.</a> Anal Chem. 83 (3), 674-677 (2011).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+visual+detection+of+highly+pathogenic+Streptococcus+suis+serotype+2+isolates+by+use+of+loop-mediated+isothermal+amplification.">Rapid visual detection of highly pathogenic Streptococcus suis serotype 2 isolates by use of loop-mediated isothermal amplification.</a> J Clin Microbiol. 51 (10), 3250-3256 (2013).</li><li>Han, F., Wang, F., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+potentially+virulent+Vibrio+vulnificus+strains+in+raw+oysters+by+quantitative+loop-mediated+isothermal+amplification.">Detecting potentially virulent Vibrio vulnificus strains in raw oysters by quantitative loop-mediated isothermal amplification.</a> Appl Environ Microbiol. 77 (8), 2589-2595 (2011).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+pathogenic+bacteria+and+screening+of+phage-derived+peptides+using+microcantilevers.">Rapid detection of pathogenic bacteria and screening of phage-derived peptides using microcantilevers.</a> Anal Chem. 86 (3), 1671-1678 (2014).</li><li>Banerjee, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+Magneto-fluorescent+Nanosensors+for+the+Ultrasensitive+Detection+of+Escherichia+coli+O157:H7.">Multiparametric Magneto-fluorescent Nanosensors for the Ultrasensitive Detection of Escherichia coli O157:H7.</a> ACS Infect Dis. 2 (10), 667-673 (2016).</li><li>Shelby, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+magnetic+relaxation+nanosensors:+an+unparalleled+&amp;#34;spin&amp;#34;+on+influenza+diagnosis.">Novel magnetic relaxation nanosensors: an unparalleled &amp;#34;spin&amp;#34; on influenza diagnosis.</a> Nanoscale. 8, 19605-19613 (2016).</li><li>Bui, M. 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This protocol has been designed to produce fully functional MFnS as simply as possible. However, there are many key points at which alteration of the protocol may be useful, depending on the user's end goal. For example, the use of different antibodies would allow for targeting of many other pathogens. In addition, this protocol is not limited to the use of antibodies as targeting molecules. Any molecule which has specific binding affinity for target pathogens, such as host cell receptors...

The persistent occurrence of bacterial contamination in both commercially produced food and water sources has created a need for increasingly rapid and specific diagnostic platforms.1,2 Some of the more common bacterial contaminants responsible for food and water contamination are from the Salmonella, Staphylococcus, Listeria, Vibrio, Shigella, Bacillus, and Escherichia genera.3,4 Bacterial contamination by these pathogens often results in symptoms such as fever, cholera, gastroenteritis, and diarrhea.4 Contamination of water sources often has drastic and adverse effects on communities without access to sufficiently filtered water, and food contamination has led to a great number of illnesses and product recall efforts.5,6
In order to reduce the occurrence of illnesses caused by bacterial contamination, there have been a number of efforts to develop methods by which water and food can be efficiently scanned prior to sale or consumption.3 Techniques such as PCR,1,7,8,9,10 ELISA,11,12 loop-mediated isothermal amplification (LAMP),13,14 among others,15,16,17,18,19,20,21,22,23,24 have recently been used for detection of various pathogens. Compared to traditional bacterial culturing methods, these techniques are far more efficient with regards to specificity and time. However, these techniques still struggle with false positives and negatives, complex procedures, and cost.1,3,25 It is for this very reason that multiparametric magneto-fluorescent nanosensors (MFnS) are proposed as an alternative method for bacterial detection.
These nanosensors uniquely pair together magnetic relaxation and fluorescent modalities, allowing for a dual-detection platform that is both rapid and accurate. Using E. coli O157:H7 as a sample contaminant, the ability of MFnS to detect as little as 1 CFU within minutes is demonstrated. Pathogen-specific antibodies are used to increase specificity, and the combination of both magnetic and fluorescent modalities allows for the detection and quantification of bacterial contaminants in both low- and high-contamination ranges.16 In the case of bacterial contamination, the nanosensors will swarm around the bacteria due to the targeting abilities of the pathogen-specific antibodies. The binding between the magnetic nanosensors and bacteria limits the interaction between the magnetic iron core and the surrounding water protons. This causes an increase in the T2 relaxation times, as recorded by a magnetic relaxometer. As the concentration of bacteria in solution rises, the nanosensors disperse with the increased number of bacteria, resulting in lower T2 values. Conversely, fluorescence emission will increase in proportion with the concentration of bacteria, due to the increased number of nanosensors directly bound to pathogen. Centrifugation of the samples, and isolation of the bacterial pellet, will only conserve the nanoparticles directly attached to the bacteria, removing any free-floating nanosensors, and directly correlating the fluorescence emission with the number of bacteria present in solution. A schematic representation of this mechanism is represented in Figure 1.
This MFnS platform has been designed with point-of-care screening in mind, resulting in low-cost and portable characteristics. MFnS are stable at room temperature, and are only required in very low concentrations for accurate detection of bacterial contaminants. Furthermore, after synthesis, use of the MFnS is simple and does not require the use of trained professionals in the field. Lastly, this diagnostic platform allows for highly customizable targeting, providing a means by which this one platform may be used to detect pathogens of all kinds, in many different settings.

<table border="0" cellpadding="0" cellspacing="0" width="821">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferrous Chloride Tetrahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">I90-500</td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ferric Chloride Hexahydrate</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>I88-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ammonium Hydroxide</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A669S-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Hydrochloric Acid</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">A144S-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Polyacryllic Acid</td>
			<td style="width:156px;">Sigma-Aldrich</td>
			<td style="width:197px;">323667-100G</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:239px;">EDC</td>
			<td style="width:156px;">Thermofisher Scientific</td>
			<td style="width:197px;">22980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">NHS</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td>AC157270250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O111 antibody&#160;</td>
			<td style="width:156px;">sera care</td>
			<td>5310-0352</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-E. coli O157:H7 antibody [P3C6]&#160;</td>
			<td style="width:156px;">Abcam</td>
			<td style="width:197px;">ab75244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">DiI Stain</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">D282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Nutrient Broth</td>
			<td style="width:156px;">Difco</td>
			<td style="width:197px;">233000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freeze-dried E. coli O157:H7 pellet</td>
			<td style="width:156px;">ATCC</td>
			<td style="width:197px;">700728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Relaxomteter&#160;</td>
			<td style="width:156px;">Bruker</td>
			<td style="width:197px;">mq20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Zetasizer</td>
			<td style="width:156px;">Malvern</td>
			<td style="width:197px;">NANO-ZS90</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Plate Reader&#160;</td>
			<td style="width:156px;">Tecan</td>
			<td style="width:197px;">Infinite M200 PRO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Magnetic Column&#160;</td>
			<td style="width:156px;">QuadroMACS</td>
			<td style="width:197px;">130-090-976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge</td>
			<td style="width:156px;">Eppendorf</td>
			<td style="width:197px;">5804 Series</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Centrifuge (accuSpin Micro 17)</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">13-100-676</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Floor Model Shaking Incubator</td>
			<td style="width:156px;">SHEL LAB</td>
			<td style="width:197px;">SSI5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Analytical Balance</td>
			<td style="width:156px;">Metler Toledo</td>
			<td style="width:197px;">ME104E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Digital Vortex Mixer</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-215-370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Open-Air Rocking Shaker</td>
			<td style="width:156px;">Fisher Scientific</td>
			<td style="width:197px;">02-217-765</td>
			<td></td>
		</tr>
	</tbody>
</table>

foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

The mechanism of MFnS action is represented in Figure 1. The clustering of MFnS around the surface of bacterial contaminants interferes with the interactions between the magnetic cores of the MFnS and the surrounding hydrogen nuclei. As a result of this clustering, magnetic relaxation values increase. As the concentration of bacterial contaminants increases, clustering reduces, and the change in T2 values decreases. Therefore, the addition of a fluorescent modality is crucial. As the bacteri...

Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

The main goal of this protocol is to design and synthesize dual-modal nanosensors capable of detecting bacterial contaminants such as E.coli O157:H7. Magneto-fluorescent nanosensors are synthesized by functionalizing iron oxide nanoparticles via a two-step procedure. First, targeting antibodies are conjugated to the surface of the nanoparticle.Then fluorescent dye is loaded into its coding. The paring of these modalities allows for rapid and sensitive detection of bacterial contaminants in both low and highly concentrated solutions. In the presence of a small amount of bacteria, the nanosensors will swarm around the bacteria due to the targeting of the conjugated antibodies.This swarming alters the interactions of the nanosensors magnetic core with the surrounding water protons, allowing for sensitive detection of changes in magnetic relaxation values. As the concentration of bacterial contaminants in solutions increases, swarming decreases and the magnetic modality's ability to quantify contamination is reduced. However, as the bacterial concentration increases, so does the fluorescent submission of the bio nanosensors.It is for this reason that the pairing of magnetic and fluorescent modalities is important. The combination has allowed for the detection of as little as one colony forming unit of E.coli O157:H7 within minutes. The first step in the systhesis of magneto-fluorescent nanosensors is the preparation of a iron salt solution consisting of both ferous and ferric chloride.The iron will provide the nanosensors with a magnetic core which allows for its adaptability to magnetic relaxation platforms. Additional solutions needed are polyacrylic acid and ammonium hydroxide. Hydrochloric acid is introduced to the iron salt solution, which is then added to the ammonium hydroxide solution while vortexing.Finally the polyacrylic acid solution is added, and the resulting mixture is vortexed for an additional hour while the reaction continues. The resulting solution is centrifuged in order to precipitate the larger molecules and isolate nanoparticles that are smaller than 100 nanometers in size. The solution is then dialyzed overnight for purification.The next step is the conjugation of targeting antibodies to the polyacrylic acid coding of the newly synthesized iron acid nanoparticles. Materials needed include MES buffer, EDC, NHS and the selected antibodies. EDC is first added to the nanoparticle solution, followed by NHS and finally the antibody.The mixture is then placed on a tabletop mixer for three hours while the reaction continues. The solution is purified using a magnetic column. The magnetized column captures the nanoparticles, allowing only free floating antibodies to exit.Afterwards the column is washed with PBS buffer, and the conjugated nanosensors are collected. The nanoparticles are then ready for the addition of fluorescent dye, providing the second key modality used for detection. One of the most key features of our nanosensor is the combination of the magnetic and fluorescent modalities, which is important for a number of reasons.Firstly, the combination of the modalities allows each one to sort of cross check the other one, which greatly reduces the risk of false positives and false negatives, which is one of the greatest hurdles faced by different diagnostic platforms used today. And secondly, the combination of the modalities greatly widens the range within which we can not only detect but also quantify bacterial contamination. The final step in the preparation of the nanosensors is the encapsulation of fluorescent dye in the coding of the nanoparticle.This is simply achieved by adding dye to the solution while vortexing, and then allowing further time for the dye to diffuse into the coding of the nanosensor. The fully functionalized nanosensors are then dialyzed one final time for purification. In order to characterize the nanosensors, the size and fluorescent submission are recorded using a zetasizer and a fluorescence plate reader.A small sample of the nanosensor is added to a cuvette water solution and placed in the zetasizer for the recording of size. For the fluorescence analysis a sample of the nanosensor is placed on a 96 well plate and inserted into the fluorescence plate reader. Ideally the nanosensors will be around 70 nanometers in diameter, and have a fluorescent submission of 575 nanometers.In addition to the synthesis of the nanosensors, the culturing of bacteria is necessary to provide the bacterial targets in the lab. A glycerol stock of bacteria is used to prepare a culture in nutrient broth. This solution is then allowed to incubate.Following the initial incubatory period, serial dilutions are performed in order to achieve a wide range of bacterial concentrations. One hundred microliters of each concentration are plated to an nutrient auger plate and then incubated for 24 hours. Following this incubation the colonies on each plate are counted in order to determine the colony forming unit, or CFU count per mil of each diluted stock.Now that the bacterial stock solutions have been prepared, they are able to be used in conjunction with the prepared nanosensors. And one important aspect of this particular serotype of the bacteria that distinguishes it from other serotypes is like suppose if you get infected with the other serotypes, you need to have many colony forming units to get an infection. But in this particular E.coli 10 to 100 CFUs or colony forming units are good enough to give you an infection.Our technology is established in such a way that you don't miss even a single colony bacterial contamination is there. In order to prepare solutions for reading in the magnetic relaxometer, 300 microliters of PBS is first pipetted into an Eppendorf tube. Then a sample of bacterial stock is added, followed by the addition of nanosensors.The solution is then transferred to a glass tube, and a piece of parafilm is placed on top in order to prevent evaporation. The glass tube is then placed inside a larger NMR tube and inserted into the magnetic relaxometer. A baseline solution containing no bacteria and only nanosensor and PBS is used to get a baseline T2 reading as shown.Then solutions containing various concentrations of bacteria are inserted into the magnetic relaxometer for analysis, and the changes in T2 values are caused by the binding between the nanosensors and the bacteria. As shown, the presence of as few as one bacterial CFU can be detected within minutes using this modality. However, as the bacterial concentration increases the MR reading are less quantifiable, which is why the use of fluorescent subsmission data is also tantamount to an accurate bacterial quantification.Before fluorescence data can be collected the sample must first be centrifuged. The solution is transferred from the glass tube to an Eppendorf tube and then centrifuged. This separates the bacteria and the nanosensors bound to it from free floating nanosensors in solution.The supernatent is discarded, and the bacterial pellet is resuspended in PBS. Finally the sample may be analyzed via fluorescent submission. The strength of the emission will be relative to the amount of nanosensors remaining in solution, and therefore also the amount of bacterial present.As can be seen, fluorescent submission strengthens as the bacterial concentration increases, and it becomes more sensitive as well. This is why the pairing of both magnetic and fluorescent modalities allows for the detection and quantification of bacterial contamination in both early and late developmental stages. In addition to the detection of bacteria in simple solutions such as PBS, these nanosensors also possess the capability to function in more complex media, such as lake water or milk.Furthermore, these nanosensors have been tested for the specificity in the presence of non-target bacteria and heat inactivated target bacteria. As shown magneto-fluorescent nanosensors have little to no reaction with non-targeted or non-living bacteria due to the specificity of the conjugated antibodies. This demonstrates their effectiveness for the detection of specific bacteria while in the presence of other species.Finally, it is also important to note that these nanosensors may be easily customized for the detection of other pathogens as well. You can synthesize or you can formulate these nanosensors that we did in our lab within one month now. You can do it within one month, and that one month of product can give you like 10 years of application.

foodborne-pathogen-screening-using-magneto-fluorescent-nanosensor

Research

JoVE Journal

Immunology and Infection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.