This research did not receive any specific funding from the public, commercial, or not-for profit sectors.

NOTE: C. perfringens is considered a biosafety hazard level 2 (BSL2) organism. Although not all food samples will contain C. perfringens, all cultured samples should be treated as such. All proper precautions and personnel protective equipment (PPE) should be worn at all times. Decontaminate all material prior to disposal.
1. Prepare modified RPM4,15
<ol>
	<li>Combine 30 g/L fluid thioglycolate medium, 60 g/L gelatin, 5 g/L peptone, 5 g/L glucose, 5 g/L potassium phosphate dibasic, 3 g/L yeast extract, 1.5 g/L sodium chloride, 0.5 g/L ferrous sulfate in deionized water until evenly dispersed. We typically make 1 L batches.</li>
	<li>Autoclave at 121 &#176;C for 15 min.</li>
	<li>Allow to cool to approximately 40 &#176;C.</li>
	<li>Once RPM is cool, add D-cycloserine to a final concentration of 440 mg/L. 
	NOTE: Stock concentrations of D-cycloserine dissolved in sterile water at 50 mg/mL may be stored at -20 &#176;C for future use.</li>
	<li>Aseptically transfer 10 mL of RPM to 15 mL conical tubes. RPM can be prepared in batches and stored at 4 &#176;C for up to one month.</li>
	<li>Warm RPM to 37 &#176;C prior to use.</li>
</ol>
2. Sample collection and RPM incubation
<ol>
	<li>Transport food to the laboratory under ambient temperatures within 1 h. Food may be transported in original packaging or sterile containers. If not testing immediately, food may be stored at -20 &#176;C until use. Make note of the type of food and the country of origin according to the packaging label, if available, as well as any other relevant information.</li>
	<li>Remove approximately 1.0&#8211;2.0 g of food and transfer to sterile Petri dish or equivalent. Finely mince with a sterile razor blade or scalpel.</li>
	<li>Inoculate into 10 mL of phosphate buffered saline (PBS) in a 15 mL conical tube. Mix well.
	<ol>
		<li>To select for vegetative cells, transfer 5 mL of the PBS-food mixture into a 15 mL conical tube containing 10 mL of RPM. Secure the lid tightly.</li>
		<li>To select for spores, heat the remaining 5 mL of PBS-food mixture at 85 &#176;C for 15 min and then transfer to a 15 mL conical tube containing 10 mL of RPM. Secure the lid tightly.</li>
	</ol>
	</li>
	<li>Vortex and invert the food-RPM cultures to ensure complete mixing.</li>
	<li>Tightly seal the conical tubes and wrap lids with paraffin film or plastic wrap to ensure an anaerobic environment.</li>
	<li>Incubate overnight (ON) at 37 &#176;C.</li>
	<li>The following morning note any turbidity or fermentation in RPM tubes.</li>
</ol>
3. TSC &#34;sandwich&#34;&#160;plating
<ol>
	<li>Inoculate ON RPM cultures into TSC agar using a &#8220;sandwich&#8221; technique (Figure 2) described below.</li>
	<li>Prepare TSC agar as per the manufacturer&#39;s instructions.
	<ol>
		<li>Prepare the base layer of the TSC plates by transferring 10 mL of molten TSC agar to sterile Petri dishes and allow to solidify. These may be prepared in advanced and stored at 4 &#176;C. Ensure plates are warmed to room temperature prior to use.</li>
		<li>For the top layer of the TSC plate, maintain the remaining molten TSC agar at 40 &#176;C. 
		NOTE: Although agar&#39;s melting point is above 85 &#176;C, molten agar solidifies around 32&#8211;45 &#176;C.</li>
	</ol>
	</li>
	<li>Carefully retrieve the ON RPM cultures and transfer 100 &#181;L to the TSC agar base. Because of fermentation, some cultures may be under pressure. Be sure to wear all appropriate PPE.</li>
	<li>Spread the RPM inoculum using sterilized glass beads or cell spreader.</li>
	<li>Allow RPM to be &#34;absorbed&#34;&#160;into TSC agar for 5&#8211;10 min at room temperature.</li>
	<li>Carefully transfer 20 mL of molten, 40 &#176;C TSC agar to plate using a serological pipette.</li>
	<li>Replace the Petri dish lid and allow TSC agar to completely solidify at room temperature.</li>
	<li>Invert the Petri dish and incubate at 37 &#176;C ON.</li>
	<li>If serial dilutions are desired, perform dilutions of the ON RPM cultures into fresh, pre-warmed RPM prior to inoculation into TSC agar.</li>
</ol>
4. Sub-culturing of sulfite-reducing colonies
<ol>
	<li>The following morning, remove plates from incubator and examine for bacterial growth. Aerobic bacteria may be present on the surface of the agar. Anaerobic bacteria will be found growing embedded within the agar. Sulfite-reducing bacteria will turn surrounding agar black. Possible C. perfringens colonies will be black and embedded in the agar.</li>
	<li>C. perfringens suspected colonies will be positive for sulfite reduction and will appear black, embedded within the agar. Using a sterile, single-use eyedropper, &#34;pluck&#34;&#160;the black colonies from the agar and transfer to 10 mL of fresh RPM in a 15 mL conical tube. It is important that the air from the eyedropper be expelled prior to piercing agar.</li>
	<li>If there is a dense amount of aerobic bacterial growth on the surface of the plate, use a sterile cell scraper to remove colonies from selected areas. Multiple C. perfringens suspected colonies can be sampled from the same TSC plate into separate RPM cultures.</li>
	<li>Tightly secure conical tube lids and wrap in paraffin film or plastic wrap. Incubate ON at 37 &#176;C.</li>
</ol>
5. DNA Extraction
<ol>
	<li>Remove ON RPM cultures and examine for signs of growth including turbidity and fermentation.</li>
	<li>Gently invert RPM culture to disperse any settled bacteria and carefully open. Transfer 1 mL of the culture to a microcentrifuge tube.</li>
	<li>Centrifuge at top speed (approximately 15,000 x g) for 10 min to pellet bacteria.</li>
	<li>Wash pellet with 1 mL of sterile PBS.
	<ol>
		<li>In some circumstances, undigested gelatin will settle on top of pelleted bacteria. To remove gelatin, &#34;fluff&#34;&#160;off gelatin by gently agitating with PBS using a micropipette. This will &#34;fluff-up&#34;&#160;the gelatin while leaving the bacterial pellet intact.</li>
		<li>Remove the PBS-gelatin mixture with gentle aspiration. Re-suspend the remaining bacterial pellet with 1 mL of fresh PBS.</li>
	</ol>
	</li>
	<li>Centrifuge the resuspended bacterial pellet at top speed for 10 min and carefully aspirate the supernatant.</li>
	<li>Immediately perform DNA extraction using a DNA extraction kit and a protocol specifically created for extraction DNA from Gram-positive bacteria (see the Table of Materials).</li>
	<li>Use the extracted DNA immediately or store at -20 &#176;C until PCR analysis.</li>
</ol>
6. Detection of C. perfringens via PCR genotyping
<ol>
	<li>To determine if cultures are positive for C. perfringens toxinotypes, examine extracted DNA for different toxin genes via PCR. 
	NOTE: Because we are most interested in the epsilon toxin producing strains type B and D C. perfringens, we evaluate for the presence of the alpha, beta, and epsilon toxin genes. Primers are listed in Table 2. Primers for 16s ribosomal DNA are used as a DNA extraction control. Additional primers can be used to test for toxinotypes A through G and can be found in published literature2,12,13.</li>
	<li>Use previously extracted C. perfringens DNA as a positive control. This control ensures that all components of the PCR reactions are working. We use DNA extracted from C. perfringens type B (ATCC 3626) as our positive control. Grow ATCC 3636 ON in RPM at 37 &#176;C and extract DNA using the same methods described in steps 5.1&#8211;5.7. Store extracted DNA at -20 &#176;C until use.</li>
	<li>Set PCR conditions as follows: 94.0 &#176;C for 3 min; 94.0 &#176;C for 30 s; 47.9 &#176;C for 30 s, 72.0 &#176;C for 1 min (Repeat steps 2&#8211;4 for 35 cycles); 72.0 &#176;C for 10 min.</li>
	<li>To confirm PCR products, run PCR reactions on a 1.8 g/100 mL agarose gel using standard techniques. Expected PCR product sizes are listed in Table 2. Typical PCR results are displayed in Figure 3.</li>
</ol>

<ol>
	<li>Petit, L., Gibert, M., Popoff, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+perfringens:+toxinotype+and+genotype.">Clostridium perfringens: toxinotype and genotype.</a> Trends in Microbiology. 7, 104-110 (1999).</li><li>Rood, J. I., 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=Expansion+of+the+Clostridium+perfringens+toxin-based+typing+scheme.">Expansion of the Clostridium perfringens toxin-based typing scheme.</a> Anaerobe. , (2018).</li><li>Murrell, T. G., O'Donoghue, P. J., Ellis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+sheep-multiple+sclerosis+connection.">A review of the sheep-multiple sclerosis connection.</a> Medical Hypotheses. 19, 27-39 (1986).</li><li>Rumah, K. R., Linden, J., Fischetti, V. A., Vartanian, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Clostridium+perfringens+type+B+in+an+individual+at+first+clinical+presentation+of+multiple+sclerosis+provides+clues+for+environmental+triggers+of+the+disease.">Isolation of Clostridium perfringens type B in an individual at first clinical presentation of multiple sclerosis provides clues for environmental triggers of the disease.</a> PLoS One. 8, 76359 (2013).</li><li>Wagley, S., 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=Evidence+of+Clostridium+perfringens+epsilon+toxin+associated+with+multiple+sclerosis.">Evidence of Clostridium perfringens epsilon toxin associated with multiple sclerosis.</a> Multiple Sclerosis. , (2018).</li><li>Linden, J. R., 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=Clostridium+perfringens+Epsilon+Toxin+Causes+Selective+Death+of+Mature+Oligodendrocytes+and+Central+Nervous+System+Demyelination.">Clostridium perfringens Epsilon Toxin Causes Selective Death of Mature Oligodendrocytes and Central Nervous System Demyelination.</a> MBio. 6, 02513 (2015).</li><li>Popoff, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epsilon+toxin:+a+fascinating+pore-forming+toxin.">Epsilon toxin: a fascinating pore-forming toxin.</a> FEBS Journal. 278, 4602-4615 (2011).</li><li>Lee, C. A., Labbe, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+Enterotoxin-+and+Epsilon-Positive+Clostridium+perfringens+Spores+in+U.S.+Retail+Spices.">Distribution of Enterotoxin- and Epsilon-Positive Clostridium perfringens Spores in U.S. Retail Spices.</a> Journal of Food Protection. 81, 394-399 (2018).</li><li>Wen, Q., McClane, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+enterotoxigenic+Clostridium+perfringens+type+A+isolates+in+American+retail+foods.">Detection of enterotoxigenic Clostridium perfringens type A isolates in American retail foods.</a> Applied and Environmental Microbiology. 70, 2685-2691 (2004).</li><li>Cooper, K. K., Bueschel, D. M., Songer, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+Clostridium+perfringens+in+retail+chicken+livers.">Presence of Clostridium perfringens in retail chicken livers.</a> Anaerobe. 21, 67-68 (2013).</li><li>Strong, D. H., Canada, J. C., Griffiths, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+Clostridium+perfringens+in+American+foods.">Incidence of Clostridium perfringens in American foods.</a> Applied and Environmental Microbiology. 11, 42-44 (1963).</li><li>Buogo, C., Capaul, S., Hani, H., Frey, J., Nicolet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+Clostridium+perfringens+type+C+enteritis+in+pigs+using+a+DNA+amplification+technique+(PCR).">Diagnosis of Clostridium perfringens type C enteritis in pigs using a DNA amplification technique (PCR).</a> Journal of Veterinary Medicine, Series B. 42, 51-58 (1995).</li><li>Yamagishi, T., Sugitani, K., Tanishima, K., Nakamura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction+test+for+differentiation+of+five+toxin+types+of+Clostridium+perfringens.">Polymerase chain reaction test for differentiation of five toxin types of Clostridium perfringens.</a> Microbiology and Immunology. 41, 295-299 (1997).</li><li>Johansson, A., Engstrom, B. E., Frey, J., Johansson, K. E., Baverud, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+clostridium+perfringens+during+simulated+transport+and+stability+of+some+plasmid-borne+toxin+genes+under+aerobic+conditions.">Survival of clostridium perfringens during simulated transport and stability of some plasmid-borne toxin genes under aerobic conditions.</a> Acta Veterinaria Scandinavica. 46, 241-247 (2005).</li><li>Erickson, J. E., Deibel, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+medium+for+rapid+screening+and+enumeration+of+Clostridium+perfringens+in+foods.">New medium for rapid screening and enumeration of Clostridium perfringens in foods.</a> Applied and Environmental Microbiology. 36, 567-571 (1978).</li><li>Regan, S. B., 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=Identification+of+epsilon+toxin-producing+Clostridium+perfringens+strains+in+American+retail+food.">Identification of epsilon toxin-producing Clostridium perfringens strains in American retail food.</a> Anaerobe. 54, 124-127 (2018).</li></ol>

No disclosures.

Here we describe a method to identify C. perfringens prevalence in retail food samples with limited subculturing and without use of an anaerobic chamber system. This method uses a combination of techniques to increase identification of C. perfringens from food samples. By using a modified version of RPM media, we allow for the selective growth of C. perfringens. By sandwiching the inoculated RPM in between layers of TSC agar, we are able to identify and isolate anaerobic, sulfite-reducing bacteria characteristic of C. perfringens. To confirm the presence of C. perfringens, sulfite-reducing colonies are sub-cultured into fresh RPM. The modified version or RPM allows us to easily extract DNA from cultures, enabling PCR confirmation of specific toxin genes. Confirmation of C. perfringens contaminated food samples can be achieved within three days.
In early experiments, food samples were simply inoculated into RPM and DNA extracted from ON cultures. This method resulted in detection of C. perfringens in a limited number of samples (data not shown). Although RPM is selective for C. perfringens growth, it is not exclusive for C. perfringens growth. Other, gram-positive, D-cycolserine resistant bacteria can still grow in RPM. We hypothesized that contamination by other bacterial species may have decreased our detection of C. perfringens strains by decreasing the sensitivity of our PCR analysis in our first ON RPM culture. A critical step in increasing the detection of C. perfringens was the inclusion of the TSC agar &#8220;sandwich&#8221; technique. This allowed us to differentiate and select for anaerobic, sulfite-reducing colonies, characteristic of C. perfringens. A key step in this process is ensuring that the top layer of molten TSC agar is at 40 &#176;C. Although some C. perfringens strains can grow at increased temperatures (46&#8211;48 &#176;C)15,16, addition of molten agar at increased temperatures greatly reduces the amount of cultures recovered, mostly likely due to cell death.
There are several potential limitations to this method. As mentioned previously, neither the RPM nor TSC agar selects or differentiates for C. perfringens exclusively, allowing for growth of other bacterial species present in food samples. This may reduce the sensitivity of the assay to select for C. perfringens only. However, this is a common limitation in almost all culturing techniques. Genotyping confirmation of purified isolates is the best method for definitively identifying C. perfringens and other bacterial species. Another limitation of this study is that we do not test the purified isolates. We purposely did this to limit the amount of subculturing, as repeated subculturing is feared to result in plasmid loss. Because we do not isolate to purity, it is possible that multiple C. perfringens toxinotypes may be present in the same sample or subculture. If researchers wish to obtain purified isolates, standard purification methods can be used on the last RPM culture described in this method; this typically requires the use of anaerobic chambers. Although originally used to isolate C. perfringens from food, this method can be used to identify and isolate C. perfringens from a multitude of sources. Specifically, one such application of this method is to test fecal samples from humans (or animals) that are suspected to be infected with C. perfringens and toxinotype the bacteria to better understand the source of infection.

Clostridium perfringens (C. perfringens) is a Gram positive, anaerobic, spore-forming, rod shaped bacterium that is found ubiquitously in the environment. This species of bacteria carries genes that encode for over 17 toxins and, historically, has been characterized into five toxinotypes (A-E) based on the presence of four different toxin genes: alpha, beta, epsilon, and iota toxin (Table 1)1. Recently, it has been suggested that this typing-scheme needs to be expanded to include types F and G, which harbor the C. perfringens enterotoxin (CPE) and NetB toxin, respectively2. However, more research is needed before this scheming system is formally accepted. While the alpha toxin gene is strictly chromosomally located, the CPE gene can be found both on the chromosome and plasmids. In comparison, the remaining toxins&#39;&#160;genes are found on various differently-sized plasmids. We are particularly interested in the prevalence of C. perfringens types B and D as these strains produce epsilon toxin, an extremely potent, pore-forming toxin, which has been suggested to play a role in triggering multiple sclerosis (MS) in humans3,4,5,6,7. How people become infected or colonized by these strains is unknown. One possible explanation is through consumption of contaminated food products. To help answer this question, we sought to determine the prevalence of different C. perfringens toxinotypes in American food samples.
The presence of C. perfringens toxinotypes in American food samples is understudied and often requires use of anaerobic container systems and numerous sub-culturing steps8,9,10,11. Although numerous sub-culturing steps are needed to obtain purified isolates, this method can lead to loss of plasmids over time12,13,14, possibly affecting the detection of plasmid-borne toxin genes including the epsilon toxin gene. We sought to develop an easy method, with fewer sub-culturing steps, to selectively culture C. perfringens without the use of anaerobic chambers, jars, or bags. Briefly, food samples are inoculated into Rapid Perfringens Media (RPM) overnight (ON), then &#34;sandwiched&#34;&#160;into TSC agar and incubated ON. Colonies suspected to be C. perfringens are then sub-cultured into RPM and incubated again ON. DNA is extracted and PCR performed to determine genotype (Figure 1). We chose to use RPM as it has been demonstrated to increase the recovery of C. perfringens strains from food samples compared to other more standard media15. In addition, RPM was successfully used to isolate an epsilon toxin producing type B strain from an MS patient4. We use a modified version of RPM instead of the original version to allow easy DNA extraction. While this method allows easy identification of toxin genes within samples, it is possible that an individual sample will contain more than one C. perfringens toxinotype. Because our method does not isolate purified strains using multiple rounds of purification, identification of multiple toxinotypes from one sample is not possible. However, standard purification techniques (typically streaking onto TSC plates or blood agar plates) can be applied at the end of our protocol to achieve purified cultures.

<table>
	<tbody>
		<tr>
			<td>D-Cycloserine</td>
			<td>Sigma-Aldrich</td>
			<td>C6880</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Sigma Life Science</td>
			<td>D9434-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Transfer Pipets</td>
			<td>any brand</td>
			<td></td>
			<td>Select one with slim tip like Thermo Scientific Disposable Transfer Pipets 137116M/EMD</td>
		</tr>
		<tr>
			<td>DNeasy Blood &#38; Tissue Kits</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td>Note: numerous DNA and plasmid extractions kits were evaluated, this kit gave the most desirable results.</td>
		</tr>
		<tr>
			<td>Dry Incubator</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluid thioglycolate medium</td>
			<td>Remel</td>
			<td>R453452</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma Life Science</td>
			<td>G1890-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Individual Primers</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (II) sulfate heptahydrate</td>
			<td>Sigma Life Science</td>
			<td>F8633-250 G</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme from chicken egg white</td>
			<td>Sigma-Aldrich</td>
			<td>L6876</td>
			<td>Needed for DNA extraction, not provided in kit</td>
		</tr>
		<tr>
			<td>microcentrifuge tube</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>parafilm or plastic wrap</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone from casein and other animal proteins</td>
			<td>Sigma-Aldrich</td>
			<td>70173-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfringens Agar Base (TSC + SFP)</td>
			<td>Oxoid</td>
			<td>CM0587</td>
			<td>Make TSC agar according to instructions</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>P2222-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S-7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Cell Scraper</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell Spreader</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile petri dishes</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Supplies and equipment for gel electrophoresis</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>table top centrifuge</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Taq&#160;PCR Master Mix Kit</td>
			<td>Qiagen</td>
			<td>201443</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler for PCR reaction</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>water bath</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sigma-Aldrich</td>
			<td>70161-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

enrichment and detection of clostridium perfringens toxinotypes in retail food samples

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized into five different toxinotypes, A through E, based on the carriage of four major toxin genes. The prevalence and distribution of these various toxinotypes is understudied, especially their pervasiveness in American retail food. Of particular interest to us are the type B and D strains, which produce epsilon toxin, an extremely lethal toxin suggested to be the environmental trigger of multiple sclerosis in humans. To evaluate the presence of different C. perfringens toxinotypes in various food samples, we developed an easy method to selectively culture these bacteria without the use of an anaerobic container system only involving three culturing steps. Food is purchased from local grocery stores and transported to the laboratory under ambient conditions. Samples are minced and inoculated into modified rapid perfringens media (RPM) and incubated overnight at 37 &#176;C in a sealed, airtight conical tube. Overnight cultures are inoculated onto a bottom layer of solid Tryptose Sulfite Cycloserine (TSC) agar, and then overlaid with a top layer of molten TSC agar, creating a &#34;sandwiched&#34;, anaerobic environment. Agar plates are incubated overnight at 37 &#176;C and then evaluated for appearance of black, sulfite-reducing colonies. C. perfringens-suspected colonies are removed from the TSC agar using sterile eye droppers, and inoculated into RPM and sub-cultured overnight at 37 &#176;C in an airtight conical tube. DNA is extracted from the RPM subculture, and then analyzed for the presence of C. perfringens toxin genes via polymerase chain reaction (PCR). Depending on the type of food sampled, typically 15&#8211;20% of samples test positive for C. perfringens.

Using this method, 15&#8211;20% of our sampled foods test positive for C. perfringens. While most strains are positive for toxinotype A, we have successfully detected both Type B and D in food samples. In a previously published paper we tested a total of 216 food samples purchased from New York retail stores16 (Table 3). These samples included various meat samples (beef, lamb, pork and lamb), poultry samples (chicken and turkey), and seafood samples (cod, salmon, shellfish, snapper, flounder, squid, tilapia, tuna, and various other fishes). Produce and dairy samples were also tested. Of 216 samples, 34 (16%) were positive for C. perfringens. Of the 34 C. perfringens positive samples, 31 samples (91.2%) contained the alpha toxin, one sample (2.9%) contained the alpha, beta and epsilon toxin, and two samples (5.9%) contained the alpha and epsilon toxin.
Interestingly, we also discovered that C. perfringens was more prevalent as vegetative cells compared to spores. Twenty-five samples were compared for the presence of vegetative C. perfringens cells or spores. Spores were selected for by heat shocking samples at 85 &#176;C for 15 min. Of the 25 samples tested, 16% were positive for vegetative C. perfringens strains versus 4% for spores. This indicates that it may be more cost effective to test for only vegetative cells instead of both.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59931/59931fig01.jpg" /> 
Figure 1: Overview of procedure. 
Food samples are minced and diluted into sterile PBS. Half of the PBS-food sample is inoculated into RPM to select for vegetative cells. The remaining PBS-food sample is heat shocked at 85 &#176;C for 15 min to select for spores prior to inoculation in RPM. Cultures are incubated ON at 37 &#176;C then plated into TSC agar. TSC agar is incubated ON at 37 &#176;C and black, sulfite-reducing cultures are sub-cultured into fresh RPM. Sub-cultured RPM cultures are incubated ON at 37 &#176;C and DNA is extracted to perform genotyping via PCR. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59931/59931fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59931/59931fig02.jpg" /> 
Figure 2: Schematic of TSC agar sandwich technique. 
RPM media containing the C. perfringens bacteria are plated between two layers of TSC agar in order to promote an anaerobic environment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59931/59931fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59931/59931fig03.jpg" /> 
Figure 3: Selected PCR results. 
Example images of the genotyping results of C. perfringens from seven different types of food, and a positive control of C. perfringens type B. A molecular weight ladder (first lane of each gel) was used to approximate the size of PCR results in base pairs (bp). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59931/59931fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Toxinotype</td>
			<td>Alpha</td>
			<td>Beta</td>
			<td>Epsilon</td>
			<td>Iota</td>
			<td>CPE</td>
			<td>NetB</td>
		</tr>
		<tr>
			<td rowspan="5">Established</td>
			<td>A</td>
			<td>+</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>B</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>C</td>
			<td>+</td>
			<td>+</td>
			<td>-</td>
			<td>-</td>
			<td>+</td>
			<td>-</td>
		</tr>
		<tr>
			<td>D</td>
			<td>+</td>
			<td>-</td>
			<td>+</td>
			<td>-</td>
			<td>+</td>
			<td>-</td>
		</tr>
		<tr>
			<td>E</td>
			<td>+</td>
			<td>-</td>
			<td>-</td>
			<td>+</td>
			<td>+</td>
			<td>-</td>
		</tr>
		<tr>
			<td rowspan="2">Proposed</td>
			<td>F</td>
			<td>+</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>+</td>
			<td>-</td>
		</tr>
		<tr>
			<td>G</td>
			<td>+</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>+</td>
		</tr>
		<tr>
			<td colspan="8"></td>
		</tr>
		<tr>
			<td colspan="8">+ present 
			- not present 
			+/- may or may not be present</td>
		</tr>
	</tbody>
</table>
Table 1: Overview of C. perfringens genotypes. A chart of the combinations of toxins produced by each C. perfringens toxinotype.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Target</td>
			<td>Primer Pairs</td>
			<td>Expected PCR Product (bp)</td>
		</tr>
		<tr>
			<td>alpha</td>
			<td>F: GCT AAT GTT ACT GCC GTT GA 
			R: CCT CTG ATA CAT CGT GTA AG</td>
			<td>325</td>
		</tr>
		<tr>
			<td>beta</td>
			<td>F: GCG AAT ATG CTG AAT CAT CTA 
			R: GCA GGA ACA TTA GTA TAT CTT C</td>
			<td>196</td>
		</tr>
		<tr>
			<td>epsilon</td>
			<td>F: GCG GTG ATA TCC ATC TAT TC 
			R: CCA CTT ACT TGT CCT ACT AAC</td>
			<td>655</td>
		</tr>
		<tr>
			<td>16s RNA</td>
			<td>F: AGA GTT TGA TCC TGG CTC A 
			R: GGT TAC CTT GTT ACG ACT T</td>
			<td>~1300</td>
		</tr>
	</tbody>
</table>
Table 2: Primers and expected PCR products for selected toxin genes. Primers used in the PCR genotyping step.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3" rowspan="2">Food Type</td>
			<td colspan="2">Type A</td>
			<td colspan="2">Type B</td>
			<td colspan="2">Type C</td>
			<td colspan="2">Type D</td>
			<td colspan="2">C. perf +</td>
		</tr>
		<tr>
			<td colspan="2">alpha toxin positive</td>
			<td colspan="2">alpha, beta, and epsilon toxin positive</td>
			<td colspan="2">alpha and beta toxin positive</td>
			<td colspan="2">alpha and epsilon toxin positive</td>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td colspan="2"></td>
			<td>n</td>
			<td>n</td>
			<td>%</td>
			<td>n</td>
			<td>%</td>
			<td>n</td>
			<td>%</td>
			<td>n</td>
			<td>%</td>
			<td>n</td>
			<td>%</td>
		</tr>
		<tr>
			<td rowspan="5">Meat</td>
			<td>beef</td>
			<td>38</td>
			<td>8</td>
			<td>21%</td>
			<td>1</td>
			<td>3%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>9</td>
			<td>24%</td>
		</tr>
		<tr>
			<td>lamb</td>
			<td>10</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>pork</td>
			<td>15</td>
			<td>2</td>
			<td>13%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>2</td>
			<td>13%</td>
		</tr>
		<tr>
			<td>mixed</td>
			<td>1</td>
			<td>1</td>
			<td>100%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>1</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>subtotal</td>
			<td>64</td>
			<td>11</td>
			<td>17%</td>
			<td>1</td>
			<td>2%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>12</td>
			<td>19%</td>
		</tr>
		<tr>
			<td rowspan="3">Poultry</td>
			<td>chicken</td>
			<td>19</td>
			<td>5</td>
			<td>26%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>5</td>
			<td>26%</td>
		</tr>
		<tr>
			<td>turkey</td>
			<td>7</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>subtotal</td>
			<td>26</td>
			<td>5</td>
			<td>19%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>5</td>
			<td>19%</td>
		</tr>
		<tr>
			<td rowspan="11">Seafood</td>
			<td>cod</td>
			<td>4</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>mixed</td>
			<td>1</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>salmon</td>
			<td>11</td>
			<td>2</td>
			<td>18%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>2</td>
			<td>18%</td>
		</tr>
		<tr>
			<td>shelfish</td>
			<td>32</td>
			<td>1</td>
			<td>3%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>1</td>
			<td>3%</td>
		</tr>
		<tr>
			<td>snapper</td>
			<td>4</td>
			<td>3</td>
			<td>75%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>3</td>
			<td>75%</td>
		</tr>
		<tr>
			<td>flounder</td>
			<td>12</td>
			<td>4</td>
			<td>33%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>4</td>
			<td>33%</td>
		</tr>
		<tr>
			<td>squid</td>
			<td>4</td>
			<td>1</td>
			<td>25%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>1</td>
			<td>25%</td>
		</tr>
		<tr>
			<td>tilapia</td>
			<td>21</td>
			<td>2</td>
			<td>10%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>2</td>
			<td>10%</td>
			<td>4</td>
			<td>19%</td>
		</tr>
		<tr>
			<td>tuna</td>
			<td>3</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>other</td>
			<td>8</td>
			<td>1</td>
			<td>13%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>1</td>
			<td>13%</td>
		</tr>
		<tr>
			<td>subtotal</td>
			<td>100</td>
			<td>14</td>
			<td>14%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>2</td>
			<td>2%</td>
			<td>16</td>
			<td>16%</td>
		</tr>
		<tr>
			<td rowspan="4">Dairy</td>
			<td>cow</td>
			<td>3</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>goat</td>
			<td>4</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>milk</td>
			<td>3</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>subtotal</td>
			<td>10</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td rowspan="4">Produce</td>
			<td>vegetable</td>
			<td>12</td>
			<td>1</td>
			<td>8%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>1</td>
			<td>8%</td>
		</tr>
		<tr>
			<td>fruit</td>
			<td>1</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>herb</td>
			<td>3</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>subtotal</td>
			<td>16</td>
			<td>1</td>
			<td>6%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>0</td>
			<td>0%</td>
			<td>1</td>
			<td>6%</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td>216</td>
			<td>31</td>
			<td>14%</td>
			<td>1</td>
			<td>0.50%</td>
			<td>0</td>
			<td>0%</td>
			<td>2</td>
			<td>0.90%</td>
			<td>34</td>
			<td>16%</td>
		</tr>
	</tbody>
</table>
Table 3: Prevalence of different C. perfringens toxinotypes in 216 food samples. An example of the results obtained when using this method to test retail food for C. perfringens. This table has been modified from the previously published manuscript Regan et al.16.

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Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

The objective of this protocol is to detect different Clostridium perfringens toxinotypes in locally purchased foods, particularly epsilon toxin producing strain types B and D, without the use of anaerobic chambers.

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized ...

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19.7K Views.  Weill Cornell Medical College. The objective of this protocol is to detect different Clostridium perfringens toxinotypes in locally purchased foods, particularly epsilon toxin producing strain types B and D, without the use of anaerobic chambers.

Video: Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g.&#160;small farms), hospitals, nursing homes, cruise ships, and hotels.

The overall goal of this study was to demonstrate the potential cross contamination mechanism of foodborne pathogen Listeria monocytogenes in a retail deli setting. This methodology may be applied to a variety of different environments to track pathogen contamination. &#160;

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne ...

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf mottling, vein yellowing, defoliation, root decay, and ultimately, the death of the citrus plant. When infected, the citrus plants have stunted growth and produce flowers out of season. These flowers rarely yield fruit, and those that do yield small, bitter, irregularly shaped citrus fruit that are not desirable. This disease is spread by the Asian citrus psyllid, Diaphorina citri, and by the grafting of infected citrus tissue. The pathogen has a long and variable incubation period within the citrus plant&#8212;sometimes years, before symptoms appear. Attempts to culture this pathogen in vitro have been unsuccessful, possibly due to the low and uneven concentration of the pathogen within infected citrus tissue, or because it is difficult to replicate the environmental conditions conducive to growth of the pathogen. It is very difficult to identify the disease before it has spread, due to its long incubation period and researchers&#39; inability to culture the pathogen. As a result, the disease only becomes apparent after suddenly destroying a citrus farmer&#39;s entire yield. Presented here is a method for the accurate and specific detection of the citrus greening pathogen, Candidatus liberibacter spp. using a genomic DNA extraction kit and PCR. This method is simple, efficient, cost effective, and adaptable for quantitative analysis. This method can be adapted for use on any citrus tissue; however, it is potentially limited by the amount of pathogen present in the tissue. Nevertheless, this method will allow citrus farmers to identify infected citrus plants earlier, and curb the spread of this destructive disease before it can further spread.

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus Greening is a particularly destructive disease affecting citrus crops globally. Presented here is a simple method using PCR and genomic DNA extraction of citrus leaf tissue for the accurate and precise identification of the citrus greening pathogen, Candidatus liberibacter spp.

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

Measuring Stolons and Rhizomes of Turfgrasses Using a Digital Image Analysis System

Length and diameter of stolons or rhizomes are usually measured using simple rulers and calipers. This procedure is slow and laborious, so it is often used on a limited number of stolons or rhizomes. For this reason, these traits are limited in their use for morphological characterization of plants. The use of digital image analysis software technology may overcome measurement errors due to human mistakes, which tend to increase as the number and size of samples also increase. The protocol can be used for any kind of crop but is particularly suitable for forage or grasses, where plants are small and numerous. Turf samples consist of aboveground biomass and an upper soil layer to the depth of maximum rhizome development, depending on the species of interest. In studies, samples are washed from the soil, and stolons/rhizomes are cleaned by hand before analysis by digital image analysis software. The samples are further dried in a laboratory heating oven to measure dry weight; therefore, for each sample, the resultant data are total length, total dry weight, and average diameter. Scanned images can be corrected before analysis by excluding visible extraneous parts, such as remaining roots or leaves not removed with the cleaning process. Indeed, these fragments normally have much smaller diameters than stolons or rhizomes, so they can be easily excluded from analysis by fixing the minimum diameter below which objects are not considered. Stolon or rhizome density per unit area can then be calculated based on sample size. The advantage of this method is quick and efficient measurement of the length and average diameter of large sample numbers of stolons or rhizomes.

A software-based image analysis system provides an alternative method to study the morphology of stoloniferous and rhizomatous species. This protocol permits measurement of the length and diameter of stolons and rhizomes and can be applied to samples with a large amount of biomass and to a wide variety of species.

Length and diameter of stolons or rhizomes are usually measured using simple rulers and calipers. This procedure is slow and laborious, so it is often ...

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are potent carcinogens that substantially reduce crop value in maize and other oil rich crops like peanut besides posing serious threat to human and animal health. Different approaches, including traditional breeding, transgenic expression of resistance associated proteins, and RNA interference (RNAi)-based host-induced gene silencing of critical A. flavus gene targets, are being evaluated to increase aflatoxin resistance in susceptible crops. Past studies have shown an important role of &#945;-amylase in A. flavus pathogenesis and aflatoxin production, suggesting this gene/enzyme is a potential target to reduce both A. flavus growth and aflatoxin production. In this regard, the current study was undertaken to evaluate heterologous expression (under control of the constitutive CaMV 35S promoter) of a Lablab purpureus L. &#945;-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is a competitive inhibitor of A. flavus &#945;-amylase enzyme and belongs to the lectin&#8211;arcelin&#8211;&#945;-amylase inhibitor protein family in common bean. In vitro studies prior to the current work had demonstrated the role of AILP in inhibition of A. flavus &#945;-amylase activity and fungal growth. Fungal growth and aflatoxin production in mature kernels were monitored in real time using a GFP-expressing A. flavus strain. This kernel screening assay (KSA) is very simple to set up and provides reliable and reproducible data on infection and the extent of spread that could be quantified for evaluation of germplasm and transgenic lines. The fluorescence from the GFP strain is closely correlated to fungal growth and, by extension, it is well-correlated to aflatoxin values.&#160; The goal of the current work was to implement this previous knowledge in a commercially important crop like maize to increase aflatoxin resistance. Our results show a 35%&#8211;72% reduction in A. flavus growth in AILP-expressing transgenic maize kernels which, in turn, translated into a 62%&#8211;88% reduction in aflatoxin levels.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Here we present a protocol to analyze Aspergillus flavus growth and aflatoxin production in maize kernels expressing an antifungal protein.&#160; Using a GFP-expressing A. flavus strain we monitored the infection and spread of the fungus in mature kernels in real time. The assay is rapid, reliable, and reproducible.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are ...