We kindly thank Sabine Reckinger for excellent technical assistance and support.

1. Preparations
<ol>
	<li>Sample collection 
	Note: The magnetic stirrer method can be used for single or pooled muscle samples.
	<ol>
		<li>Collect samples from the appropriate predilection sites and in an appropriate amount9. Refer to the country requirements or to the recommendations of ICT, OIE or ISO.
		<ol>
			<li>For the European Union, for example, take a 1 g sample from a diaphragm pillar at the transition to the sinewy part of domestic pigs. Ensure that all muscle samples are free of all fat and fascia.</li>
		</ol>
		</li>
		<li>If the tongue is tested, remove the surface layer prior to testing.</li>
		<li>Take care if samples are frozen, as most Trichinella larvae are not freeze resistant and uncoil after death, are less resistant to digestion and tend to stick to the container walls, resulting in decreased assay sensitivity. 
		Note: If frozen, double the sample size at least and increase the sedimentation time (see below).</li>
	</ol>
	</li>
	<li>Preparation of digestion fluid
	<ol>
		<li>Add 25% HCl (16 &#177; 0.5 mL) to 2 L of tap water preheated at 46 - 48 &#176;C into a 3 L glass beaker. Too low of a temperature results in incomplete digestion; too high of a temperature deactivates the enzymatic properties of pepsin.</li>
		<li>Place a stirring rod in the beaker, and stir the solution on a preheated plate (46 - 48 &#176;C).</li>
		<li>Add 10 &#177; 0.2 g of pepsin (1:10,000 NF, 1:12,500 BP, 2,000 FIP) to the acidic solution. 
		Note: The quality of the pepsin is of utmost importance and enzyme activity must be certified by the producer. For laboratory safety reasons and to maintain the pepsin activity, the sequence of adding digestion fluid components must be adhered to.</li>
	</ol>
	</li>
	<li>Muscle sample preparation
	<ol>
		<li>Chop up to 100 g of muscle samples in a blender or grinder. To facilitate blending, add 2 mL preheated water to the samples and blend at max speed for 2 - 3 s.</li>
		<li>Before the second blending, open the blender and transfer any muscle sample at the top of the blending bowl edge margin to the bottom and repeat blending. Continue blending with bursts of 2 - 3 s until no visible pieces of meat remain. 
		Note: Too little blending may result in incomplete digestion, while too much blending can damage the Trichinella larvae present in the samples6.</li>
	</ol>
	</li>
</ol>
2. Procedure
<ol>
	<li>Muscle sample digestion
	<ol>
		<li>Transfer 1 L of the digestion fluid into a cylinder. Transfer the ground meat into the beaker and disseminate in the digestion fluid with a spoon or fork. Thoroughly rinse the blender lid, blade and blender bowl with 500 mL of the preheated digestion fluid into the 3 L beaker. 
		Note: Incomplete rinsing can result in the loss of sensitivity.</li>
		<li>Cover the beaker with aluminum foil and keep a constant temperature of 44 - 46 &#176;C and monitor with a thermometer.</li>
		<li>Stir the digestion fluid for 30 min to create a deep vortex without splashing until the meat particles disappear. Depending on the type of meat tested (e.g. meat of wild animals), increase the digestion time until a maximum of 60 min.</li>
	</ol>
	</li>
	<li>Filtration and sedimentation of the digestion fluid
	<ol>
		<li>To determine the amount of undigested tissue, adjust the scale to zero, weigh the sieve before use, and note its weight. The sieve should be clean and devoid of any tissue particles, which could hinder the Trichinella larvae reaching the sedimentation funnel. Check that the separation funnel is leveled vertically.</li>
		<li>After digestion, carefully pour the digestion fluid through a 180 &#181;m sieve into a separation glass funnel. The width of the separation glass funnel should not be larger than 55% of the length to allow good larva sedimentation. Take care to avoid any overflow.</li>
		<li>To avoid loss of sensitivity, thoroughly rinse the glass beaker and the sieve with approximately 200 mL of tap water from a squeeze wash bottle and transfer the rinsing water into the sedimentation glass funnel. Take care to carefully rinse the edges of the 3 L beaker. Lift the sieve and rinse the area under the sieve thoroughly.</li>
	</ol>
	</li>
	<li>Determination of undigested tissue amount 
	Note: Due to errors in the execution of the method, muscle tissue can remain on the sieve, as well fascia and connective tissue which are indigestible. The amount of undigested tissue is determined during the first 10 min sedimentation step (see 2.5). This allows approximately 30 min for the sieve to dry, as residual water can influence the determined weight of the undigested tissue.
	<ol>
		<li>Place a paper towel on a scale, and weigh the sieve with the undigested tissues.</li>
		<li>Subtract the sieve weight before filtration from the sieve weight with undigested tissues. The digestion process is considered satisfactory if not more than 5% of the starting sample weight remains on the sieve (i.e. 5 g/100 g starting material).</li>
		<li>If the amount of undigested tissue exceeds 5%, repeat the test using fresh muscle samples. If the remaining tissue is predominantly comprised of undigested muscle tissue and fresh samples are not available, digest the undigested remains again and examine in addition to the original samples.</li>
	</ol>
	</li>
	<li>Sedimentation of the digestion fluid
	<ol>
		<li>Keep the filtered digestion fluid in the separatory funnel for 30 min. If left undisturbed, the Trichinella larvae will settle at the bottom of the funnel.</li>
		<li>If frozen samples are used, increase the sedimentation time to a maximum of 60 min.</li>
		<li>Once sedimentation is complete, fully open the stopcock of the separatory funnel to let at least 40 mL of digestion fluid quickly run off into a measuring cylinder (80 mL if muscle samples had been previously frozen).</li>
		<li>First, let half of the fluid into the cylinder and then fully open the stopcock in the opposite direction to allow 10 mL of digestion fluid to pass. Finally, open the stopcock in the opposite direction for another 10 mL. This procedure ensures that Trichinella larvae are not trapped in the stopcock. 
		Note: Sufficient speed is needed to avoid larvae remaining in the separatory funnel, decreasing sensitivity.</li>
	</ol>
	</li>
	<li>Sedimentation of the digestion fluid in the cylinder
	<ol>
		<li>Leave the sediment in the cylinder for 10 min to allow larvae to sediment. Remove 30 mL supernatant by suctioning with a pipette without disturbing the 10 mL sediment.</li>
		<li>To reduce the amount of tissue fibers in the sample, add 30 mL tap water to the sediment and leave for another 10 min. Repeat until the liquid is clear.</li>
		<li>Remove supernatant and transfer the 10 mL washed sediment to a gridded Petri dish or larval counting basin. Rinse the cylinder with 10 mL tap water and then add the water to the sample. 
		Note: As large amounts of debris in the sample can prevent the identification of the Trichinella larvae (Figure 1), further washing steps should be performed, if required.</li>
	</ol>
	</li>
	<li>Microscopic examination
	<ol>
		<li>Use a trichinoscope or a stereo-microscope with a sub-stage transmitted light source of adjustable intensity at a 15 to 20X magnification to examine the samples immediately after the washing steps.</li>
		<li>After pouring the sample into the gridded Petri dish, examine the dish/basin systematically grid by grid. If no suspect structures are found, gently move the fluid in the gridded Petri dish or larval counting basin in a circular fashion to allow any Trichinella larvae, which were potentially overlooked, to accumulate in the center of the Petri dish. Repeat the examination.</li>
		<li>If a suspect structure is found, increase magnification to 40 - 100X. Remove Trichinella larvae from the dish/basin with a pipette and collect in a tube with 90% ethanol.</li>
		<li>After the complete dish has been examined, repeat the procedure, starting from the gentle shaking of the dish/basin. Repeat the procedure at least three times or until no more larvae are found.</li>
		<li>Count the number of larvae. Correlate to the amount of muscle tissue tested and present as larvae per gram (lpg).</li>
		<li>If too many larvae are present in the digestion fluid to determine the larval number reliably, return the fluid containing the larvae to the measuring cylinder, rinse with tap water from a squeeze wash bottle, and leave larvae to settle to the bottom.</li>
		<li>After a 10 min sedimentation time, reduce the supernatant to a defined volume (e.g. 10 mL). To determine the exact volume, transfer the supernatant into a new cylinder with a pipette.</li>
		<li>Suspend larvae homogenously in the fluid by vigorous shaking. During the shaking motion, add 12 drops of 20 &#181;L larval suspension onto a Petri dish.</li>
		<li>Examine each drop by microscope. Determine the number of larvae in 240 &#181;L and delete the highest and lowest count. Extrapolate the number of larvae to the total volume (e.g. 10 mL) of supernatant. 
		Note: The quality of the stereo-microscope is of paramount importance for the identification of Trichinella larvae.</li>
		<li>When larvae are collected in a tube by a pipette, check carefully that the larvae did not stick on the pipette wall but are transferred to the tube. 
		Note: Positive Trichinella findings from pooled samples must be traced back from the pool to the carcass of origin. Here, the pool size should progressively be decreased until the positive carcass is identified. The sample size can also be increased during this process to increase sensitivity. Where examination of a collective sample produces a positive or uncertain result, a further 20 g sample is taken from each pig. The 20 g samples from five pigs are pooled and examined using the described method. In this way samples from 20 groups of five pigs will be examined. When Trichinella is detected in a pooled sample from five pigs, 20 g samples are collected from the individual pigs in the group and each is examined separately until the positive carcass of origin is detected.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Gottstein, B., Pozio, E., N&#246;ckler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology,+Diagnosis,+Treatment,+and+Control+of+Trichinellosis.">Epidemiology, Diagnosis, Treatment, and Control of Trichinellosis.</a> Clin. Microbiol. Rev. 22, 127-145 (2009).</li><li>Pozio, E., Murrell, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematics+and+epidemiology+of+Trichinella.">Systematics and epidemiology of Trichinella.</a> Adv. Parasitol. 63, 367-439 (2006).</li><li>9 CFR Part 318.10 - Prescribed treatment of pork and products containing pork to destroy trichinae. U.S. Government Publishing Office Available from: <a target="_blank" href="https://www.gpo.gov/fdsys/pkg/CFR-2016-title9-vol2/pdf/CFR-2016-title9-vol2-sec318-10.pdf">https://www.gpo.gov/fdsys/pkg/CFR-2016-title9-vol2/pdf/CFR-2016-title9-vol2-sec318-10.pdf</a> (2012)</li><li>Gamble, H. 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=International+Commission+on+Trichinellosis:+recommendations+on+methods+for+the+control+of+Trichinella+in+domestic+and+wild+animals+intended+for+human+consumption.">International Commission on Trichinellosis: recommendations on methods for the control of Trichinella in domestic and wild animals intended for human consumption.</a> Vet. Parasitol. 93 (3-4), 393-408 (2000).</li><li>Gajadhar, A. A., Forbes, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+internationally+recognized+quality+assurance+system+for+diagnostic+parasitology+in+animal+health+and+food+safety,+with+example+data+on+trichinellosis.">An internationally recognized quality assurance system for diagnostic parasitology in animal health and food safety, with example data on trichinellosis.</a> Vet. Parasitol. 103, 133-140 (2002).</li><li>N&#246;ckler, K., Kapel, C. M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3,+Detection+and+surveillance+for+Trichinella:+meat+inspection+and+hygiene,+and+legislation.">Chapter 3, Detection and surveillance for Trichinella: meat inspection and hygiene, and legislation.</a> FAO/WHO/OIE Guidelines for the surveillance, management, prevention and control of trichinellosis. , 69-98 (2007).</li><li>Boch, J., Schnieder, T., Supperer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Veterin&#228;rmedizinische Parasitologie. , (2006).</li><li>Despommier, D. D., M&#252;ller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stichosome+and+its+secretion+granules+in+the+mature+muscle+larva+of+Trichinella+spiralis.">The stichosome and its secretion granules in the mature muscle larva of Trichinella spiralis.</a> J. Parasitol. 62 (5), 775-785 (1976).</li><li>Marucci, G., Interisano, M., La Rosa, G., Pozio, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+identification+of+nematode+larvae+different+from+those+of+the+Trichinella+genus+detected+by+muscle+digestion.">Molecular identification of nematode larvae different from those of the Trichinella genus detected by muscle digestion.</a> Vet Parasitol. 194, 117-120 (2013).</li><li>Forbes, L. B., Gajadhar, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+validated+Trichinella+digestion+assay+and+an+associated+sampling+and+quality+assurance+system+for+use+in+testing+pork+and+horse+meat.">A validated Trichinella digestion assay and an associated sampling and quality assurance system for use in testing pork and horse meat.</a> J. Food Prot. 62, 1308-1313 (1999).</li><li>Malakauskas, A., 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=Molecular+epidemiology+of+Trichinella+spp.+in+three+Baltic+countries:+Lithuania,+Latvia,+and+Estonia.">Molecular epidemiology of Trichinella spp. in three Baltic countries: Lithuania, Latvia, and Estonia.</a> Parasitol. Res. 100 (4), 687-693 (2007).</li><li>Kapel, C. M., Webster, P., Gamble, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+distribution+of+sylvatic+and+domestic+Trichinella+larvae+in+production+animals+and+wildlife.">Muscle distribution of sylvatic and domestic Trichinella larvae in production animals and wildlife.</a> Vet. Parasitol. 132 (1-2), 101-105 (2005).</li><li>Interisano, M., 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=Validation+of+a+latex+agglutination+test+for+the+detection+of+Trichinella+infections+in+pigs.">Validation of a latex agglutination test for the detection of Trichinella infections in pigs.</a> Vet. Parasitol. 194, 121-124 (2013).</li><li>Pozio, E., La Rosa, ., G, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR-derived+methods+for+the+identification+of+Trichinella+parasites+from+animal+and+human+samples.">PCR-derived methods for the identification of Trichinella parasites from animal and human samples.</a> Methods Mol. Biol. 216, 299-309 (2003).</li></ol>

The authors declare that they have no competing financial interests.

Although the magnetic stirrer method can be easily performed, not strictly adhering to technical details leads to insufficient sensitivity, thus endangering consumer health. The critical control points have been highlighted in the text above. In addition, the use of appropriate equipment is crucial for the successful outcome of the test. All vessels should be made of glass and pipette tips coated with silicon to reduce adherence of larvae to the surface.
The sensitivity of the digestion method depends on the larval density in the muscles and the amount of muscle sample tested. For a larval density of 3 - 5 lpg of muscle tissues, a sensitivity of 100% was reported, but below 1 lpg the sensitivity dropped to 40%13. Depending on the tested host animal species, the sensitivity of the test can be improved by increasing the amount of sample used. The infection burden in wildlife is usually low, therefore larger samples sizes are used14. Predilection sites also vary among host animals. Here, the lower digestibility of some muscle tissues such as the tongue needs to be taken into account, which can also result in larger sample sizes15.
Further, it is important to understand that the magnetic stirrer method does not include internal controls. Therefore, quality assurance management from sampling to documentation is essential to obtain accurate and precise results. The quality of laboratory performance to detect Trichinella larvae in muscle samples should be monitored by regular participation of each analyst in proficiency tests.
Further limitations of the method are that the final identification stage of muscle larvae by microscopy is highly subjective and heavily dependent on the knowledge of larval morphology by the examining analyst.
An interesting alternative method to circumvent this problem is the magnetic stirrer method/on filter isolation followed by larvae detection by a latex agglutination test. However, according to EU legislation this method is only considered equivalent for the testing of meat of domestic swine but not for animals which are at higher risk of infection such as wild boar4. The identification of the larval antigen with monoclonal antibodies makes the test more objective, but denies the laboratory the possibility of determining the number of larvae per gram of meat16.
Further variations of the magnetic stirrer method include the mechanically assisted pooled sample digestion method/sedimentation technique, the mechanically assisted pooled sample digestion method/on filter isolation technique, and the automatic digestion method for pooled samples of up to 35 g technique. These techniques are all based on the artificial digestion of the meat and the visualization and counting of Trichinella larvae, but differ in the equipment used for the filtration and sedimentation steps.
The isolated larvae can be further identified at the species level by a molecular test (e.g., multiplex PCR)17. According to EU legislation, all positive samples must be forwarded to the national reference laboratory or to EURLP for the Trichinella species determination.

Nematodes of the genus Trichinella can be detected in striated muscles of carnivore and omnivore mammals, birds, and reptiles worldwide. These zoonotic nematodes can reach human beings when raw or semi-raw meat and meat derived products from swine, horse, and game animals are ingested. This zoonosis can be a serious disease in humans characterized by pathognomonic signs and symptoms, e.g. diarrhea, fever, periorbital edema and myalgia and possible complications such as myocarditis, thromboembolic disease and encephalitis1.
Trichinella spiralis is the most widespread etiological agent of Trichinella infection in wild and domestic animals and causes most of the human infections worldwide. In Europe, North and West Africa, and Western Asia, Trichinella britovi is another source of human infections. In addition, ten other taxa are less commonly reported as the causative agents of the human disease and are found in different regions of the world, usually in wild animals2. In addition to wild animals such as wild boar, bears, walruses and badgers, pork represents the most important source of human infection worldwide.
The best way to prevent trichinellosis is to refrain from eating raw meat products and to cook any meat, especially game meat to safe temperatures (&#62; 65 &#176;C for 1 min, i.e. to change the meat color from pink to brown in the core of the meat product)3. The European Union and USDA have specified freezing and cooking times and temperatures for pork products that can be used to kill T. spiralis larvae in meat4,5. Furthermore, recommendations for the inactivation of Trichinella in meat and meat products were published by the International Commission on Trichinellosis6. In Europe, in addition to the introduction of controlled housing conditions in commercial swine herds where the risk of Trichinella infection is negligible, the prevention of human trichinellosis is based on the laboratory examination of muscle samples from susceptible animals4.
For food safety purposes, digestion assays are the only reliable procedures for the direct detection of Trichinella larvae in meat3,7. Even if there are several variations of the digestion assay, the magnetic stirrer method is the internationally accepted reference method3,4,7. This assay is based on the detection of Trichinella larvae in striated muscle tissues. After the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps, Trichinella larvae are identified by microscopy. Depending on trade obligations and national legislation, there are a multitude of small variations in the general protocol of the magnetic stirrer method. Here, technical details are based on EU requirements4, ISO specifications8, ICT guidelines6, and the experience of the European Union Reference Laboratory for Parasites (EURLP) and the German Reference Laboratory for Trichinella.

<table border="0" cellpadding="0" cellspacing="0" width="1019">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Knife,Scissors, Tweezers&#160;</td>
			<td style="width:356px;"></td>
			<td style="width:80px;"></td>
			<td style="width:363px;">Cutting samples and removing non-digestible tissue</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Blender&#160;</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">With a sharp chopping blade</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Magnetic stirrer</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">With thermostatically controlled heating plate</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Stirring Rod</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Teflon-coated, approximately 5 cm long</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:220px;">Conical glass separation funnel</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Capacity of at least 2 litres, preferably fitted with Teflon safety plugs. The width should not be larger than 55% of the length to allow a good larva sedimentation</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Stands, rings and clamps</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:220px;">Sieve</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Mesh size 180 microns, external diameter 11 cm, with stainless steel mesh</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Funnel</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Internal diameter not less than 12 cm, to support the sieves</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Glass beaker</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Capacity 3 litres</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Glass measuring cylinder</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Capacity 50 to 100 ml, or centrifuge tube</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:220px;">Stereo-microscope</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">With a substage transmitted light source of adjustable intensity or trichinoscope with a horizontal table</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:220px;">Petri dishes</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">9 cm diameter, marked on their undersides into 10 &#215; 10 mm square examination areas using a pointed instrument or a larval counting basin for the tricinoscope</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Aluminium foil</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Alternatively a lid to cover the beakers</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">25 % hydrochloric acid</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:220px;">Pepsin</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Strength: 1:10 000 NF (US National Formulary) corresponding to 1:12 500 BP (British Pharmacopoeia) and to 2 000 FIP (F&#233;d&#233;ration internationale de pharmacie), or stabilised liquid pepsin with minimum 660 European Pharmacopoeia units/ml</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Ethanol</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">70-90% ethyl alcohol</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Tap water</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Heated to 46-48 &#176;C before use</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Balance</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Accurate to at least 0.1 g</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:220px;">Metal tray</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Capacity 10 to 15 litres, to collect the remaining digestive juice</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Pipettes and pipette holder</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Different sizes (1, 10 and 25 ml)</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:220px;">Thermometer&#160;</td>
			<td style="width:356px;"></td>
			<td></td>
			<td style="width:363px;">Accurate to 0.5 &#176;C, temperature range at least from 20 &#176; C to 70 &#176; C</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:220px;">Siphon&#160;</td>
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magnetic stirrer method for the detection of trichinella larvae in muscle samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

After digestion, the shape of the infective muscle larvae can vary, making identification more difficult. Typical forms include tightly coiled larvae, lightly coiled larvae or c-shaped or completely uncoiled larvae (Figures 2B - 2C). The number of larvae found per gram can vary considerably and can range from one larva to a few hundred larvae.
The morphology of the infective muscle larva is shown in Figure 2A. Trichinella larvae are 0.7 to 1.5 mm long and approximately 0.3 mm in width. The esophagus is narrow and has a slightly rounded end. The cuticle is smooth. In the anterior half of the body cavity, the stichosome, a structure constituted of a long slender tube surrounded by a row of 45 to 55 large cells (stichocytes), can be observed. The rectum is also rounded without any projections or appendages10,11.
Other structures, besides Trichinella larvae, can be found after muscle digestion. Some examples are shown in Figures 3A - 3F. Wild animals are often infected with other parasites or the muscle samples available for testing are contaminated during the evisceration process by animate or inanimate structures/organisms. The length of the larvae, the appearance of the anterior and posterior ends and the occurrence of the stichosome help to discriminate between the different nematode genera12.
<img alt="Figure 1" src="/files/ftp_upload/55354/55354fig1.jpg" /> 
Figure 1: Microscopic Examination of Trichinella Larvae After (A) Insufficient and (B) Sufficient Rinsing Steps. Scale bars indicate 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55354/55354fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55354/55354fig2.jpg" /> 
Figure 2: Morphology of Infective Trichinella Larvae Showing the Rectum, Stichosome and Esophagus of the Larva (A). Possible shapes of the muscle larvae after digestion showing (B) tightly coiled and lightly coiled moving larvae and (C) uncoiled larvae. Scale bars indicate 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55354/55354fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55354/55354fig3.jpg" /> 
Figure 3: Examples of Incidental Findings after Digestion of Muscle Samples with the Magnetic Stirrer Method. (A) bristle-like hair of earth worm found in wild boar; (B) Alaria alata from wild boar; (C) muscle fiber from wild boar; (D) Metastrongylus sp. from wild boar; (E) plant fiber found in wild boar (F): Toxocara sp. larva from wild boar. Scale bars indicate 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55354/55354fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples at JoVE.com

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

magnetic-stirrer-method-for-detection-trichinella-larvae-muscle

Research

JoVE Journal

Immunology and Infection

28.5K Views.  Federal Institute for Risk Assessment. The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Video: Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

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 ...

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to the best of these authors' knowledge, no method has been developed to allow for the in situ visualization of the 
bacteria in histological sections.
While bacterial localization has been determined in vivo in previous studies using plate counts 3, radiometric labeled 4, 
or bioluminescent bacteria 5, most of these studies have only been performed at the gross organ level, with one exception 6. This limitation 
is of particular concern because E. ictaluri has a complex infection cycle 1,7, and it has a variety of virulence factors 8,9. 
The complex interaction of E. ictaluri with its host is similar in many respects to Salmonella typhi 10, which is in the same taxonomic family.
Here we describe a technique allowing for the detection of bacteria using indirect immuno-histochemistry using the monoclonal Ed9 antibody described by Ainsworth et al.11.
Briefly, a blocking serum is applied to paraffin embedded histological sections to prevent non-specific biding. Then, the 
sections are incubated with the primary antibody: E. ictaluri specific monoclonal antibody Ed9. Excess antibodies are rinsed away and the FitC 
labeled secondary antibodies are added. After rinsing, the sections are mounted with a fluorescent specific mounting medium. 
This allowed for the detection of E. ictaluri in situ in histological sections of channel catfish tissues.

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

Here we describe a procedure allowing the labeling of Edwardsiella ictaluri in situ in histological sections from channel catfish Ictalurus punctatus using indirect immunohistochemistry with monoclonal antibodies Ed9 as a primary, and fluorescent FitC labeled antibodies as a secondary. This allowed for the detection of the bacterium using fluorescent microscopy.

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to ...

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

A Method for Targeted 16S Sequencing of Human Milk Samples

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.

A semi-automated workflow is presented for targeted sequencing of 16S rRNA from human milk and other low-biomass sample types.

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as ...

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 ...