Die Autoren danken den Mitgliedern des FDA Microbiology Methods Validation Subcommittee (MMVS) und des Bacteriological Analytical Manual (BAM) Council für die kritische Überprüfung von Salmonella LAMP-Methodenvalidierungsstudien.

<ol>
	<li>Notomi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+of+DNA.">Loop-mediated isothermal amplification of DNA.</a> Nucleic Acids Research. 28 (12), 63 (2000).</li><li>Kaneko, H., Kawana, T., Fukushima, E., Suzutani, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+of+loop-mediated+isothermal+amplification+to+a+culture+medium+and+biological+substances.">Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances.</a> Journal of Biochemical and Biophysical Methods. 70 (3), 499-501 (2007).</li><li>Francois, P., 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=Robustness+of+a+loop-mediated+isothermal+amplification+reaction+for+diagnostic+applications.">Robustness of a loop-mediated isothermal amplification reaction for diagnostic applications.</a> FEMS Immunology and Medical Microbiology. 62 (1), 41-48 (2011).</li><li>Yang, Q., Wang, F., Prinyawiwatkul, W., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robustness+of+Salmonella+loop-mediated+isothermal+amplification+assays+for+food+applications.">Robustness of Salmonella loop-mediated isothermal amplification assays for food applications.</a> Journal of Applied Microbiology. 116 (1), 81-88 (2014).</li><li>Nagamine, K., Watanabe, K., Ohtsuka, K., Hase, T., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+reaction+using+a+nondenatured+template.">Loop-mediated isothermal amplification reaction using a nondenatured template.</a> Clinical Chemistry. 47 (9), 1742-1743 (2001).</li><li>Zhang, X., Lowe, S. B., Gooding, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+review+of+monitoring+methods+for+loop-mediated+isothermal+amplification+(LAMP).">Brief review of monitoring methods for loop-mediated isothermal amplification (LAMP).</a> Biosensors &amp; Bioelectronics. 61, 491-499 (2014).</li><li>Nagamine, K., Hase, T., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+reaction+by+loop-mediated+isothermal+amplification+using+loop+primers.">Accelerated reaction by loop-mediated isothermal amplification using loop primers.</a> Molecular and Cellular Probes. 16 (3), 223-229 (2002).</li><li>Yang, Q., Domesle, K. J., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+for+Salmonella+detection+in+food+and+feed:+Current+applications+and+future+directions.">Loop-mediated isothermal amplification for Salmonella detection in food and feed: Current applications and future directions.</a> Foodborne Pathogens and Disease. 15 (6), 309-331 (2018).</li><li>Mori, Y., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP):+Expansion+of+its+practical+application+as+a+tool+to+achieve+universal+health+coverage.">Loop-mediated isothermal amplification (LAMP): Expansion of its practical application as a tool to achieve universal health coverage.</a> Journal of Infection and Chemotherapy. 26 (1), 13-17 (2020).</li><li>Mansour, S. M., Ali, H., Chase, C. C., Cepica, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+for+diagnosis+of+18+World+Organization+for+Animal+Health+(OIE)+notifiable+viral+diseases+of+ruminants,+swine+and+poultry.">Loop-mediated isothermal amplification for diagnosis of 18 World Organization for Animal Health (OIE) notifiable viral diseases of ruminants, swine and poultry.</a> Animal Health Research Reviews. 16 (2), 89-106 (2015).</li><li>Kumar, Y., Bansal, S., Jaiswal, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP):+A+rapid+and+sensitive+tool+for+quality+assessment+of+meat+products.">Loop-mediated isothermal amplification (LAMP): A rapid and sensitive tool for quality assessment of meat products.</a> Comprehensive Reviews in Food Science and Food Safety. 16 (6), 1359-1378 (2017).</li><li>Kundapur, R. R., Nema, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification:+Beyond+microbial+identification.">Loop-mediated isothermal amplification: Beyond microbial identification.</a> Cogent Biology. 2, 1137110 (2016).</li><li>. Compliance Policy Guide Sec. 690.800 Salmonella in Food for Animals Available from: <a target="_blank" href="https://www.fda.gov/downloads/iceci/compliancemanuals/compliancepolicyguidancemanual/ucm361105.pdf">https://www.fda.gov/downloads/iceci/compliancemanuals/compliancepolicyguidancemanual/ucm361105.pdf</a> (2013)</li><li>Bird, P., 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=Evaluation+of+the+3M+molecular+detection+assay+(MDA)+2+-+Salmonella+for+the+detection+of+Salmonella+spp.+in+select+foods+and+environmental+surfaces:+collaborative+study,+first+action+2016.01.">Evaluation of the 3M molecular detection assay (MDA) 2 - Salmonella for the detection of Salmonella spp. in select foods and environmental surfaces: collaborative study, first action 2016.01.</a> Journal of AOAC International. 99 (4), 980-997 (2016).</li><li>D'Agostino, 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+loop-mediated+amplification/ISO+6579-based+method+for+analysing+soya+meal+for+the+presence+of+Salmonella+enterica.">Validation of a loop-mediated amplification/ISO 6579-based method for analysing soya meal for the presence of Salmonella enterica.</a> Food Analytical Methods. 9 (11), 2979-2985 (2016).</li><li>Ge, 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=Multi-laboratory+validation+of+a+loop-mediated+isothermal+amplification+method+for+screening+Salmonella+in+animal+food.">Multi-laboratory validation of a loop-mediated isothermal amplification method for screening Salmonella in animal food.</a> Frontiers in Microbiology. 10, 562 (2019).</li><li>D'Agostino, M., Diez-Valcarce, M., Robles, S., Losilla-Garcia, B., Cook, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+loop-mediated+isothermal+amplification-based+method+for+analysing+animal+feed+for+the+presence+of+Salmonella.">A loop-mediated isothermal amplification-based method for analysing animal feed for the presence of Salmonella.</a> Food Analytical Methods. 8 (10), 2409-2416 (2015).</li><li>Galan, J. E., Ginocchio, C., Costeas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+functional+characterization+of+the+Salmonella+invasion+gene+invA:+homology+of+InvA+to+members+of+a+new+protein+family.">Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family.</a> Journal of Bacteriology. 174 (13), 4338-4349 (1992).</li><li>Chen, S., Wang, F., Beaulieu, J. C., Stein, R. E., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+viable+salmonellae+in+produce+by+coupling+propidium+monoazide+with+loop-mediated+isothermal+amplification.">Rapid detection of viable salmonellae in produce by coupling propidium monoazide with loop-mediated isothermal amplification.</a> Applied and Environmental Microbiology. 77 (12), 4008-4016 (2011).</li><li>Yang, Q., Chen, S., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+Salmonella+serovars+in+shell+eggs+by+loop-mediated+isothermal+amplification.">Detecting Salmonella serovars in shell eggs by loop-mediated isothermal amplification.</a> Journal of Food Protection. 76 (10), 1790-1796 (2013).</li><li>Yang, Q., 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=Evaluation+of+loop-mediated+isothermal+amplification+for+the+rapid,+reliable,+and+robust+detection+of+Salmonella+in+produce.">Evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of Salmonella in produce.</a> Food Microbiology. 46, 485-493 (2015).</li><li>Yang, Q., Domesle, K. J., Wang, F., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+Salmonella+in+food+and+feed+by+coupling+loop-mediated+isothermal+amplification+with+bioluminescent+assay+in+real-time.">Rapid detection of Salmonella in food and feed by coupling loop-mediated isothermal amplification with bioluminescent assay in real-time.</a> BMC Microbiology. 16 (1), 112 (2016).</li><li>Domesle, K. J., Yang, Q., Hammack, T. S., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+Salmonella+loop-mediated+isothermal+amplification+assay+in+animal+food.">Validation of a Salmonella loop-mediated isothermal amplification assay in animal food.</a> International Journal of Food Microbiology. 264, 63-76 (2018).</li><li>Bustin, S. 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=The+MIQE+guidelines:+minimum+information+for+publication+of+quantitative+real-time+PCR+experiments.">The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.</a> Clinical Chemistry. 55 (4), 611-622 (2009).</li></ol>

Die Autoren erklären, dass sie keine konkurrierenden finanziellen Interessen haben. Die in diesem Manuskript zum Ausdruck gebrachten Ansichten sind die der Autoren und spiegeln nicht unbedingt die offizielle Politik des Department of Health and Human Services, der U.S. Food and Drug Administration oder der US-Regierung wider. Die Bezugnahme auf kommerzielle Materialien, Ausrüstungen oder Verfahren stellt in keiner Weise eine Genehmigung, Billigung oder Empfehlung der Food and Drug Administration dar.

Wir haben hier eine einfache, schnelle, spezifische und empfindliche LAMP-Methode zum Screening und zur Bestätigung von Salmonellen in Tierfutter bzw. reiner Kultur vorgestellt. Mit dem Komfort eines isothermen Master-Mix, der vier Schlüsselreagenzien und einen gebrauchsfertigen, hausinternen Primer-Mix enthält, erfordert die Montage einer LAMP-Reaktion nur wenige Pipettierschritte(Abbildung 1). Die Gesamtlaufzeit einschließlich Amplifikation und Annealphase beträgt weniger als 38 min(Abbildung 2A,B und Abbildung 3A,B). Positive Ergebnisse werden über Echtzeitfluoreszenz überwacht (Abbildung 2C und Abbildung 3C,D) und können bereits in 5 min26nachgewiesen werden. Die Nealphase dient als zusätzliche Bestätigung der LAMP-Spezifität, da nur Proben mit korrektem Tm (ca. 90 °C) als positiv gemeldet werden(Abbildung 2D,E und Abbildung 3E-G). Empfindlichkeiten von 1 Salmonellenzelle in Reinkultur und < 1 KBE/25 g in Tierfutter wurden bereits26berichtet.
Da LAMP sehr effektiv ist und eine große Menge an DNA1erzeugt, ist es wichtig, dass die besten Laborpraktiken verwendet werden, um Kreuzkontaminationen zu verhindern, die die Bereiche für die Vorbereitung des LAMP-Master-Mix physisch trennen und DNA-Vorlagen hinzufügen können, die Erzeugung von Aerosolen zu vermeiden, Filterpipettenspitzen zu verwenden, Handschuhe häufig zu wechseln und auf das Öffnen von LAMP-Reaktionsröhren nach der Amplifikation zu verzichten.
Die Spezifität dieser Salmonella LAMP-Methode wurde zuvor mit 300 Bakterienstämmen (247 Salmonellen von 185 Serovars und 53 Nicht-Salmonellen) getestet und nachweislich 100% spezifisch26. Insbesondere wurden signifikante Unterschiede in Tmax zwischen den beiden Salmonellenarten S. enterica und Salmonella bongoriund zwischen s. enterica Subspecies, insbesondere subsp. arizonae (IIIa)26beobachtet. Dennoch waren dies nach wie vor gültige positive Ergebnisse nach den Regeln für die Interpretation der LAMP-Ergebnisse. In unserer multilaborativen kollaborativen Studie in trockener Hundenahrung, an der 14 Analytiker19beteiligt waren, wurden gelegentlich Proben mit inkonsistenten Ergebnissen in doppelten LAMP-Läufen beobachtet. In der Regel handelte es sich um Proben mit verzögerten positiven Ergebnissen (Tmax > 15 min). Wiederholte beide Läufe unabhängig voneinander löste das Problem in der Regel. Seltener beobachteten wir Proben mit korrekten Tm, aber keine oder unregelmäßige Tmax Werte (< 5 min). Dies wurde in der Regel durch Luftblasen im Reaktionsrohr verursacht.
Während des gesamten Lebenszyklus der Entwicklung, Evaluierung, Präkollaborativen Studie und Multilaborierung von LAMP haben wir eine hohe Toleranz von LAMP gegenüber Inhibitoren in verschiedenen Tiernahrungs- oder Lebensmittelmatrizen und Kulturmedien4,19,22,23,24beobachtet, die die Robustheit der Methode hervorhebt und zahlreiche andere Studien auf globaler Ebene8kooperiert. Dies ist besser als PCR oder Echtzeit-PCR, die in der Regel eine interne Verstärkungskontrolle erfordert, um sicherzustellen, dass negative Ergebnisse nicht auf Matrix-Hemmung28zurückzuführen sind. Darüber hinaus zeigte LAMP in der überwiegenden Mehrheit der Studien eine ähnliche (oder überlegene) Spezifität und Empfindlichkeit im Vergleich zu PCR oder Echtzeit-PCR in der überwiegenden Mehrheit der Studien8. Die Kosten für LAMP-Reagenzien belaufen sich auf ca. 1 USD pro Reaktion. Die in diesem Protokoll verwendeten LAMP-Instrumente sind klein, wartungsarm und tragbar. Sie können jede isotherme Amplifikationsmethode handhaben, die die Zieldetektion durch Fluoreszenzmessung verwendet, LAMP inklusive. Mit der LAMP-Software können umfassende Berichte in mehreren Formaten (pdf, Text und Bild) generiert werden.
Die Methodenvalidierung ist ein wichtiger Schritt, bevor eine neue Methode für die Routineanwendung übernommen werden kann. Bemerkenswert ist, dass das hier gemeldete LAMP-Protokoll die Multilabor-Validierung erfolgreich abgeschlossen hat19. Mit der kürzlicherfolgten Aufnahme dieses LAMP-Protokolls in das BAM Chapter 5 Salmonella27der US-amerikanischen FDA wird erwartet, dass die Methode viel breiter genutzt wird, sowohl als Schnellscreening-Methode in Tiernahrung als auch als zuverlässige Bestätigungsmethode für mutmaßliche Salmonellenisolate aus allen Lebensmittelkategorien.

Loop-mediale isotherme Amplifikation (LAMP) ist ein neuartiger isothermaler Nukleinsäure-Amplifikationstest (iNAAT), der im Jahr 2000 von einer Gruppe japanischer Wissenschaftler erfunden wurde1. Durch die Bildung einer zielspezifischen Stammschleifen-DNA-Struktur in den ersten Schritten verwendet LAMP eine Strang-verdrängende DNA-Polymerase, um dieses Ausgangsmaterial quasi-exponentiell effizient zu verstärken, was zu 109 Kopien des Ziels in weniger als 1 h1führt. Im Vergleich zur Polymerase-Kettenreaktion (PCR), einem weit verbreiteten NAAT, hat LAMP mehrere Vorteile. Zunächst werden LAMP-Reaktionen unter isothermen Bedingungen durchgeführt. Dies ermuntert die Notwendigkeit eines ausgeklügelten thermischen Fahrradinstruments. Zweitens ist LAMP sehr tolerant gegenüber Kulturmedien und biologischen Substanzen2 mit Robustheit, die sowohl für klinische als auch für Lebensmittelanwendungen nachgewiesen wird3,4. Dies vereinfacht die Probenvorbereitung und minimiert falsche negative Ergebnisse5. Drittens ist LAMP für mehrere Detektionsplattformen wie Trübung, Colorimetrie, Biolumineszenz, Fluoreszenz und Mikrofluidik6zugänglich. Viertens ist LAMP sehr spezifisch, da es vier bis sechs speziell entwickelte Primer verwendet, um sechs bis acht spezifische Regionen1,7anzuvisieren. Fünftens ist LAMP ultrasensibel und zahlreiche Studien haben seine überlegene Empfindlichkeit gegenüber PCR oder Echtzeit-PCR8berichtet. Schließlich ist LAMP schneller mit vielen Assays, die jetzt eine Standardlaufzeit von 30 min anwenden, während PCR-Assays in der Regel 1-2 h8dauern.
Diese attraktiven Eigenschaften befeuerten die Anwendung von LAMP in breiten Bereichen der Pathogenerkennung, einschließlich der In-vitro-Diagnostik 9, der Tierseuchendiagnostik10und der Lebensmittel- und Umwelttests11. Insbesondere wurde eine TB-LAMP (LAMP for Mycobacterium tuberculosis) von der WHO als gültiger Ersatztest für die Sputum-Abstrichmikroskopie für Lungentuberkulose-Diagnosen in peripheren Umgebungen empfohlen12. Die LAMP-Anwendung erstreckt sich auch über die mikrobielle Identifizierung hinaus und umfasst den Nachweis von Allergenen, Tierarten, Arzneimittelresistenzen, genetisch veränderten Organismen und Pestiziden13.
Nontyphoidal Salmonella ist ein zoonotischer Erreger von erheblichem Lebensmittelsicherheit und öffentlicher Gesundheit weltweit14. Es wurde auch als eine wichtige mikrobielle Gefahr in Lebensmitteln für Tiere (d. h. Tierfutter)15,16identifiziert. Um Salmonellenkrankheiten/Ausbrüche durch kontaminierte menschliche und tierische Lebensmittel zu verhindern, ist es unerlässlich, schnelle, zuverlässige und robuste Methoden zur Prüfung von Salmonellen in einer Vielzahl von Matrizen zu haben. In den letzten zehn Jahren wurden international erhebliche Anstrengungen zur Entwicklung und Anwendung von Salmonella LAMP-Assays in einer breiten Palette von Lebensmittelmatrizen unternommen, wie kürzlich in einem ausführlichenBerichtzusammengefasst 8 . Mehrere Salmonella LAMP-Assays, darunter die hier vorgestellte, haben die Multilabor-Validierung nach etablierten internationalen Richtlinien17,18,19,20erfolgreich abgeschlossen.
Unser Salmonella LAMP-Test zielt speziell auf das Salmonella-Invasionsgen invA (GenBank-Beitrittsnummer M90846)21 ab und ist schnell, zuverlässig und robust in mehreren Lebensmittelmatrizen4,22,23,24,25,26. Die Methode wurde in sechs Tiernahrungsmatrizen in einer vorgemeinschaftlichen Studie26 und in trockener Hundefutter in einer Multilabor-Kollaborationsstudie19validiert. Infolgedessen wurde die hier vorgestellte Salmonella LAMP-Methode kürzlich in das Bacteriological Analytical Manual (BAM) Chapter 5 Salmonella27 der U.S. Food and Drug Administration (FDA) aufgenommen, um zwei Zwecken zu dienen, eine als Schnellscreening-Methode für das Vorhandensein von Salmonellen in Tiernahrung und zwei als zuverlässige Bestätigungsmethode für mutmaßliche Salmonellen, die aus allen Lebensmitteln isoliert sind.

<table>
	<tbody>
		<tr>
			<td>Brain heart infusion (BHI) broth</td>
			<td>BD Diagnostic Systems, Sparks, MD</td>
			<td>299070</td>
			<td>Liquid growth medium used in the cultivation of Salmonella.</td>
		</tr>
		<tr>
			<td>Buffered peptone water (BPW)</td>
			<td>BD Diagnostic Systems, Sparks, MD</td>
			<td>218105</td>
			<td>Preenrichment medium for the recovery of Salmonella from animal food samples.</td>
		</tr>
		<tr>
			<td>DNA AWAY</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>7010</td>
			<td>Eliminates unwanted DNA and DNase from laboratory bench, glassware, and plasticware without affecting subsequent DNA samples.</td>
		</tr>
		<tr>
			<td>Genie Explorer software</td>
			<td>OptiGene Ltd., West Sussex, United Kingdom</td>
			<td>Version 2.0.6.3</td>
			<td>Supports remote operation of Genie instruments including LAMP runs and data analysis.</td>
		</tr>
		<tr>
			<td>Genie II or Genie III (LAMP instrument)</td>
			<td>OptiGene Ltd., West Sussex, United Kingdom</td>
			<td>GEN2-02 or GEN3-02</td>
			<td>A small instrument capable of temperature control up to 100 &#176;C with &#177; 0.1 &#176;C accuracy and simultaneous fluorescence detection via the FAM channel. Genie II has 2 blocks (A and B) with 8 samples in each block. Genie III has a single block that accommodates 8 samples.</td>
		</tr>
		<tr>
			<td>Genie strip</td>
			<td>OptiGene Ltd., West Sussex, United Kingdom</td>
			<td>OP-0008</td>
			<td>8-well microtube strips with integral locking caps and a working volume of 10 to 150 &#181;l.</td>
		</tr>
		<tr>
			<td>Genie strip holder</td>
			<td>OptiGene Ltd., West Sussex, United Kingdom</td>
			<td>GBLOCK</td>
			<td>Used to hold Genie strips when setting up a LAMP reaction, the aluminum holder can also be used as a cool block.</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl) solution, 1 N</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>SA48-500</td>
			<td>Adjusts pH of animal food samples after adding BPW and prior to overnight enrichment.</td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>88-860-022</td>
			<td>Heats samples at 100 &#177; 1 oC for DNA extraction.</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>3960</td>
			<td>Standard laboratory incubator.</td>
		</tr>
		<tr>
			<td>ISO-001 isothermal master mix</td>
			<td>OptiGene Ltd., West Sussex, United Kingdom</td>
			<td>ISO-001</td>
			<td>An optimized master mix to simplify the assembly of a LAMP reaction, containing a strand-displacing GspSSD DNA polymerase large fragment from Geobacillus spp., thermostable inorganic pyrophosphatase, reaction buffer, MgSO4, dNTPs, and a double-stranded DNA binding dye (FAM detection channel).</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>A416</td>
			<td>Disinfects work surfaces.</td>
		</tr>
		<tr>
			<td>LAMP primers</td>
			<td>Integrated DNA Technologies Inc., Coralville, IA</td>
			<td>Custom</td>
			<td>LAMP primers with detailed information in Table 1.</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf North America, Hauppauge, NY</td>
			<td>22620207</td>
			<td>MiniSpin plus personal microcentrifuge.</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>05-408-129</td>
			<td>Standard microcentrifuge tubes.</td>
		</tr>
		<tr>
			<td>Molecular grade water</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>AM9938</td>
			<td>Used in making primer stocks, primer mix, and LAMP reaction mix.</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH) solution, 1 N</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>SS266-1</td>
			<td>Adjusts pH of animal food samples after adding BPW and prior to overnight enrichment.</td>
		</tr>
		<tr>
			<td>Nonselective agar (e.g., blood agar, nutrient agar, and trypticase soy agar)</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>R01202</td>
			<td>Solid growth medium used in the cultivation of Salmonella.</td>
		</tr>
		<tr>
			<td>Peptone water</td>
			<td>BD Diagnostic Systems, Sparks, MD</td>
			<td>218071</td>
			<td>Dilutes overnight Salmonella cultures to make positive control DNA.</td>
		</tr>
		<tr>
			<td>Pipettes and tips</td>
			<td>Mettler-Toledo Rainin LLC, Oakland CA</td>
			<td>Pipet Lite LTS series</td>
			<td>Standard laboratory pipettes and tips.</td>
		</tr>
		<tr>
			<td>PrepMan Ultra sample preparation reagent</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>4318930</td>
			<td>A simple kit used for the rapid preparation of DNA templates for use in a LAMP reaction.</td>
		</tr>
		<tr>
			<td>Salmonella reference strain LT2</td>
			<td>ATCC, Manassas, VA</td>
			<td>700720</td>
			<td>Salmonella reference strain used as positive control.</td>
		</tr>
		<tr>
			<td>Trypticase soy broth (TSB)</td>
			<td>BD Diagnostic Systems, Sparks, MD</td>
			<td>211768</td>
			<td>Liquid growth medium used in the cultivation of Salmonella.</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Scientific Industries, Inc., Bohemia, NY</td>
			<td>SI-0236</td>
			<td>Standard laboratory vortex mixer.</td>
		</tr>
		<tr>
			<td>Whirl-pak filter bag</td>
			<td>Nasco Sampling Brand, Fort Atkinson, WI</td>
			<td>B01318</td>
			<td>Filter bags to hold animal food samples for preenrichment.</td>
		</tr>
	</tbody>
</table>

loop-mediated isothermal amplification for screening salmonella in animal food and confirming salmonella from culture isolation

Loop-vermittelte isotherme Amplifikation (LAMP) hat sich als leistungsstarker Nukleinsäure-Amplifikationstest für den schnellen Nachweis zahlreicher bakterieller, Pilz-, Parasit- und Virusmittel herausgebildet. Salmonellen sind ein bakterieller Erreger, der weltweit für Lebensmittelsicherheit von Bedeutung ist, einschließlich Lebensmitteln für Tiere. Hier wird ein multilaboriertes Salmonella-LAMP-Protokoll vorgestellt, das verwendet werden kann, um Tiernahrung schnell auf das Vorhandensein von Salmonellenkontamination zu untersuchen, und kann auch verwendet werden, um mutmaßliche Salmonellenisolate zu bestätigen, die aus allen Lebensmittelkategorien gewonnen wurden. Der LAMP-Assay zielt speziell auf das Salmonella-Invasionsgen (invA) ab und ist schnell, empfindlich und hochspezifisch. Template DNAs werden aus Anreicherungsbrühen von Tierfutter oder reinen Kulturen von mutmaßlichen Salmonellenisolaten hergestellt. Die LAMP-Reagenzmischung wird durch die Kombination eines isothermen Master-Mixes, Primer, DNA-Schablone und Wasser hergestellt. Der LAMP-Assay läuft bei einer konstanten Temperatur von 65 °C für 30 min. Positive Ergebnisse werden über Echtzeitfluoreszenz überwacht und können bereits nach 5 min nachgewiesen werden. Der LAMP-Test weist eine hohe Toleranz gegenüber Inhibitoren in tierischen Lebensmitteln oder Kulturmedien auf und dient als schnelle, zuverlässige, robuste, kostengünstige und benutzerfreundliche Methode zum Screening und zur Bestätigung von Salmonellen. Die LAMP-Methode wurde vor kurzem in das Kapitel 5 der U.S. Food and Drug Administration es Bacteriological Analytical Manual (BAM) aufgenommen.

Abbildung 2 und Abbildung 3 zeigen repräsentative LAMP-Diagramme/Tabellen, die auf beiden Plattformen angezeigt werden. In diesem LAMP-Lauf sind die Proben S1 bis S6 10-fach serielle Verdünnungen von S. enterica serovar Infantis ATCC 51741 im Bereich von 1,1 x 106 KBE bis 11 KBE pro Reaktion. Positivist ist S. enterica serovar Typhimurium ATCC 19585 (LT2) bei 1,7 x 104 KBE pro Reaktion und NTC ist molekulares Wasser.
Wie in Abbildung 2E und Abbildung 3Gdargestellt, sind sowohl NTC- als auch PC-Bohrungen gültige Steuerungen. Der NTC-Brunnen hat leer Tmax, während Tm < 83 °C auf dem LAMP-Instrumentenpanel und leer in der LAMP-Software ist, was auf ein negatives Ergebnis hindeutet. Der PC-Brunnen hat Tmax. 7 min 45 sec und Tm von 90 °C auf beiden Plattformen, was auf ein positives Ergebnis hindeutet. Die Proben S1 bis S6 haben Tmax zwischen 6 min 30 sec und 12 min 15 sec, alle sind Salmonella-positiv.
Nach doppelten Ausführungen desselben Satzes von Stichproben werden die endgültigen LAMP-Ergebnisse für diese Stichproben gemeldet. Dieser repräsentative LAMP-Lauf zeigt, dass LAMP Salmonellen mit einer Vielzahl von Konzentrationen in den Proben erfolgreich erkennt.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61239/61239fig2v2.jpg"> Abbildung 2: Repräsentative LAMP-Ergebnisse, die auf dem LAMP-Instrumentenpanel angezeigt werden. (A) Die Registerkarte Profil zeigt das programmierte Temperaturprofil an. (B) Die Registerkarte Temperatur zeigt die tatsächlichen Temperaturen in den Probenbrunnen an, während die LAMP-Reaktion fortgesetzt wird. (C) Die Registerkarte Amplifikation zeigt Fluoreszenzwerte während der LAMP-Verstärkung. (D) Die Registerkarte Anneal zeigt Veränderungen der Fluoreszenz (Derivat) während der Nealphase. (E) Auf der Registerkarte Ergebnisse wird eine tabellarische Ansicht der LAMP-Ergebnisse angezeigt. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61239/61239fig2v2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Abbildung anzuzeigen.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61239/61239fig3v2.jpg"> Abbildung 3: Repräsentative LAMP-Ergebnisse in der LAMP-Software angezeigt. (A) Die Registerkarte Profil zeigt das programmierte Temperaturprofil an. (B) Die Registerkarte Temperatur zeigt die tatsächlichen Temperaturen in den Probenbrunnen an, während die LAMP-Reaktion fortgesetzt wird. (C) Die Registerkarte Amplifikation zeigt Fluoreszenzwerte während der LAMP-Verstärkung. (D) Die Registerkarte Amplifikationsrate zeigt Veränderungen der Fluoreszenz (Fluoreszenzverhältnis) während der LAMP-Verstärkung. (E) Die Anneal-Registerkarte zeigt Fluoreszenzwerte während der Nealphase. (F) Die Registerkarte Anneal-Derivat zeigt Veränderungen der Fluoreszenz (Derivat) während der Nealphase. (G) Die Registerkarte Ergebnis zeigt eine tabellarische Ansicht der LAMP-Ergebnisse an. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61239/61239fig3v2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Abbildung anzuzeigen.</a>

Watch this Scientific Journal Video about Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation at JoVE.com

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-vermittelte isotherme Amplifikation (LAMP) ist ein isothermer Nukleinsäure-Amplifikationstest (iNAAT), der ein breites Interesse am Nachweisvon Erreger geweckt hat. Hier präsentieren wir ein multilaboriertes Salmonella LAMP-Protokoll als schnelle, zuverlässige und robuste Methode zum Screening von Salmonellen in Tierfutter und zur Bestätigung mutmaßlicher Salmonellen aus der Kulturisolierung.

Loop-vermittelte isotherme Amplifikation zum Screening von Salmonellen in Tierfutter und Bestätigung von Salmonellen aus der Kulturisolierung

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, ...

loop-mediated-isothermal-amplification-for-screening-salmonella

Nanyang Technological University

Research

JoVE Journal

Biology

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

7.9K Aufrufe.  U.S. Food and Drug Administration. Loop-vermittelte isotherme Amplifikation (LAMP) ist ein isothermer Nukleinsäure-Amplifikationstest (iNAAT), der ein breites Interesse am Nachweisvon Erreger geweckt hat. Hier präsentieren wir ein multilaboriertes Salmonella LAMP-Protokoll als schnelle, zuverlässige und robuste Methode zum Screening von Salmonellen in Tierfutter und zur Bestätigung mutmaßlicher Salmonellen aus der Kulturisolierung.

Serie: Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Chronic Salmonella Infected Mouse Model

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo. Mice with Salmonella persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.

Chronic Salmonella Infected Mouse Model

Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.

Chronisch Salmonellen Infected Maus-Modell

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

Forschung

Biologie

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Isolierung und Primärkultur von Ratten Leberzellen

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolation und Kultur der Dental epithelialen Stammzellen aus dem erwachsenen Maus Schneidezahn

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Ein einfaches Hocheffizienzprotokoll für die Isolierung von Pankreas-Isleten von Mäusen

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also termed the hypothalamic-pituitary-gonadal axis. The reproductive axis is a key integrator of environmental and internal input adapting fertility to favorable conditions for reproduction. Prior to embarking upon a fertility study in mice and rats, sexual maturity is evaluated to exclude the possibility that the observed reproductive phenotypes are caused by delayed or absent pubertal onset. This protocol describes a non-invasive approach to assess pubertal onset in males through the determination of preputial separation, and in females through vaginal opening and first estrus. After the confirmation of the completion of puberty and the achievement of sexual maturity, a fertility study can be initiated. The procedure describes the optimal breeding conditions for mice and rats, how to set up a fertility study, and what parameters to evaluate and determine if the treatment or gene deletion has an impact on fertility.

Many treatments and genetic mutations impact the timing of sexual maturity and fertility. This protocol describes a non-invasive method to evaluate pubertal onset in mice and rats prior to setting up a fertility study in sexually mature animals.

Bestimmung der reproduktive Kompetenz durch Bestätigung pubertären Beginn und Durchführung eines Fruchtbarkeit Assays bei Mäusen und Ratten

Assessment of reproductive competence is critical for understanding the impact of a treatment or genetic manipulation on the reproductive axis, also ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

Quasi-metagenomischen Analyse Salmonella aus Lebensmitteln und Umweltproben

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolierung und Kultur von primären oralen Keratinozyten aus dem erwachsenen Mausgaumen

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

Zellkernisolierung aus der Niere adulter Mäuse für die Einzelkern-RNA-Sequenzierung

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Double-Staining Method to Detect Pectin in Plant-Fungus Interaction

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an efficient inducible mechanism to isolate the fungal haustoria from the plant cell protoplast. Conversely, pectin, one of the polymeric components of the cell wall, is a target of several pectolytic enzymes in necrotrophic interactions. Here, a protocol to detect pectin and fungal hyphae through optical microscopy is presented. The pectin-rich encapsulation in the cells of coffee leaves infected by the rust fungus Hemileia vastatrix and the mesophyll cell wall modification induced by Cercospora coffeicola are investigated. Lesioned leaf samples were fixed with the Karnovsky solution, dehydrated, and embedded in glycol methacrylate for 2-4 days. All steps were followed by vacuum-pumping to remove air in the intercellular spaces and improve the embedding process. The embedded blocks were sectioned into 5-7 &#181;m thick sections, which were deposited on a glass slide covered with water and subsequently heated at 40 &#176;C for 30 min. Next, the slides were double-stained with 5% cotton blue in lactophenol to detect the fungus and 0.05% ruthenium red in water to detect pectin (acidic groups of polyuronic acids of pectin). Fungal haustoria of Hemileia vastatrix were found to be encapsulated by pectin. In coffee cercosporiosis, mesophyll cells exhibited dissolution of cell walls, and intercellular hyphae and conidiophores were observed. The method presented here is effective to detect a pectin-associated response in the plant-fungi interaction.

This protocol describes a microscopical method to detect pectin in coffee-fungus interaction.

Doppelfärbungsmethode zum Nachweis von Pektin in der Pflanzen-Pilz-Interaktion

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an ...

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.