Yazarlar, Salmonella LAMP yöntem doğrulama çalışmalarını eleştirel bir şekilde incelediklerinden dolayı FDA'nın Mikrobiyoloji Yöntemleri Doğrulama Alt Komitesi (MMVS) ve Bakteriyolojik Analitik El Kitabı (BAM) Konseyi üyelerine teşekkür etmektedir.

<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>

Yazarlar rakip finansal çıkarları olmadığını beyan ederler. Bu yazıda ifade edilen görüşler yazarlara aittir ve Sağlık ve İnsan Hizmetleri Bakanlığı, ABD Gıda ve İlaç İdaresi veya ABD Hükümeti'nin resmi politikasını yansıtmak zorunda değildir. Herhangi bir ticari malzemeye, ekipmana veya işleme atıfta bulunulması hiçbir şekilde Gıda ve İlaç İdaresi tarafından onay, onay veya tavsiye teşkil etmez.

Burada sırasıyla hayvansal gıda ve saf kültürde Salmonella'yı taramak ve onaylamak için basit, hızlı, spesifik ve hassas bir LAMP yöntemi sunduk. Dört anahtar reaktif içeren bir izotermal ana karışımın rahatlığı ve kullanıma hazır, şirket içinde hazırlanmış bir astar karışımı ile, bir LAMP reaksiyonunun montajı sadece birkaç pipetleme adımı gerektirir (Şekil 1). Amplifikasyon ve tavlama aşamaları dahil olmak üzere toplam çalışma süresi 38 dakikadan azdır(Şekil 2A,B ve Şekil 3A,B). Olumlu sonuçlar gerçek zamanlı floresan ( Şekil2C ve Şekil 3C,D) aracılığıyla izlenir ve 5 dk26kadar erken bir tarihte tespit edilebilir. Tavlama fazı LAMP özgüllüğünün ekstra bir teyidi olarak işlev görür, çünkü sadece doğru Tm (yaklaşık 90 °C) olan numuneler pozitif olarak bildirilir (Şekil 2D,E ve Şekil 3E−G). Saf kültürde 1 Salmonella hücresinin ve hayvansal gıdalarda < 1 CFU/25 g hassasiyetlerinin daha önce26olduğu bildirilmiştir.
LAMP oldukça etkili olduğundan ve çok miktarda DNA1ürettiğinden, LAMP ana karışımını hazırlamak ve DNA şablonları eklemek için alanları fiziksel olarak ayırmayı, aerosol üretmekten kaçınmayı, filtre pipet uçlarını kullanmaktan, eldivenleri sık sık değiştirmeyi ve amplifikasyon sonrası LAMP reaksiyon tüplerini açmaktan kaçınmayı içerebilecek çapraz kontaminasyonu önlemek için en iyi laboratuvar uygulamalarının kullanılması kritik öneme sahiptir.
Bu Salmonella LAMP yönteminin özgüllüğü daha önce 300 bakteri suşu (185 serovar ve 53 Salmonella olmayan 247Salmonella)kullanılarak test edildi ve% 100 spesifik26olduğu gösterildi. Özellikle, iki Salmonella türü, S. enterica ve Salmonella bongoriarasında ve S. enterica alt türleri arasında, özellikle subsp. arizonae (IIIa)26arasında Tmax'te önemli farklılıklar gözlenmiştir. Bununla birlikte, bunlar LAMP sonuçlarını yorumlama kurallarına göre hala geçerli olumlu sonuçlardı. 14 analist 19 'u içeren kuru köpekmamasındakiçok laboratuvarlı işbirliği çalışmamızda, yinelenen LAMP çalışmalarında tutarsız sonuçlara sahip örnekler zaman zaman gözlendi. Bunlar genellikle gecikmiş pozitif sonuçlara sahip örnekleri içeriyordu(Tmax > 15 dk). Her iki çalıştırmayı da bağımsız olarak yinelemek genellikle sorunu çözmüştür. Daha nadiren, doğru Tm ancak düzensiz Tmax değerlerine (< 5 dk) sahip örnekler gözlemledik. Buna genellikle reaksiyon tüpündeki hava kabarcıkları neden oldu.
LAMP yöntemi geliştirme, değerlendirme, önkolaboratif çalışma ve çok laboratuvarlı doğrulama yaşam döngüsü boyunca, LAMP'nin çeşitli hayvansal gıda veya gıda matrislerindeki inhibitörlere ve kültür ortamlarına karşı yüksek tolerans gösterdiğini gözlemledik4,19,22,23,24, yöntemin sağlamlığını vurgulayan ve küresel ölçekte çok sayıda diğer çalışmalarla işbirliği yapmak8. Bu, PCR veya gerçek zamanlı PCR'ye kıyasla daha üstündür, bu da genellikle olumsuz sonuçların matris inhibisyonuna bağlı olmadığından emin olmak için dahili bir amplifikasyon kontrolü gerektirir28. Ayrıca LAMP, çalışmaların büyük çoğunluğunda PCR veya gerçek zamanlı PCR ile karşılaştırıldığında benzer (veya üstün) özgüllük ve hassasiyet göstermiştir8. LAMP reaktiflerinin maliyeti reaksiyon başına yaklaşık 1 $ 'dır. Bu protokolde kullanılan LAMP cihazları küçük, az bakım gerektiren ve taşınabilirdir. Lamp dahil floresan ölçümü ile hedef tespiti kullanan herhangi bir izotermal amplifikasyon yöntemini işleyebilirler. LAMP yazılımı kullanılarak, kapsamlı raporlar birden fazla biçimde (pdf, metin ve görüntü) oluşturulabilir.
Yöntem doğrulaması, rutin kullanım için yeni bir yöntemin benimsenebilmesi için kritik bir adımdır. Burada bildirilen LAMP protokolünün çoklu laboratuvar doğrulamasını başarıyla tamamlamış olması dikkat çekicidir19. Bu LAMP protokolünün abd FDA'nın BAM Bölüm 5 Salmonella27'ye yakın zamanda dahil olmasıyla, yöntemin hem hayvansal gıdalarda hızlı bir tarama yöntemi hem de tüm gıda kategorilerinden izole edilen muhtemel Salmonella için güvenilir bir onay yöntemi olarak çok daha geniş bir kullanım kazanması beklanmaktadır.

Döngü aracılı izotermal amplifikasyon (LAMP), bir grup Japon bilim adamı tarafından 2000 yılında icat edilen yeni bir izotermal nükleik asit amplifikasyon testidir1. İlk adımlar sırasında hedefe özgü bir kök döngü DNA yapısının oluşturulması yoluyla LAMP, bu başlangıç malzemesini yarı katlanarak verimli bir şekilde yükseltmek için iplikçik yerinden eden bir DNA polimeraz kullanır ve 1saat1'den daha az bir sürede 109 hedef kopyasına neden olur. Yaygın olarak kullanılan bir NAAT olan polimeraz zincir reaksiyonu (PCR) ile karşılaştırıldığında, LAMP çeşitli avantajlara sahiptir. İlk olarak, LAMP reaksiyonları izotermal koşullar altında gerçekleştirilir. Bu, sofistike bir termal bisiklet aletine olan ihtiyacı ortadan ortadan ayırmaktadır. İkinci olarak, LAMP hem klinik hem de gıda uygulamaları için gösterilen sağlamlığa sahip kültür medyasına ve biyolojik maddeler2'ye karşı oldukça hoşgörülüdür3,4. Bu, numune hazırlamayı basitleştirir ve yanlış negatif sonuçları en aza indirir5. Üçüncüsü, LAMP bulanıklık, renklendirme, biyolüminesans, floresan ve mikroakışkanlar6gibi birden fazla algılama platformuna açıktır. Dördüncü olarak, LAMP, altı ila sekiz belirli bölgeyi hedeflemek için dört ila altı özel olarak tasarlanmış astar kullandığı için son derece spesifiktir1,7. Beşinci olarak, LAMP ultra duyarlıdır ve çok sayıda çalışma PCR veya gerçek zamanlı PCR8'eüstün hassasiyetini bildirmektedir. Son olarak, LAMP artık PCR tipi tahliller genellikle 1−2 h8alırken, birçok tahlil ile 30 dakikalık standart çalışma süresini benimseyerek daha hızlıdır.
Bu çekici özellikler, in vitro tanı9,hayvan hastalığı tanılama10ve gıda ve çevre testi11dahil olmak üzere geniş patojen tespit alanlarında LAMP uygulamasını körükledi. Özellikle, periferik ortamlarda pulmoner tüberküloz tanıları için balgam-smear mikroskopisi için geçerli bir değiştirme testi olarak DSÖ tarafından bir TB-LAMP (Mycobacteriumtuberculosis için LAMP) önerilmiştir12. LAMP uygulaması ayrıca alerjenlerin, hayvan türlerinin, ilaç direncinin, genetiği değiştirilmiş organizmaların ve pestisitlerin tespitini içerecek şekilde mikrobiyal tanımlamanın ötesine genişler13.
Nontyphoidal Salmonella, dünya çapında önemli gıda güvenliği ve halk sağlığı endişesinin zoonotik bir patojenidir14. Ayrıca hayvanlar için gıdalarda (yani hayvansal gıdalarda) önemli bir mikrobiyal tehlike olarak tanımlanmıştır15,16. Salmonella hastalıklarının/salgınlarının kontamine insan gıdası ve hayvansal gıdalardan uzak olmasını önlemek için, Salmonella'yı çeşitli matrislerde test etmek için hızlı, güvenilir ve sağlam yöntemlere sahip olmak zorunludur. Son on yılda, salmonella LAMP tahlillerinin geliştirilmesi ve uygulanması konusunda uluslararası alanda önemli çabalar sarf edilmiştir, son zamanlarda kapsamlı bir incelemede özetlendiğiniz gibi8. Burada sunulan da dahil olmak üzere birkaç Salmonella LAMP tahlilleri, köklü uluslararası yönergeler 17 , 18 ,19,20'yitakiben çok laboratuvarlı doğrulamayı başarıyla tamamlamıştır.
Salmonella LAMP testimiz özellikle Salmonella istila gen invA (GenBank katılım numarası M90846)21'i hedef alıyor ve birden fazla gıda matrisindehızlı,güvenilir ve sağlam 4 , 22,23,24,25,26. Yöntem, önkolaboratif bir çalışmada altı hayvansal gıda matrisinde26 ve kuru köpek mamasında çok laboratuvarlı bir işbirliği çalışmasında doğrulanmıştır19. Sonuç olarak, burada sunulan Salmonella LAMP yöntemi yakın zamanda ABD Gıda ve İlaç İdaresi'nin (FDA) Bakteriyolojik Analitik El Kitabı (BAM) Bölüm 5 Salmonella27'ye, biri hayvansal gıdalarda Salmonella varlığı için hızlı bir tarama yöntemi ve iki tüm gıdalardan izole edilmiş varsayımsal Salmonella için güvenilir bir onay yöntemi olarak iki amaca hizmet etmek üzere dahil edilmiştir.

<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

Döngü aracılı izotermal amplifikasyon (LAMP), çok sayıda bakteriyel, mantar, parazitik ve viral ajanın hızlı tespiti için güçlü bir nükleik asit amplifikasyon testi olarak ortaya çıkmıştır. Salmonella, hayvanlar için yiyecekler de dahil olmak üzere dünya çapında gıda güvenliği endişesi olan bakteriyel bir patojendir. Burada sunulan, Salmonella kontaminasyonunun varlığı için hayvansal yiyecekleri hızlı bir şekilde taramak için kullanılabilecek ve ayrıca tüm gıda kategorilerinden kurtarılan varsayılan Salmonella izolelerini doğrulamak için de kullanılabilen çok laboratuvar onaylı bir Salmonella LAMP protokolüdür. LAMP tahlili özellikle Salmonella istila genini(invA)hedefler ve hızlı, hassas ve son derece spesifiktir. Şablon DNA'lar, hayvansal gıdaların zenginleştirme et sularından veya varsayılan Salmonella izolelerinin saf kültürlerinden hazırlanır. LAMP reaktif karışımı bir izotermal ana karışım, astarlar, DNA şablonu ve su birleştirilerek hazırlanır. LAMP tahlili 30 dakika boyunca 65 °C sabit bir sıcaklıkta çalışır. Pozitif sonuçlar gerçek zamanlı floresan ile izlenir ve 5 dk gibi erken bir sürede tespit edilebilir. LAMP tahlili, hayvansal gıda veya kültür ortamındaki inhibitörlere karşı yüksek tolerans göstererek Salmonella'yıtaramak ve onaylamak için hızlı, güvenilir, sağlam, uygun maliyetli ve kullanıcı dostu bir yöntem olarak hizmet eder. LAMP yöntemi yakın zamanda ABD Gıda ve İlaç İdaresi'nin Bakteriyolojik Analitik El Kitabı (BAM) Bölüm 5'e dahil edilmiştir.

Şekil 2 ve Şekil 3 her iki platformda görüntülenen temsili LAMP grafiklerini/tablolarını göstermektedir. Bu LAMP çalışmasında, S1 ila S6 örnekleri, reaksiyon başına 1.1 x 106 CFU ila 11 CFU arasında değişen S. enterica serovar Infantis ATCC 51741'in 10 kat seri seyreltmeleridir. Pozitif kontrol S. enterica serovar Typhimurium ATCC 19585 (LT2) reaksiyon başına 1.7 x 104 CFU ve NTC moleküler dereceli sudur.
Şekil 2E ve Şekil 3G'degösterildiği gibi, hem NTC hem de PC kuyuları geçerli kontrollerdir. NTC kuyusu boş Tmax'e sahipken Tm, LAMP gösterge panelinde < 83 °C ve LAMP yazılımında boştur, bu da olumsuz bir sonuç olduğunu gösterir. PC kuyusu her iki platformda da Tmax 7 dk 45 sn ve Tm ~ 90 °C'ye sahiptir, bu da olumlu bir sonuç olduğunu gösterir. S1 ila S6 örnekleri, hepsi Salmonella-pozitif olmak üzere 6 dk 30 sn ile 12 dk 15 sn arasında Tmax'e sahiptir.
Aynı örnek kümesinin yinelenen çalıştırmalarını takiben, bu örnekler için son LAMP sonuçları bildirilmektedir. Bu temsili LAMP çalışması, LAMP'in salmonella'nın numunelerde çok çeşitli konsantrasyonlarla başarıyla tespit ettiğini göstermektedir.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61239/61239fig2v2.jpg"> Şekil 2: LAMP gösterge panelinde görüntülenen temsili LAMP sonuçları. (A) Profil sekmesinde programlanmış sıcaklık profili gösterilir. (B) Sıcaklık sekmesi, LAMP reaksiyon devam ederken numune kuyularındaki gerçek sıcaklıkları gösterir. (C) Amplifikasyon sekmesi LAMP amplifikasyonu sırasında floresan okumalarını gösterir. (D) Tavlama sekmesi, tavlama aşamasında floresan (türev) değişiklikleri gösterir. (E) Sonuçlar sekmesi LAMP sonuçlarının tablolu bir görünümünü gösterir. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61239/61239fig2v2large.jpg" target="_blank">Bu rakamın daha büyük bir sürümünü görüntülemek için lütfen buraya tıklayın.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61239/61239fig3v2.jpg"> Şekil 3: Lamp yazılımında görüntülenen temsili LAMP sonuçları. (A) Profil sekmesinde programlanmış sıcaklık profili gösterilir. (B) Sıcaklık sekmesi, LAMP reaksiyon devam ederken numune kuyularındaki gerçek sıcaklıkları gösterir. (C) Amplifikasyon sekmesi LAMP amplifikasyonu sırasında floresan okumalarını gösterir. (D) Amplifikasyon Oranı sekmesi, LAMP amplifikasyonu sırasında floresan (floresan oranı) değişiklikleri gösterir. (E) Tavlama sekmesi, tavlama aşamasında floresan okumalarını gösterir. (F) Tav türevi sekmesi, tavlama aşamasında floresan (türev) değişiklikleri gösterir. (G) Sonuç sekmesi LAMP sonuçlarının tablolu bir görünümünü gösterir. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61239/61239fig3v2large.jpg" target="_blank">Bu rakamın daha büyük bir sürümünü görüntülemek için lütfen buraya tıklayın.</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

Döngü aracılı izotermal amplifikasyon (LAMP), patojen tespit alanında geniş ilgi gören bir izotermal nükleik asit amplifikasyon testidir (iNAAT). Burada, Salmonella'yı hayvansal gıdalarda taramak ve tahmin edilen Salmonella'yı kültür izolasyonundan doğrulamak için hızlı, güvenilir ve sağlam bir yöntem olarak çok laboratuvar onaylı bir Salmonella LAMP protokolü sunuyoruz.

Hayvansal Gıdalarda Salmonella Taramak ve Salmonella'yı Kültür İzolasyonundan Doğrulamak için Döngü Aracılı İzotermal Amplifikasyon

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 Görüntüleme.  U.S. Food and Drug Administration. Döngü aracılı izotermal amplifikasyon (LAMP), patojen tespit alanında geniş ilgi gören bir izotermal nükleik asit amplifikasyon testidir (iNAAT). Burada, Salmonella'yı hayvansal gıdalarda taramak ve tahmin edilen Salmonella'yı kültür izolasyonundan doğrulamak için hızlı, güvenilir ve sağlam bir yöntem olarak çok laboratuvar onaylı bir Salmonella LAMP protokolü sunuyoruz.

Video: 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.

Kronik Salmonella Infected Fare Modeli

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

Biyoloji

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%.

Sıçan Karaciğer Hücreleri İzolasyon ve İlköğretim Kültür

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.

Yetişkin Fare insizör Diş Epitel Kök Hücreleri İzolasyon ve Kültür

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.

Farelerden Pankreas Adacık İzolasyon için Basit Bir Yüksek Verimlilik Protokolü

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.

Pubertal başlangıçlı teyit ve doğurganlık tahlil fare ve sıçanlar üreme yetkinlik belirlenmesi

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;

Metagenomic yarı analiz Salmonella gıda ve çevre örnekleri

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.

Yetişkin Fare Damak Tadından Birincil Oral Keratinositlerin İzolasyonu ve Kültürü

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.

Tek Çekirdekli RNA Dizilimi için Yetişkin Fare Böbreğinden Çekirdek İzolasyonu

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.

Bitki-Mantar Etkileşiminde Pektini Tespit Etmek için Çift Boyama Yöntemi

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.