Os autores agradecem aos membros do Subcomitê de Validação de Métodos de Microbiologia da FDA (MMVS) e do Bacteriological Analytical Manual (BAM) Council por revisar criticamente os estudos de validação do método Salmonella LAMP.

<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>

Os autores declaram que não têm interesses financeiros concorrentes. As opiniões expressas neste manuscrito são dos autores e não refletem necessariamente a política oficial do Departamento de Saúde e Serviços Humanos, da Administração de Alimentos e Medicamentos dos EUA ou do Governo dos EUA. A referência a quaisquer materiais comerciais, equipamentos ou processos não constitui de forma alguma aprovação, endosso ou recomendação da Food and Drug Administration.

Apresentamos aqui um método lâmpada simples, rápido, específico e sensível para triagem e confirmação de Salmonella em alimentos animais e cultura pura, respectivamente. Com a conveniência de uma mistura isotemal mestre que contém quatro reagentes-chave, e uma mistura de primer pronta para uso, em casa, a montagem de uma reação LAMP requer apenas alguns passos de pipetação(Figura 1). O tempo total de execução, incluindo fases de amplificação e anneal, é inferior a 38 min (Figura 2A,B e Figura 3A,B). Os resultados positivos são monitorados via fluorescência em tempo real (Figura 2C e Figura 3C,D) e podem ser detectados a partir de 5 min26. A fase anneal serve como uma confirmação extra da especificidade da LÂMPADA, uma vez que apenas amostras com Tm correto (em torno de 90 °C) são relatadas como positivas (Figura 2D,E e Figura 3E−G). Sensibilidades de 1 célula salmonela em cultura pura e < 1 UFC/25 g em alimentos de origem animal foram relatadas anteriormente26.
Como a LÂMPADA é bastante eficaz e gera uma grande quantidade de DNA1,é fundamental que as melhores práticas laboratoriais sejam usadas para evitar a contaminação cruzada, o que pode incluir separar fisicamente as áreas para preparar a mistura mestre da LÂMPADA e adicionar modelos de DNA, evitar gerar aerossóis, usar pontas de pipeta de filtro, trocar luvas com frequência e abster-se de abrir tubos de reação lampião após a amplificação.
A especificidade deste método Salmonella LAMP foi previamente testada utilizando 300 cepas bacterianas (247 Salmonella de 185 serovars e 53 não-Salmonella ) e demonstrou ser 100% específica26. Notavelmente, foram observadas diferenças significativas em Tmax entre as duas espécies Salmonella, S. enterica e Salmonella bongori, e entre as subespécies de S. enterica, especialmente subsp. arizonae (IIIa)26. No entanto, estes ainda foram resultados positivos válidos de acordo com as regras de interpretação dos resultados da LÂMPADA. Em nosso estudo colaborativo multi-laboratorial em alimentos para cães secos que envolveu 14 analistas19, amostras com resultados inconsistentes em corridas duplicadas de LÂMPADAs foram ocasionalmente observadas. Estas geralmente envolviam amostras com resultados positivos atrasados (Tmax > 15 min). Repetir ambas as corridas de forma independente geralmente resolveu o problema. Mais raramente, observamos amostras com valores corretos de Tm, mas não ou valoresmáximos Tirregulares (< 5 min). Isso geralmente era causado por bolhas de ar no tubo de reação.
Ao longo do ciclo de vida do desenvolvimento do método LAMP, avaliação, estudo pré-colaborativo e validação multi-laboratorial, observamos alta tolerância da LÂMPADA aos inibidores em diversos alimentos animais ou matrizes alimentares e mídia cultural4,19,22,23,24, destacando a robustez do método e colaborando inúmeros outros estudos em escala global8. Isso é superior em comparação com PCR ou PCR em tempo real, que geralmente requer um controle de amplificação interna para garantir que os resultados negativos não sejam devido à inibição matricial28. Além disso, lamp demonstrou especificidade e sensibilidade semelhantes (ou superiores) em comparação com PCR ou PCR em tempo real na grande maioria dos estudos8. O custo dos reagentes LAMP é de cerca de US $ 1 por reação. Os instrumentos LAMP utilizados neste protocolo são pequenos, de baixa manutenção e portáteis. Eles podem lidar com qualquer método de amplificação isoteérmica que emprega detecção de alvo por medição de fluorescência, lamp incluído. Usando o software LAMP, relatórios abrangentes podem ser gerados em vários formatos (pdf, texto e imagem).
A validação do método é um passo crítico antes que um novo método possa ser adotado para uso rotineiro. Vale ressaltar que o protocolo LAMP aqui relatado concluiu com sucesso a validação multi-laboratorial19. Com a recente incorporação deste protocolo LAMP no BAM Capítulo 5 Salmonella27da FDA dos EUA, espera-se que o método ganhe um uso muito mais amplo, tanto como um método de triagem rápida em alimentos animais quanto como um método de confirmação confiável para os presuntivos isolados salmonella de todas as categorias alimentares.

Amplificação isotérmica mediada por loop (LAMP) é um novo teste isotémico de amplificação de ácido nucleico (iNAAT) inventado em 2000 por um grupo de cientistas japoneses1. Através da formação de uma estrutura de DNA específica para o alvo durante as etapas iniciais, a LAMP usa uma polimerase de DNA deslocada para amplificar eficientemente este material inicial quase exponencialmente, resultando em 109 cópias do alvo em menos de 1 h1. Comparado com a reação em cadeia de polimerase (PCR), um NAAT amplamente utilizado, o LAMP possui várias vantagens. Em primeiro lugar, as reações lampiães são realizadas em condições istermais. Isso evita a necessidade de um sofisticado instrumento de ciclismo térmico. Em segundo lugar, o LAMP é altamente tolerante à mídia cultural e às substâncias biológicas2 com robustez demonstrada tanto para aplicações clínicas quanto alimentares3,4. Isso simplifica a preparação da amostra e minimiza os resultados negativos falsos5. Em terceiro lugar, o LAMP é amenável a múltiplas plataformas de detecção, como turbidez, colorimetria, bioluminescência, fluorescência e microfluidos6. Em quarto lugar, o LAMP é altamente específico, pois usa de quatro a seis primers especialmente projetados para atingir de seis a oito regiões específicas1,7. Em quinto lugar, lamp é ultrasensível e inúmeros estudos relataram sua sensibilidade superior ao PCR ou PCR8em tempo real . Finalmente, o LAMP é mais rápido com muitos ensaios agora adotando um tempo de execução padrão de 30 minutos, enquanto os ensaios do tipo PCR geralmente levam 1−2 h8.
Essas características atraentes alimentaram a aplicação de LÂMPADA em amplas áreas de detecção de patógenos, incluindo diagnóstico in vitro 9, diagnóstico de doenças animais10e testes alimentares e ambientais11. Notavelmente, uma LÂMPADA de TB (LAMP for Mycobacterium tuberculosis)tem sido recomendada pela OMS como um teste de substituição válido para microscopia de esfutum-difamação para diagnósticos de tuberculose pulmonar em ambientes periféricos12. A aplicação de LÂMPADAs também se expande além da identificação microbiana para incluir a detecção de alérgenos, espécies animais, resistência a medicamentos, organismos geneticamente modificados e pesticidas13.
A Salmonella Não ticidal é um patógeno zoonótico de preocupação substancial com segurança alimentar e saúde pública em todo o mundo14. Também foi identificado como um importante risco microbiano em alimentos para animais (ou seja, alimentos de animais)15,16. Para prevenir doenças/surtos de Salmonella de alimentos humanos e animais contaminados, é imperativo ter métodos rápidos, confiáveis e robustos para testar Salmonella em uma variedade de matrizes. Na última década, esforços consideráveis foram feitos internacionalmente no desenvolvimento e aplicação de ensaios salmonella LAMP em uma ampla gama de matrizes alimentares, como recentemente resumido em uma extensa revisão8. Vários ensaios da Salmonella LAMP, incluindo o apresentado aqui, concluíram com sucesso a validação multi-laboratorial seguindo diretrizes internacionais bem estabelecidas17,18,19,20.
Nosso ensaio Salmonella LAMP tem como alvo especificamente o gene de invasão salmonela invA (número de adesão do GenBank M90846)21 e é rápido, confiável e robusto em múltiplas matrizes alimentares4,22,23,24,25,26. O método foi validado em seis matrizes de alimentos de origem animal em um estudo pré-colaborativo26 e em alimentos para cães secos em um estudo colaborativo multi-laboratorial19. Como resultado, o método Salmonella LAMP apresentado aqui foi recentemente incorporado à Food and Drug Administration (FDA) do Manual Analítico Bacteriológico (BAM) Capítulo 5 Salmonella27 para servir a dois propósitos, um como método de triagem rápida para a presença de Salmonella em alimentos animais e dois como um método de confirmação confiável para a salmonela isolada de todos os alimentos.

<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

A amplificação isotérmica mediada por loop (LAMP) emergiu como um poderoso teste de amplificação de ácido nucleico para a rápida detecção de numerosos agentes bacterianos, fúngicos, parasitários e virais. Salmonela é um patógeno bacteriano da preocupação mundial com a segurança alimentar, incluindo alimentos para animais. Apresentado aqui é um protocolo salmonella LAMP validado em vários laboratórios que pode ser usado para rastrear rapidamente alimentos de origem animal para a presença de contaminação salmonela e também pode ser usado para confirmar os presuntivos isolados salmonella recuperados de todas as categorias alimentares. O ensaio LAMP tem como alvo especificamente o gene de invasão salmonela (invA)e é rápido, sensível e altamente específico. Modelo DNAs são preparados a partir de caldos de enriquecimento de alimentos de origem animal ou culturas puras de isolados de Salmonella presunçosos. A mistura de reagente LAMP é preparada combinando uma mistura isotemal mestre, primers, modelo de DNA e água. O ensaio LAMP funciona a uma temperatura constante de 65 °C por 30 min. Os resultados positivos são monitorados via fluorescência em tempo real e podem ser detectados em até 5 minutos. O ensaio LAMP exibe alta tolerância aos inibidores em alimentos ou meios de cultura animal, servindo como um método rápido, confiável, robusto, econômico e fácil de usar para triagem e confirmação de Salmonella. O método LAMP foi recentemente incorporado ao Manual Analítico Bacteriológico (BAM) da Administração de Alimentos e Medicamentos dos EUA, capítulo 5.

As figuras 2 e a figura 3 mostram gráficos/tabelas lampiões representativas exibidos em ambas as plataformas. Nesta execução da LÂMPADA, as amostras S1 a S6 são diluições seriais de S. enterica serovar Infantis ATCC 51741 variando de 1,1 x 106 UFC a 11 UFC por reação. Controle positivo é S. enterica serovar Typhimurium ATCC 19585 (LT2) a 1,7 x 104 UFC por reação e NTC é água de grau molecular.
Como mostrado na Figura 2E e Figura 3G,tanto os poços NTC quanto o PC são controles válidos. O poço NTC tem Tmax em branco enquanto Tm é < 83 °C no painel de instrumentos LAMP e em branco no software LAMP, sugerindo um resultado negativo. O PC bem tem Tmax de 7 min 45 seg e Tm de ~ 90 °C em ambas as plataformas, sugerindo um resultado positivo. Amostras S1 a S6 têm Tmax entre 6 min 30 seg e 12 min 15 seg, todas sendo Salmonella-positivo.
Após as duplicatas do mesmo conjunto de amostras, os resultados finais da LÂMPADA são relatados para estas amostras. Esta execução de LÂMPADA representativa mostra que a LAMP detecta com sucesso Salmonella com uma ampla gama de concentrações nas amostras.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61239/61239fig2v2.jpg"> Figura 2: Resultados representativos da LÂMPADA exibidos no painel de instrumentos LAMP. (A) A guia Perfil mostra o perfil de temperatura programado. (B) A guia temperatura mostra temperaturas reais nos poços de amostra e a reação da LÂMPADA prossegue. (C) A guia Amplificação mostra leituras de fluorescência durante a amplificação da LÂMPADA. (D) A aba Anneal apresenta alterações na fluorescência (derivada) durante a fase anneal. (E) A guia Resultados mostra uma exibição tabular dos resultados da LÂMPADA. <a href="https://www.jove.com/files/ftp_upload/61239/61239fig2v2large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61239/61239fig3v2.jpg"> Figura 3: Resultados de LÂMPADA representativos visualizados no software LAMP. (A) A guia Perfil mostra o perfil de temperatura programado. (B) A guia temperatura mostra temperaturas reais nos poços de amostra e a reação da LÂMPADA prossegue. (C) A guia Amplificação mostra leituras de fluorescência durante a amplificação da LÂMPADA. (D) A guia Taxa de Amplificação apresenta alterações na fluorescência (relação fluorescência) durante a amplificação da LÂMPADA. (E) A aba Anneal apresenta leituras de fluorescência durante a fase anneal. (F) A aba Derivada De Anneal apresenta alterações na fluorescência (derivada) durante a fase anneal. (G) A guia Resultado mostra uma exibição tabular dos resultados do LAMP. <a href="https://www.jove.com/files/ftp_upload/61239/61239fig3v2large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</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

Amplificação isotérmica mediada por loop (LAMP) é um teste isotémico de amplificação de ácido nucleico (iNAAT) que tem atraído amplo interesse no campo de detecção de patógenos. Aqui, apresentamos um protocolo salmonella LAMP validado em vários laboratórios como um método rápido, confiável e robusto para a triagem de Salmonella em alimentos animais e confirmando salmonela presuntiva do isolamento cultural.

Amplificação isotérmica mediada por loop para triagem de salmonela em alimentos animais e confirmando salmonela do isolamento cultural

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

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JoVE Journal

Biology

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

7.9K visualizações.  U.S. Food and Drug Administration. Amplificação isotérmica mediada por loop (LAMP) é um teste isotémico de amplificação de ácido nucleico (iNAAT) que tem atraído amplo interesse no campo de detecção de patógenos. Aqui, apresentamos um protocolo salmonella LAMP validado em vários laboratórios como um método rápido, confiável e robusto para a triagem de Salmonella em alimentos animais e confirmando salmonela presuntiva do isolamento cultural.

Vídeo: 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.

Crônico Salmonella Modelo de camundongos infectados

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

Biologia

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.

Determinação da competência reprodutiva, confirmando o início puberal e realizando um ensaio de fertilidade em camundongos e ratos

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-metagenomic análise de Salmonella de alimentos e amostras ambientais

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.

Isolamento e Cultura de Queratinócitos Orais Primários do Paladar do Rato Adulto

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

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.

Isolamento e Triagem da Biodiversidade do Solo para Fungos Envolvidos na Degradação de Materiais Recalcitrantes

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

Preparação de Suspensão de Célula Única do Intestino de Tilápia do Nilo para Sequenciamento de Célula Única

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

Usando análise colorimétrica e RT-LAMP para detectar SARS-CoV-2 

Detecting SARS-CoV-2 Virus by Reverse Transcription-Loop-Mediated Isothermal Amplification

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern society because many people die as a result of infection. The disease is diagnosed using serologic and molecular tests, such as the gold standard real-time polymerase chain reaction (RT-PCR). The last has several disadvantages because it requires specialized infrastructure, costly equipment, and trained personnel. Here, we present a protocol outlining the steps required to detect the SARS-CoV-2 virus using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) in human samples. The protocol includes instructions for designing primers in silico, preparing reagents, amplification, and visualization. Once standardized, this method can be easily implemented and adapted to any laboratory or point-of-care within 60 min at a low cost and using inexpensive equipment. It is adaptable to detecting different pathogens. Thus, it can potentially be used in the field and in health centers to carry out timely epidemiological surveillance.

Autor em destaque: Avançando no diagnóstico de patógenos com LAMP padronizado

Here we provide a complete protocol to standardize and implement the method for detecting the SARS-CoV-2 virus in human samples by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method, done within 60 min, could be adapted to any laboratory or point-of-care at a low cost and using inexpensive equipment.

Detecção do vírus SARS-CoV-2 por amplificação isotérmica mediada por loop de transcrição reversa

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern ...

Usando LPS bacteriano para eliminar bacteriófagos de culturas de Salmonella

Bacteriophage Removal from Infected Salmonella Cultures

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and interactions. These organisms are viruses that infect and replicate within bacterial hosts. Phages are ubiquitous on Earth, highly diverse, and very abundant. While bacteriophages have valuable roles in different environments and are a key area of research in microbiology and ecology, their presence can be undesirable in certain industrial processes or products. Considering the abundance and ubiquity of bacteriophages on Earth, the design of procedures for the removal of bacteriophages from bacterial cultures is crucial in diverse laboratory and industrial applications to preserve the integrity of the cultures and ensure accurate experimental results or product quality. Here, we have fine-tuned a protocol to eliminate the bacteriophages from infected Salmonella enterica cultures, using a strategy based on the use of lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria. Bacterial LPS plays an important role in host recognition by phages, and we make use of this property to design an effective procedure for the removal of phages, which use LPS as a receptor, in Salmonella bacterial cultures.

Autor em destaque: Eliminação eficiente de bacteriófagos de culturas de Salmonella infectadas usando lipopolissacarídeos

Bacteriophages, ubiquitous and diverse on Earth, infect and replicate within bacterial hosts, playing a crucial role in microbial ecosystems. Despite their importance, their presence may disrupt industrial processes. We have developed a method using bacterial lipopolysaccharides to eliminate bacteriophages from Salmonella cultures.