저자는 살모넬라 LAMP 방법 검증 연구를 비판적으로 검토하기 위한 FDA의 미생물학 방법 검증 소위원회 (MMVS) 및 세균성 분석 매뉴얼 (BAM) 위원회의 회원들에게 감사드립니다.

<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>

저자는 경쟁적인 재정적 이익이 없다고 선언합니다. 이 원고에 표현 된 견해는 저자의 견해이며 반드시 보건 복지부, 미국 식품 의약국 또는 미국 정부의 공식 정책을 반영하지 는 않습니다. 식품의약품안전청의 승인, 보증 또는 권고에 따라 상업적 재료, 장비 또는 공정에 대한 언급은 어떠한 방식으로도 구성되지 않습니다.

동물성 음식과 순수한 문화에서 살모넬라를 선별하고 확인하는 간단하고 신속하고 구체적이며 민감한 LAMP 방법을 소개했습니다. 4개의 키 시약이 포함된 이더스말 마스터 믹스의 편리함과 함께, 기성용, 사내 준비된 프라이머 믹스, LAMP 반응을 조립하려면 몇 개의 파이펫팅단계(도 1)만필요하다. 증폭 및 음막 단계를 포함한 총 실행 시간은 38분 미만입니다(그림2A, B 및 도 3A, B). 양수 결과는 실시간 형광(도2C 및 도 3C,D)을통해 모니터링되며 5분26분으로조기에 검출될 수 있다. 멸음 단계는 정확한 Tm(약 90°C)을 가진 샘플만 양성(도2D, E 및 도 3E−G)으로보고되기 때문에 LAMP 특이성의 추가 확인 역할을 한다. 순수한 배양에서 살모넬라 세포 1개 및 동물사료의 CFU/25 g1의 민감성은 이전에26개에보고되었다.
LAMP는 매우 효과적이며 다량의 DNA1을생성하기 때문에, 최고의 실험실 관행은 가로등 마스터 믹스를 준비하고 DNA 템플릿을 추가하기 위한 영역을 물리적으로 분리하고, 에어로졸 생성을 피하고, 필터 파이펫 팁을 사용하고, 장갑을 자주 변경하고, 증폭 후 LAMP 반응 튜브를 열지 않도록 하는 것을 포함하는 교차 오염을 방지하는 데 가장 좋은 실험실 관행을 사용하는 것이 중요합니다.
본 살모넬라 LAMP 방법의 특이성은 이전에 300개의 세균균종(185개의 세로바르의 살모넬라 247개 및 53개의비살모넬라)을사용하여 시험하고 100% 특이적26인것으로 나타났다. 특히, T맥스의 현저한 차이는 두 살모넬라종, S. 엔테리카와 살모넬라 봉고리,그리고 S. 엔테리카 아종, 특히 아취종(IIIa)26종사이에서 관찰되었다. 그럼에도 불구하고, 이들은 여전히 LAMP 결과를 해석하기위한 규칙에 따라 유효한 긍정적 인 결과였다. 14명의분석가19명이참여한 건식 개 사료에 대한 다연구소 협력 연구에서 중복 램프 런에서 일관되지 않은 결과를 갖는 샘플이 때때로 관찰되었습니다. 이들은 일반적으로 지연된 양성 결과를 가진 견본을 관련시켰습니다(Tmax > 15 분). 두 실행 모두 독립적으로 반복하여 일반적으로 문제를 해결했습니다. 더 드물게 정확한Tm을 가진 샘플을 관찰했지만 불규칙한 T최대 값(&lt; 5분)이 없습니다. 이것은 일반적으로 반응 관에 있는 기포에 기인했습니다.
LAMP 방법의 수명주기 전반에 걸쳐 다양한 동물성 식품 또는 식품 매트릭스 및 배양 매체4,19,22,23,24에서램프의 억제제에 대한 높은 관용을 관찰하여, 방법의 견고성을 강조하고 글로벌 스케일8에대한 수많은 다른 연구를 협력하였다. 이는 일반적으로 부정적인 결과가 매트릭스억제(28)로인한 것이 아님을 보장하기 위해 내부 증폭 제어가 필요한 PCR 또는 실시간 PCR에 비해 우수하다. 또한, LAMP는 대부분의 연구에서 PCR 또는 실시간 PCR에 비해 유사한(또는 우수한) 특이성 및민감도를 입증하였다 8. LAMP 시약의 비용은 반응 당 약 $1입니다. 이 프로토콜에 사용되는 LAMP 계측기는 작고 유지 보수가 적으며 휴대용입니다. 그들은 형광 측정, LAMP 포함에 의해 표적 검출을 사용하는 모든 이체 증폭 방법을 처리 할 수 있습니다. LAMP 소프트웨어를 사용하면 포괄적인 보고서를 여러 형식(pdf, 텍스트 및 이미지)으로 생성할 수 있습니다.
메서드 유효성 검사는 일상적인 사용을 위해 새 메서드를 채택하기 전에 중요한 단계입니다. 여기에 보고된 LAMP 프로토콜이 다중 실험실 유효성검사(19)를성공적으로 완료했다는 점은 주목할 만하다. 최근 미국 FDA의 BAM 챕터 5 살모넬라27에이 LAMP 프로토콜이 통합됨에 따라 동물사료의 신속한 선별 방법과 모든 식품 범주에서 분리된 추정 살모넬라 분리를 위한 신뢰할 수 있는 확인 방법으로 훨씬 더 폭넓게 사용될 것으로 기대된다.

루프 매개 이더스말 증폭(LAMP)은 일본 과학자1의그룹에 의해 2000년에 발명된 새로운 이더실 핵산 증폭 시험(iNAAT)이다. 초기 단계에서 표적 특이적 줄기 루프 DNA 구조의 형성을 통해 LAMP는 가닥 변위 DNA 폴리머라아제를 사용하여 이 시작 물질준기하수수체를 효율적으로 증폭시켜 1h1미만의 표적109카피를 생성한다. 널리 사용되는 NAAT인 폴리머라제 연쇄 반응(PCR)과 비교하여 LAMP는 몇 가지 장점을 가지고 있습니다. 첫째, 램프 반응은 이소성 조건하에서 수행됩니다. 이것은 정교한 열 사이클링 장비의 필요성을 없애. 둘째, LAMP는 임상 및 식품 응용 분야3,4모두에 대해 입증된 견고성을 갖춘 배양 매체 및 생물학적 물질2에 매우관대하다. 이렇게 하면 샘플 준비를 단순화하고 잘못된 음수 결과5를최소화합니다. 셋째, LAMP는 탁도, 색도, 생물발광, 형광, 및미세유체6와같은 다중 검출 플랫폼에 순종한다. 넷째, LAMP는 특별히 설계된 프라이머 4~6개에 사용하여 6~8개의 특정 영역1,7을대상으로 하기 때문에 매우 특이적이다. 다섯째, LAMP는 매우 민감하며 수많은 연구에서 PCR 또는 실시간 PCR8에대한 우수한 민감성을 보고했습니다. 마지막으로, LAMP는 PCR 형 분석이 일반적으로 1-2 h8을취하는 동안 30 분 표준 런타임을 채택하는 많은 분석으로 빠릅니다.
이러한 매력적인 특징은 체외 진단9,동물질환 진단10,식품 및 환경 검사11을포함한 광범위한 병원체 검출 영역에서 LAMP의 적용을 촉진시켰다. 특히, 결핵결핵을 위한결핵(LAMP)은 WHO가 말초 설정에서 폐결핵 진단을 위한 가래-얼룩 현미경검사에 대한 유효한 대체 시험으로서 추천되었다(12) LAMP 응용 프로그램은 또한 알러지 유발 물질, 동물 종, 약물 내성, 유전자 변형 유기체 및 살충제13의검출을 포함하도록 미생물 식별을 넘어 확장됩니다.
nontyphoidal 살모넬라는 전 세계적으로14의실질적인 식품 안전 및 공중 보건 문제의 동물성 병원체입니다. 또한 동물(즉, 동물성 식품)15,16을위한 식품의 중요한 미생물 위험으로 확인되었다. 살모넬라 병/발병이 오염된 인간 음식과 동물 사료로부터 발생하지 않도록 하기 위해 다양한 행렬에서 살모넬라를 테스트하기 위한 신속하고 신뢰할 수 있으며 강력한 방법을 갖는 것이 필수적입니다. 지난 10 년 동안, 최근 광범위한 검토8에요약된 바와 같이, 다양한 식품 행렬에서 살모넬라 LAMP 에세이의 개발 및 적용에 국제적으로 상당한 노력이 이루어졌습니다. 여기에 제시된 것을 포함하여 몇몇 살모넬라 LAMP 소는,잘 확립된 국제 지침17,18,19,20에따라 다중 실험실 검증을 성공적으로 완료했습니다.
당사의 살모넬라 램프 분석은 특별히 살모넬라 침공 유전자 invA(GenBank 가입 번호 M90846)21을 대상으로 하며, 여러 식품 행렬4,22,23,24,25,26에서빠르고 신뢰할 수 있고 견고하다. 이 방법은 사전 협업 연구26및 다연구소 협력 연구에서 건식 개사료(19)에서6개의 동물사료 행렬에서 검증되었다. 그 결과, 여기에 제시된 살모넬라 램프 방법은 최근 미국 식품의약국(FDA)의 세균성 분석 매뉴얼(BAM) 제5장 살모넬라(27)에 통합되어 동물식품에 살모넬라의 존재에 대한 신속한 검진 방법으로, 2개는 모든 식품에서 분리된 살모넬라를 위한 신뢰할 수 있는 확인 방법으로 사용된다.

<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

루프 매개 이더스말 증폭(LAMP)은 수많은 세균성, 곰팡이, 기생및 바이러스 성 제제의 신속한 검출을 위한 강력한 핵산 증폭 시험으로 부상하고 있다. 살모넬라는 동물을 위한 음식을 포함하여 세계적인 음식 안전 관심사의 세균성 병원체입니다. 여기에 제시된 다중 실험실 검증 살모넬라 LAMP 프로토콜은 살모넬라 오염의 존재를 위해 동물 사료를 신속하게 선별하는 데 사용될 수 있으며 모든 식품 범주에서 회수된 추정 살모넬라 분리를 확인하는 데 사용할 수 있습니다. LAMP 분석은 특히 살모넬라 침략유전자(invA)를대상으로 하며 신속하고 민감하며 매우 특이적입니다. 템플릿 DNA는 동물 사료의 농축 국물 또는 추정 살모넬라 분리의 순수한 문화에서 제조됩니다. LAMP 시약 혼합물은 이더스 말 마스터 믹스, 프라이머, DNA 템플릿 및 물을 결합하여 제조됩니다. LAMP 분석은 30 분 동안 65 °C의 일정한 온도에서 실행됩니다. 긍정적인 결과는 실시간 형광을 통해 감시되고 5 분 일찍 검출될 수 있습니다. LAMP 분석법은 동물성 식품 또는 배양 배지의 억제제에 대한 높은 내성을 나타내며, 살모넬라를선별하고 확인하기 위한 신속하고 신뢰할 수 있고 견고하며 비용 효율적이며 사용자 친화적인 방법으로 사용된다. LAMP 방법은 최근 미국 식품의약국의 세균분석 매뉴얼(BAM) 제5장에 통합되었습니다.

그림 2 및 그림 3은 두 플랫폼에 표시된 대표 램프 그래프/테이블을 표시합니다. 이 LAMP 실행에서, 샘플 S1 에서 S6은 반응당 1.1 x 106 CFU에서 11 CFU에 이르기까지 S. enterica 세로바 인판티스 ATCC 51741의 10 배 직렬 희석이다. 양성 대조군은 반응당 1.7 x 104 CFU에서 S. 엔테리카 세로바 티피무륨 ATCC 19585(LT2)이며 NTC는 분자등급 수이다.
도 2E 및 도 3G에도시된 바와 같이 NTC 및 PC 웰은 모두 유효한 컨트롤입니다. NTC 우물은 빈 T맥스를 가지고 있으며,Tm은 LAMP 계기판에 83°C이고 LAMP 소프트웨어의 공백은 부정적인 결과를 시사합니다. PC우물은 두 플랫폼에서 7분 45초, Tm의 Tm을 모두 플랫폼에 90°C로 가지고 있어 긍정적인 결과를 시사한다. 샘플 S1 에서 S6까지의 T최대값은 6분 30초에서 12분 15초 사이이며, 모두 살모넬라양성입니다.
동일한 샘플 집합의 중복 된 실행에 따라 이러한 샘플에 대해 최종 LAMP 결과가 보고됩니다. 이 대표적인 LAMP 실행은 LAMP가 샘플에서 광범위한 농도로 살모넬라를 성공적으로 감지한다는 것을 보여줍니다.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61239/61239fig2v2.jpg"> 그림 2: LAMP 계기판에 표시된 대표적인 램프 결과. (A) 프로파일 탭에는 프로그래밍된 온도 프로파일이 표시됩니다. (B) 온도 탭은 램프 반응이 진행됨에 따라 샘플 우물의 실제 온도를 나타낸다. (C) 증폭 탭은 램프 증폭 시 형광 측정값을 나타낸다. (D) 멸막 탭은 멸음 단계 동안 형광(유도체)의 변화를 나타낸다. (E) 결과 탭에는 LAMP 결과의 표 모양이 표시됩니다. <a href="https://www.jove.com/files/ftp_upload/61239/61239fig2v2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61239/61239fig3v2.jpg"> 그림 3: LAMP 소프트웨어에서 볼 수 있는 대표적인 램프 결과. (A) 프로파일 탭에는 프로그래밍된 온도 프로파일이 표시됩니다. (B) 온도 탭은 램프 반응이 진행됨에 따라 샘플 우물의 실제 온도를 나타낸다. (C) 증폭 탭은 램프 증폭 시 형광 측정값을 나타낸다. (D) 증폭 속도 탭은 램프 증폭 시 형광(형광비)의 변화를 나타낸다. (E) 담낭 탭은 막음 단계 동안 형광 측정값을 나타낸다. (F) 절전막 유도체 탭은 막음 단계 동안 형광(유도체)의 변화를 나타낸다. (G) 결과 탭에는 LAMP 결과의 표 모양뷰가 표시됩니다. <a href="https://www.jove.com/files/ftp_upload/61239/61239fig3v2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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

루프 매개 이소성 증폭(LAMP)은 병원체 검출 분야에 대한 폭넓은 관심을 받고 있는 이더스말 핵산 증폭 시험(iNAAT)이다. 여기서, 우리는 동물성 식품에서 살모넬라를 선별하고 문화 격리에서 추정 살모넬라를 확인하는 신속하고 신뢰할 수 있고 강력한 방법으로 다연구소 검증 살모넬라 LAMP 프로토콜을 제시합니다.

동물성 식품에서 살모넬라를 선별하고 문화 격리에서 살모넬라를 확인하기 위한 루프 중재 이더말 증폭

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

Universidad San Sebastian

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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 조회수.  U.S. Food and Drug Administration. 루프 매개 이소성 증폭(LAMP)은 병원체 검출 분야에 대한 폭넓은 관심을 받고 있는 이더스말 핵산 증폭 시험(iNAAT)이다. 여기서, 우리는 동물성 식품에서 살모넬라를 선별하고 문화 격리에서 추정 살모넬라를 확인하는 신속하고 신뢰할 수 있고 강력한 방법으로 다연구소 검증 살모넬라 LAMP 프로토콜을 제시합니다.

비디오: 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.

만성의 살모넬라균 감염된 마우스 모델

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

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생물학

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%.

쥐 간장 세포의 분리 및 차 문화

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.

성인 마우스 앞니의 치아 상피 줄기 세포의 분리 및 문화

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.

마우스에서 췌장 췌도 격리를 위한 간단한 고효율 프로토콜

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 발병을 확인 하 고 쥐 및 쥐에 다 산 분석 결과 수행 하 여 생식 능력의 결정

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;

음식과 환경 샘플에서 살 모 넬 라 의 준 metagenomic 분석

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.

성인 마우스 입천장에서 1 차 구강 각막 세포의 격리 및 문화

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.

석회화 물질의 분해에 관여하는 곰팡이에 대한 토양 생물 다양성으로부터의 분리 및 스크리닝

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.

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 ...

SARS-CoV-2를 검출하기 위해 비색 및 RT-LAMP 분석 사용 

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.

저자 스포트라이트: Advancing Pathogen Diagnostics with Standardized LAMP(표준화된 LAMP를 통한 병원균 진단의 발전)

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.

역전사 루프 매개 등온 증폭을 통한 SARS-CoV-2 바이러스 검출

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