著者らは、FDAの微生物学的方法検証小委員会(MMVS)および細菌学的分析マニュアル(BAM)評議会のメンバーに、 サルモネラ LAMP法検証研究を批判的に見直してくれたことに感謝する。

<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>

著者らは、競合する財政的利益はないと宣言している。この原稿に記載されている見解は著者のものであり、必ずしも保健福祉省、米国食品医薬品局、または米国政府の公式政策を反映しているわけではありません。商業用資材、設備、またはプロセスへの言及は、食品医薬品局による承認、承認、または勧告を構成するものではありません。

ここでは、動物性食品および純粋な培養物におけるサルモネラ菌のスクリーニングと確認のための、シンプルで迅速かつ特異的で敏感なLAMP法を紹介しました。4つの主要試薬を含む等温マスターミックスの利便性と、すぐに使用できる社内で準備されたプライマーミックスにより、LAMP反応を組み立てるには、わずかなピペット化ステップしか必要としません(図1)。増幅およびアニール相を含む合計実行時間は38分未満である(図2A、Bおよび図3A,B)。陽性の結果はリアルタイム蛍光(図2Cおよび図3C,D)を介して監視され、早ければ5分26分に検出することができる。正しいTm(約90°C)のサンプルのみが陽性として報告されるため、アニール相はLAMP特異性の追加確認として機能します(図2D、E、図3E-G)。純粋な培養における1サルモネラ細胞の感受性および<動物性食品中の1 CFU/25 gは、以前26個報告されている。
LAMPは非常に効果的で大量のDNA1を生成するため、LAMPマスターミックスの準備とDNAテンプレートの追加、エアロゾルの発生の回避、フィルターピペットチップの使用、手袋の交換、増幅後のLAMP反応チューブの開封を控えるなど、クロスコンタミネーションを防ぐために最善の実験室慣行が使用されることが重要です。
このサルモネラランプ法の特異性は、以前に300種の細菌株(247サルモネラ菌185種のサルモネラ菌および53非サルモネラ菌)を用いて試験し、100%特異的な26であることが実証された。特に、Tmaxの有意差は、2つのサルモネラ種、S.エンテラとサルモネラボンゴリ、およびS.腸内亜種の間、特にアリゾナ(IIIa)26の間で有意な差が認められた。それにもかかわらず、これらはLAMPの結果を解釈するための規則に従って有効な肯定的な結果であった。14人のアナリスト19人が関与したドライドッグフードの多実験室共同研究では、LAMPランが重複して一貫性のない結果を有するサンプルが時折観察された。これらは通常、遅延陽性結果を有するサンプルを含む(T最大>15分)。両方の実行を個別に繰り返す場合は、通常、問題を解決しました。よりまれに、我々は正しいTmを有するが、または不規則なT最大値(<5分)を有するサンプルを観察した。これは通常、反応管内の気泡によって引き起こされた。
LAMP法の開発、評価、事前共同研究、および多実験室検証のライフサイクルを通じて、我々は、様々な動物性食品または食品マトリックスおよび培養メディア4、19、22、23、24における阻害剤に対するLAMPの高い耐性を観察し、この方法の堅牢性を強調し、世界規模での他の多くの研究を共同研究した。これはPCRまたはリアルタイムPCRに比べて優れていますが、通常、負の結果がマトリックス阻害によるものではないことを保証するために内部増幅制御が必要です。さらに、LAMPは、研究の大半でPCRまたはリアルタイムPCRと比較して同様の(または優れた)特異性と感度を実証しました 8.LAMP試薬のコストは、反応ごとに約$ 1です。このプロトコルで使用されるLAMPの器械は小さく、低い維持および携帯用である。それらは蛍光測定によってターゲット検出を採用するあらゆる等温増幅方法を扱うことができる、LAMP含まれている。LAMPソフトウェアを使用すると、包括的なレポートを複数の形式(pdf、テキスト、画像)で生成できます。
メソッドの検証は、ルーチンで使用するために新しいメソッドを採用する前に重要なステップです。ここで報告されたLAMPプロトコルが、マルチラボ検証19を正常に完了したことは注目に値します。このLAMPプロトコルが米国FDAのBAM第5章サルモネラ27に最近組み込まれているので、動物性食品の迅速なスクリーニング方法として、そしてすべての食品カテゴリーからの推定サルモネラ分離物の信頼できる確認方法として、この方法ははるかに広い使用を得ることが期待される。

ループ媒介等温増幅(LAMP)は、日本の科学者グループによって2000年に発明された新しい等温核酸増幅試験(iNAAT)1である。初期段階で標的特異的なステムループDNA構造の形成を通じて、LAMPは、この出発物質を指数関数的に効率的に増幅するために、ストランド変位DNAポリメラーゼを使用し、1h1未満で109コピーの標的を生じる。広く使用されているNAATであるポリメラーゼ連鎖反応(PCR)と比較して、LAMPはいくつかの利点を有する。まず、LAMP反応は等温条件下で行われる。これは洗練された熱サイクリング器械のための必要性を取り消す。第二に、LAMPは、臨床および食品用途3、4の両方に対して実証された堅牢性を有する培養培地および生物学的物質2に対して非常に耐性である。これにより、サンプルの準備が簡素化され、偽陰性の結果が最小限に抑えられます 5。第3に、LAMPは、濁度、着色度、生物発光、蛍光、マイクロ流体など、複数の検出プラットフォームに適しています6。第4に、LAMPは、特別に設計されたプライマーを4~6個使用して、6~8個の特定の領域1、7をターゲットにするため、非常に具体的です。第5に、LAMPは超高感度であり、多くの研究はPCRまたはリアルタイムPCR8に対する優れた感度を報告している。最後に、LAMPは、現在、PCRタイプのアッセイは通常1-2時間8を取るが、現在30分の標準実行時間を採用している多くのアッセイで高速です。
これらの魅力的な特徴は、 体外 診断9、動物病診断10、食品および環境試験11を含む広い病原体検出領域におけるLAMPの適用を促進した。特に、結核ランプ(結核 菌用LAMP)は、末梢設定12における肺結核診断のための痰・スミア顕微鏡の有効な代替試験としてWHOによって推奨されている。LAMPアプリケーションはまた、アレルゲン、動物種、薬剤耐性、遺伝子組換え生物、および農薬13の検出を含むように微生物同定を超えて拡大する。
ノンチホイドサルモネラは、世界中の実質的な食品安全と公衆衛生上の懸念の人獣共通病原体である14.また、動物(すなわち、動物性食品)15,16の食品における重要な微生物ハザードとして同定されている。汚染されたヒト食品や動物性食品によるサルモネラの病気/発生を防ぐためには、サルモネラ菌を様々なマトリックスで検査するための迅速で信頼性の高い堅牢な方法が不可欠です。過去10年間で、最近の広範なレビュー8に要約されるように、幅広い種類の食品マトリックスにおけるサルモネラLAMPアッセイの開発と適用に関して、国際的にかなりの努力がなされている。ここに示されたものを含むいくつかのサルモネラLAMPアッセイは、確立された国際ガイドライン17、18、19、20に従って、マルチラボ検証を正常に完了しました。
当社のサルモネラランプアッセイは、具体的にはサルモネラ侵入遺伝子invA(GenBank加盟番号M90846)21を標的とし、複数の食品マトリックス4、22、23、24、25、26で迅速、信頼性、堅牢性を備えています。この方法は、事前共同研究26および多実験室共同研究19のドライドッグフードにおいて6つの動物食品マトリックスで検証されている。その結果、ここで提示されたサルモネラランプ法は、最近、米国食品医薬品局(FDA)の細菌学的分析マニュアル(BAM)第5章サルモネラ27に組み込まれ、動物性食品中のサルモネラ菌の存在を迅速にスクリーニングする方法として、そして2つは、すべての食品から単離された推定サルモネラ菌の信頼できる確認方法として組み込まれました。

<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

ループ媒介等温増幅(LAMP)は、多数の細菌、真菌、寄生、およびウイルス剤を迅速に検出するための強力な核酸増幅試験として登場しました。 サルモネラ菌 は、動物のための食品を含む世界的な食品安全上の懸念の細菌病原体です。ここに提示される多実験室で検証された サルモネラ ランププロトコルは 、サルモネラ 汚染の存在のために動物の食品を迅速にスクリーニングするために使用することができ、また、すべての食品カテゴリーから回収された推定 サルモネラ 分離株を確認するために使用することができる。LAMPアッセイは 、サルモネラ 属の浸潤遺伝子(invA)を特異的に標的とし、迅速、感受性、および非常に特異的である。テンプレートDNAは、動物性食品の濃縮スープまたは推定 サルモネラ 分離物の純粋な培養物から調製される。LAMP試薬混合物は等温マスターミックス、プライマー、DNAテンプレートおよび水を組み合わせることによって製造される。LAMPアッセイは、一定温度の65°Cで30分間実行されます。陽性の結果はリアルタイム蛍光によって監視され、早ければ5分で検出することができる。LAMPアッセイは、動物性食品または培養培地中の阻害剤に対して高い耐性を示し、 サルモネラ菌をスクリーニングおよび確認するための迅速で信頼性の高い、堅牢で、費用対効果の高い、ユーザーフレンドリーな方法として機能する。LAMP法は、米国食品医薬品局の 細菌学分析マニュアル (BAM)第5章に最近組み込まれています。

図 2と図 3は、両方のプラットフォームに表示される代表的な LAMP グラフ/テーブルを示しています。このLAMPランでは、サンプルS1〜S6は、1.1 x 106 CFUから11 CFUまでのS.エンテラビカ・インファンティスATCC 51741の10倍の連続希釈液である。陽性対照は、1回の反応で1.7 x 104 CFUでS.エンテロケロバーティフィムリウムATCC 19585(LT2)であり、NTCは分子グレードの水である。
図 2Eおよび図 3Gに示すように、NTC と PC の両方のウェルが有効なコントロールです。NTCウェルはブランクT最大、Tmはランプ計器パネルの<83°C、LAMPソフトウェアではブランクであり、否定的な結果を示唆している。PCウェルは、両方のプラットフォームで7分45秒のTマックスとTmの~90°Cを有し、肯定的な結果を示唆している。サンプルS1〜S6は、TMaxを6分30秒~15秒の間で有し、すべてサルモネラ陽性である。
同じサンプルセットの重複実行の後、これらのサンプルに対して最終的なLAMP結果が報告されます。この代表的なLAMPの実行は、LAMPがサンプル中の濃度の広い範囲で サルモネラ菌 を正常に検出することを示しています。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61239/61239fig2v2.jpg"> 図2:LAMPインストルメントパネルに表示されるLAMPの代表的な結果(A)プロファイルタブには、プログラムされた温度プロファイルが表示されます。(B)温度タブは、LAMP 反応が進行するにつれてサンプルウェル内の実際の温度を示します。(C) [増幅]タブは、LAMP増幅中の蛍光測定値を示します。(D)アニールタブは、アニール期の蛍光(誘導体)の変化を示す。(E)結果タブには、LAMP 結果の表形式ビューが表示されます。<a href="https://www.jove.com/files/ftp_upload/61239/61239fig2v2large.jpg" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61239/61239fig3v2.jpg"> 図3:LAMPソフトウェアで見たLAMPの代表的な結果(A)プロファイルタブには、プログラムされた温度プロファイルが表示されます。(B)温度タブは、LAMP 反応が進行するにつれてサンプルウェル内の実際の温度を示します。(C) [増幅]タブは、LAMP増幅中の蛍光測定値を示します。(D) [増幅率]タブには、LAMP増幅中の蛍光(蛍光比)の変化が表示されます。(E)アニールタブは、アニール期の蛍光測定値を示す。(F)アニール誘導体タブは、アニール期における蛍光(誘導体)の変化を示す。(G)結果タブには、LAMP 結果の表形式ビューが表示されます。<a href="https://www.jove.com/files/ftp_upload/61239/61239fig3v2large.jpg" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</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

ループ媒介等温増幅(LAMP)は、病原体検出分野に広く関心を集めている等温核酸増幅試験(iNAAT)です。ここでは、動物性食品中のサルモネラ菌をスクリーニングし、培養分離から推定サルモネラ菌を確認するための迅速で信頼性の高い、堅牢な方法として、マルチラボ検証されたサルモネラLAMPプロトコルを提示します。

動物性食品中の サルモネラ菌 をスクリーニングし、培養分離から サルモネラ菌 を確認するためのループ媒介等温増幅

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

Research

JoVE Journal

Biology

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

7.9K ビュー.  U.S. Food and Drug Administration. ループ媒介等温増幅(LAMP)は、病原体検出分野に広く関心を集めている等温核酸増幅試験(iNAAT)です。ここでは、動物性食品中のサルモネラ菌をスクリーニングし、培養分離から推定サルモネラ菌を確認するための迅速で信頼性の高い、堅牢な方法として、マルチラボ検証されたサルモネラLAMPプロトコルを提示します。

ビデオ: 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.

慢性サルモネラ感染マウスモデル

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

生物学

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%.

ラット肝細胞の単離及び初代培養

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.

成体マウス切歯から歯科上皮幹細胞の単離および培養

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.

マウスからの膵島分離のための簡単な高効率プロトコル

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.

思春期の発症を確認マウスおよびラットの不妊治療法を実行することで生殖能力の決定

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;

食品および環境試料からのサルモネラの準メタゲノム解析

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.

成人マウス口蓋からの一次口腔角化細胞の分離と培養

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.

単核RNAシーケンシングのための成体マウス腎臓からの核分離

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.

植物-真菌相互作用におけるペクチンを検出する二重染色法

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.

動物性食品中のサルモネラ菌をスクリーニングし、培養分離からサルモネラ菌を確認するためのループ媒介等温増幅

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慢性サルモネラ感染マウスモデル

ラット肝細胞の単離及び初代培養

成体マウス切歯から歯科上皮幹細胞の単離および培養

マウスからの膵島分離のための簡単な高効率プロトコル

思春期の発症を確認マウスおよびラットの不妊治療法を実行することで生殖能力の決定

食品および環境試料からのサルモネラの準メタゲノム解析

成人マウス口蓋からの一次口腔角化細胞の分離と培養

単核RNAシーケンシングのための成体マウス腎臓からの核分離

植物-真菌相互作用におけるペクチンを検出する二重染色法

難治性物質の分解に関与する菌類の土壌生物多様性からの単離とスクリーニング

動物性食品中の サルモネラ菌 をスクリーニングし、培養分離から サルモネラ菌 を確認するためのループ媒介等温増幅

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慢性サルモネラ感染マウスモデル

ラット肝細胞の単離及び初代培養

成体マウス切歯から歯科上皮幹細胞の単離および培養

マウスからの膵島分離のための簡単な高効率プロトコル

思春期の発症を確認マウスおよびラットの不妊治療法を実行することで生殖能力の決定

食品および環境試料からのサルモネラの準メタゲノム解析

成人マウス口蓋からの一次口腔角化細胞の分離と培養

単核RNAシーケンシングのための成体マウス腎臓からの核分離

植物-真菌相互作用におけるペクチンを検出する二重染色法

難治性物質の分解に関与する菌類の土壌生物多様性からの単離とスクリーニング

動物性食品中のサルモネラ菌をスクリーニングし、培養分離からサルモネラ菌を確認するためのループ媒介等温増幅