Name: 著者スポットライト:標準化されたLAMPによる病原体診断の進歩
Uploaded: 2023-09-08T13:55:00
Description: Read this Scientific Article Protocol about Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP at JoVE.com

この研究は、コロンビアのSistema General de Regalías(助成金番号BPIN 2020000100092)とUniversidad Icesi - Convocatoria Interna(助成金番号CA0413119)によって資金提供されました。MFVTは、ロスアンデス大学の助教授基金からも資金提供を受けました。資金提供機関は、原稿の設計、活動の実行、データ収集、データ分析、および準備には参加していません。SARS-CoV-2サンプルからのウイルスRNAを提供してくださったUniversity Hospital Fundación Valle del Liliと、原稿へのコメントを提供してくださったAlvaro Barrera-Ocampo博士に感謝します。

使用されたサンプルは、Fundación Valle del Lili大学病院の臨床検査室から提供され、RT-qPCR技術を使用してCOVID-19の検査で陽性となった患者の精製されたRNAに対応しました。すべての患者が研究にインフォームドコンセントを提供し、この研究は、Fundación Valle del Lili大学病院のヒト研究の生命倫理委員会によって承認されました。
1. RT-LAMPプライマーの設計と調製</...

比色分析とRT-LAMP分析を使用したSARS-CoV-2の検出 

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Who coronavirus (COVID-19) dashboard (no date). , (2023).</li><li>Ibrahim, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiologic+surveillance+for+controlling+Covid-19+pandemic:+types,+challenges+and+implications.">Epidemiologic surveillance for controlling Covid-19 pandemic: types, challenges and implications.</a> Journal of Infection and Public Health. 13 (11), 1630-1638 (2020).</li><li>Rojas-Gallardo, D. 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=COVID-19+in+Latin+America:+Contrasting+phylodynamic+inference+with+epidemiological+surveillance.+(Molecular+epidemiology+of+COVID-19+in+Latin+America).">COVID-19 in Latin America: Contrasting phylodynamic inference with epidemiological surveillance. (Molecular epidemiology of COVID-19 in Latin America).</a> medRxiv. , (2020).</li><li>Liu, R., 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=Positive+rate+of+RT-PCR+detection+of+SARS-CoV-2+infection+in+4880+cases+from+one+hospital+in+Wuhan,+China,+from+Jan+to+Feb+2020.">Positive rate of RT-PCR detection of SARS-CoV-2 infection in 4880 cases from one hospital in Wuhan, China, from Jan to Feb 2020.</a> Clinica Chimica Acta. 505, 172-175 (2020).</li><li>Kevadiya, B. D., 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=Diagnostics+for+SARS-CoV-2+infections.">Diagnostics for SARS-CoV-2 infections.</a> Nature Materials. 20 (5), 593-605 (2021).</li><li>Tomita, N., Mori, Y., Kanda, H., 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)+of+gene+sequences+and+simple+visual+detection+of+products.">Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products.</a> Nature Protocols. 3 (5), 877-882 (2008).</li><li>Li, Y., Fan, P., Zhou, S., Zhang, L. <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+novel+rapid+detection+platform+for+pathogens.">Loop-mediated isothermal amplification (LAMP): A novel rapid detection platform for pathogens.</a> Microbial Pathogenesis. 107, 54-61 (2017).</li><li>Notomi, T., Mori, Y., Tomita, N., Kanda, H. <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):+principle,+features,+and+future+prospects.">Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects.</a> Journal of Microbiology. 53 (1), 1-5 (2015).</li><li>Augustine, R., 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+(LAMP):+A+rapid,+sensitive,+specific,+and+cost-effective+point-of-care+test+for+coronaviruses+in+the+context+of+COVID-19+pandemic.">Loop-mediated isothermal amplification (LAMP): A rapid, sensitive, specific, and cost-effective point-of-care test for coronaviruses in the context of COVID-19 pandemic.</a> Biology (Basel). 9 (8), 182 (2020).</li><li>. Nextstrain Available from: <a target="_blank" href="https://nextstrain.org/">https://nextstrain.org/</a> (2023)</li><li>. Neb Lamp, NEB LAMP Available from: <a target="_blank" href="https://lamp.neb.com/">https://lamp.neb.com/</a> (2023)</li><li>. Blast: Basic local alignment search tool (no date) Available from: <a target="_blank" href="https://blast.ncbi.nlm.nih.gov/">https://blast.ncbi.nlm.nih.gov/</a> (2023)</li><li>Zhang, Y., 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=Rapid+molecular+detection+of+SARS-CoV-2+(COVID-19)+virus+RNA+using+colorimetric+LAMP.">Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP.</a> medRxiv. , (2020).</li><li>Lu, R., 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=Development+of+a+novel+reverse+transcription+loop-mediated+isothermal+amplification+method+for+rapid+detection+of+SARS-CoV-2.">Development of a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of SARS-CoV-2.</a> Virologica Sinica. 35 (3), 344-347 (2020).</li><li>Najafov, A., Hoxhaj, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PCR Guru. , (2017).</li><li>Zhang, Y., 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=Enhancing+colorimetric+loop-mediated+isothermal+amplification+speed+and+sensitivity+with+guanidine+chloride.">Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride.</a> Biotechniques. 69 (3), 178-185 (2020).</li><li>Ram&#237;rez-Chavarr&#237;a, R. G., 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=Automatic+analysis+of+isothermal+amplification+via+impedance+time-constant-domain+spectroscopy:+A+SARS-CoV-2+case+study.">Automatic analysis of isothermal amplification via impedance time-constant-domain spectroscopy: A SARS-CoV-2 case study.</a> Chemosensors. 11 (4), 230 (2023).</li><li>Haque, M. F. U., 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=A+novel+RdRp-based+colorimetric+RT-LAMP+assay+for+rapid+and+sensitive+detection+of+SARS-CoV-2+in+clinical+and+sewage+samples+from+Pakistan.">A novel RdRp-based colorimetric RT-LAMP assay for rapid and sensitive detection of SARS-CoV-2 in clinical and sewage samples from Pakistan.</a> Virus Research. 302, 198484 (2021).</li><li>Donia, 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=Integration+of+RT-LAMP+and+microfluidic+technology+for+detection+of+SARS-CoV-2+in+wastewater+as+an+advanced+point-of-care+platform.">Integration of RT-LAMP and microfluidic technology for detection of SARS-CoV-2 in wastewater as an advanced point-of-care platform.</a> Food and Environmental Virology. 14, 364-373 (2022).</li></ol>

RT-LAMPは、分子診断を実行するための補完的な方法論と見なされていますが、プロトコルを標準化して実装する際に考慮しなければならないいくつかの制限と重要なステップもあります。
SARS-CoV-2の検出のためのLAMP標準化では、マスターミックスの次のパラメーターと成分が評価されました:(a)プライマーのアライメントの濃度と温度。(b)酵素の濃度。(c)マグネシウム濃度?...

重症急性呼吸器症候群コロナウイルス2(SARS-CoV-2)は、コロナウイルス病2019(COVID-19)を引き起こします。世界保健機関(WHO)は、2020年1月30日に国際的に懸念される公衆衛生上の緊急事態を宣言し、2020年3月11日にパンデミックを宣言しました。パンデミックにより、この記事が書かれた時点で7億6,000万人以上の症例と687万人の死亡者が出ています1。
このウイルスの影響により、感染症の検出と制御を改善するために、より優れた、より正確で、より速く、より広く利用可能な監視ツールの必要性が浮き彫りになりました2,3。パンデミック時には、SARS-CoV-2の診断検査は核酸、抗体、タンパク質の検出が基本でしたが、核酸のRT-PCR検出はゴールドスタンダードです4。ただし、RT-PCRにはいくつかの制限があります。これには、分子生物学の訓練を受けた特殊な機器、インフラストラクチャ、および人員が必要であり、その適用は専門の研究所に限定されています。さらに、それは時間がかかり(4〜6時間)、標本を実験室に輸送する時間を含まない、これには5日かかることがあります。これらの制約により、効率的なサンプル処理や、緊急時対応計画や疫学管理に必要な情報の取得が妨げられます。
逆転写ループ媒介等温増幅法(RT-LAMP)は、RT-PCRに比べていくつかの利点があり、特にリソースに制約のある環境で、将来のポイントオブケア診断検査(POCT)を設計するための魅力的な戦略となっています6。まず、DNAやRNAなど、標的配列の6〜8つの領域を認識する4〜6つのプライマーを使用するため、非常に特異的です7,8。次に、一定の温度で動作するため、増幅を生成するためにリアルタイムのサーマルサイクラーなどの高度な機器を必要とせず、高度な訓練を受けた人員を操作する必要もありません。第三に、反応時間が非常に短く(~60分)、あまり専門化されていない試薬が使用されるため、費用対効果の高いツールになります6。上記とCOVID-19のパンデミックによって引き起こされた健康上の緊急事態を考えると、この技術は、迅速、安価、かつ簡単に実施できる代替診断方法と見なすことができる9。
この記事では、サーモサイクラーとウォーターバスを使用した比色法によりSARS-CoV-2を検出するためのRT-LAMPを標準化および実装するためのプロトコルについて説明します(図1)。重要なポイント、その制限、およびそれらを進めるための代替案について説明します。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65662/65662fig01.jpg"> 図1:RT-LAMP技術を使用してSARS-CoV-2を増幅するためのプロトコルのスキーム。 この <a href="https://www.jove.com/files/ftp_upload/65662/65662fig01large.jpg" target="_blank">図の拡大版を表示するには、ここをクリックしてください。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 kb DNA Ladder</td>
			<td>SOLIS BIODYNE</td>
			<td>07-12-00050</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>50x TAE Electrophoresis Buffer</td>
			<td>ThermoScientific</td>
			<td>B49</td>
			<td>Store at roome temperature</td>
		</tr>
		<tr>
			<td>Accuris High Fidelity Polymerase</td>
			<td>ACCURIS LIFE SCIENCE REAGENTS</td>
			<td>PR1000-HF-200</td>
			<td>It can be used in case Q5 High-Fidelity DNA polymerase cannot be purchased. For the enzyme, make aliquots of an appropriate volume and store at -20 &#176;C until use.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>PanReacAppliChem</td>
			<td>A8963,0100</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Bst 3.0 DNA Polymerase 8000 IU/mL</td>
			<td>New England BioLabs</td>
			<td>M0374S/M0374L</td>
			<td>For the enzyme, make aliquots of an appropriate volume and store at -20 &#176;C until use.</td>
		</tr>
		<tr>
			<td>Deoxynucleotide (dNTP) Solution Set</td>
			<td>New England BioLabs</td>
			<td>N0446S</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Diethyl pyrocarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>159220-25G&#160;</td>
			<td>Handle it with caution under an extraction cabinet</td>
		</tr>
		<tr>
			<td>GeneRuler 100 bp Plus DNA Ladder, ready-to-use</td>
			<td>ThermoScientific</td>
			<td>SM0322</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Hydroxy naphthol blue disodium salt</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-215156B</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Q5 High-Fidelity DNA polymerase 2000 IU/mL&#160;</td>
			<td>New England BioLabs</td>
			<td>M0491S/M0491L</td>
			<td>For the enzyme, make aliquots of an appropriate volume and store at -20 &#176;C until use.</td>
		</tr>
		<tr>
			<td>WarmStart RTx Reverse Transcriptase 15000 IU/mL</td>
			<td>New England BioLabs</td>
			<td>M0380S/M0380L</td>
			<td>For the enzyme, make aliquots of an appropriate volume and store at -20 &#176;C until use.</td>
		</tr>
	</tbody>
</table>

detecting sars-cov-2 virus by reverse transcription-loop-mediated isothermal amplification

重症急性呼吸器症候群コロナウイルス2(SARS-CoV-2)ウイルスは、人間の健康に劇的な影響を与えています。感染によって多くの人が亡くなるため、現代社会にとって脅威であり続けています。この疾患は、ゴールドスタンダードのリアルタイムポリメラーゼ連鎖反応(RT-PCR)などの血清学的および分子学的検査を使用して診断されます。後者には、特殊なインフラストラクチャ、高価な機器、および訓練を受けた人員が必要になるため、いくつかの欠点があります。ここでは、ヒトサンプルの逆転写ループ媒介等温増幅法(RT-LAMP)を使用してSARS-CoV-2ウイルスを検出するために必要な手順を概説したプロトコルを示します。このプロトコールには、 プライマーin silicoの設計、試薬の調製、増幅、および可視化の指示が含まれています。標準化されると、この方法は簡単に実装でき、60分以内に低コストで安価な機器を使用して、あらゆるラボやポイントオブケアに適応させることができます。さまざまな病原体の検出に適応できます。したがって、現場や保健センターでタイムリーな疫学監視を実施するために使用できる可能性があります。

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). The World Health Organization declared a public health emergency of international concern on 30 January 2020 and a pandemic on 11 March 2020. The pandemic resulted in over 760 million cases and 6.87 million deaths as of the date this article was written1.
The impact of this virus has highlighted the need for better, more accurate, faster, and more widely available surveillance tools to improve infectious disease detection and control2,3. During the pandemic, SARS-CoV-2 diagnostic tests were based on detecting nucleic acid, antibodies, and proteins, but RT-PCR detection of nucleic acid is the gold standard4. However, RT-PCR has some limitations; it requires specialized equipment, infrastructure, and personnel trained in molecular biology, limiting its application to specialized laboratories. Further, it is time-consuming (4-6 h), not including the time to transport the specimens to the laboratory, which can take days5. These constraints prevent efficient sample processing and obtaining the information required for contingency planning and epidemiological management.
Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) has several advantages over RT-PCR, making it an appealing strategy for designing future point-of-care diagnostic tests (POCT), particularly in resource-constrained settings6. First, it is greatly specific because it uses between four and six primers that recognize six to eight areas in the target sequence, be it DNA or RNA7,8. Second, because it operates at a constant temperature, it does not require sophisticated equipment such as real-time thermal cyclers to generate the amplification, nor does it necessitate highly trained personnel to operate it. Third, the reaction time is very short (~60 min), and reagents that are not very specialized are employed, which makes it a cost-effective tool6. Given the foregoing and the health emergency caused by the COVID-19 pandemic, this technique can be viewed as an alternative diagnostic method that is quick, inexpensive, and simple to implement in any research laboratory9.
The protocol for standardizing and implementing an RT-LAMP to detect SARS-CoV-2 by colorimetric methods using a thermocycler and a water bath is described in this article (Figure 1). Critical points, their limitations, and alternatives to advance them are discussed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65662/65662fig01.jpg" /> 
Figure 1: Scheme of the protocol for amplifying SARS-CoV-2 using the RT-LAMP technique. <a href="https://www.jove.com/files/ftp_upload/65662/65662fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

著者スポットライト:標準化されたLAMPによる病原体診断の進歩

逆転写ループを介した等温増幅によるSARS-CoV-2ウイルスの検出

Our research aims to standardize LAMP as a prediagnostic test, suitable for field or primary care settings lacking advanced laboratory infrastructure. We seek to promote the use of locally-producible buffers and alternative dyes, reducing dependence on imported commercial kits. Currently, CRISPR-Cas is an efficient system employed in the development of fast, specific, and sensitive diagnostic methods for pathogens.Now, this system has a broad range of and supplements to detect RNA or DNA targets in different samples of interest. The current experimental challenges involve preventing non-target amplification resulting from amplicon contamination and primary cell especially with lengthy primers prone to dimer formation. Additionally, we aim to determine optimal reaction conditions to achieve a visually distinguishable color change in the dye.Once it's standardized and fully validated, this method can be easily implemented and adapt to any laboratory or point-of-care within 60 minutes, at low cost and using inexpensive equipment, to detect almost any pathogen. Thus, it can potentially be used in primary care centers to carry out timely, epidemiological surveillance. Our multidisciplinary group is focused on the discovery of molecules from native Colombia plants, trying to identify the potential to inhibit the replication of viruses such as dengue, Zika, or SARS-COVID-2.In addition, we are conducting studies to develop molecular diagnostic methods for these viruses.

プロトコールの実施は、上記のプロトコールに従って各標的遺伝子のプライマーのセットを設計することから始まります。2020年6月、NextStrainデータベースから5,000のSARS-CoV-2ゲノムが取得され、コロンビアのゲノムの代表性は10%でした。これらの配列は、プライマー設計プロセスで使用されたコンセンサス配列を得るためにアラインメントされました。 表1 に、プライマーRdRp/Helと...

Read this Scientific Article Protocol about Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP at JoVE.com

ここでは、逆転写ループ媒介等温増幅(RT-LAMP)によりヒトサンプル中のSARS-CoV-2ウイルスを検出する方法を標準化し、実装するための完全なプロトコルを提供します。この方法は60分以内に完了し、低コストで安価な機器を使用して、あらゆる検査室やポイントオブケアに適応できます。

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

私たちの研究は、高度な検査インフラが不足しているフィールドケアやプライマリケアの環境に適した、診断前検査としてLAMPを標準化することを目指しています。私たちは、現地で生産可能なバッファーと代替染料の使用を促進し、輸入された商用キットへの依存を減らすことを目指しています。現在、CRISPR-Casは、病原体の迅速、特異的、高感度な診断法の開発に採用されている効率的なシステムです。現在、このシステムには、関心のあるさまざまなサンプルのRNAまたはDNAターゲットを検出するための幅広いサプリメントがあります。現在の実験上の課題は、アンプリコン汚染や初代細胞、特に二量体形成しやすい長いプライマーによる非標的増幅の防止です。さらに、色素の視覚的に識別可能な色の変化を達成するための最適な反応条件を決定することを目指しています。標準化され、完全に検証されると、この方法は簡単に実装でき、60分以内に低コストで安価な機器を使用して、ほぼすべての病原体を検出できます。したがって、プライマリケアセンターでタイムリーな疫学的監視を実施するために使用できる可能性があります。私たちの学際的なグループは、デング熱、ジカ熱、SARS-COVID-2などのウイルスの複製を阻害する可能性を特定しようとして、コロンビア原産の植物からの分子の発見に焦点を当てています。また、これらのウイルスの分子診断法を開発するための研究も進めています。

detecting-sars-cov-2-virus-reverse-transcription-loop-mediated

Research

JoVE Journal

Biology

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.

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.

生物学

Reverse Transcriptase-Loop Mediated Isothermal Amplification Reaction (RT-LAMP) for SARS-CoV-2 Detection

To begin, switch on the laminar airflow cabinet, allowing it to stabilize for three minutes. Aseptically clean and sanitize the cabinet's internal surfaces using the following disinfection agents. After disinfecting any material that will enter the cabinet, place napkins and nitrile gloves into the cabinet.Then switch off the cabinet and expose it to ultraviolet light for 15 minutes. After the irradiation, restart the cabinet. Place the primer design reagents in an ice-filled cooler.After sanitizing it with 70%ethanol, place the container inside the cabinet. Prepare the loop mediated isothermal amplification, or LAMP mix, in a 0.6 milliliter microcentrifuge tube. Pipette the mixture to homogenize it.Next, incubate the closed tubes in a heating block at 95 degrees Celsius for five minutes. Then cool the tubes in an ice-filled polystyrene cooler for five minutes. After cooling, place the tubes in the laminar flow cabinet.To complete the LAMP mix, add four microliters of DNA polymerase, followed by one microliter of reverse transcriptase. To perform colorimetric detection, add the dihydroxynaphthol blue. After reagent addition, pipette each solution to properly mix the LAMP reagents.Next, pipette 22 microliters of the solution into PCR tubes, being careful not to create bubbles. For the negative control, add three microliters of 0.1%diethyl pyrocarbonate water. Keep aside any remaining tubes for the addition of genetic material.Next, remove all materials from the cabinet. After sanitizing the cabinet surfaces with 70%ethanol, shut it off. In a separate area, add three microliters of the sample to each PCR tube and pipette well with a 20 microliter micropipette to thoroughly homogenize.Before initiating the colorimetric reaction, use a high quality camera to take pictures of the PCR tubes. Now, carry out the reaction using a thermal cycler and a water bath. For the thermal cycler, place the tubes into the reaction block and set up thermo profile on the equipment.If using the water bath, securely place the tubes in circular containers, then place these containers in the water bath at 66.3 degrees Celsius for 60 minutes. After the reaction time, remove the tubes and store them at four degrees Celsius until use. If a colorimetric reaction was performed, capture images of the PCR with a high quality camera.A temperature gradient demonstrated that the optimal melting temperature was 66.3 degrees Celsius. The concentration of BST 3.0 enzyme was optimized to 3.2 international units per microliter. To define the colorimetric strategy, phenol red in neutral red dyes were evaluated.However, no color change was observed after amplification. Standardization of different concentrations of hydroxynaphthol blue was performed with the optimum change occurring with 125 micromoles of hydroxynaphthol blue. The amplified samples demonstrated a change in the color in accordance with their concentration.Sample amplification was further confirmed by electrophoresis.