Name: 作者聚焦：使用标准化 LAMP 推进病原体诊断
Uploaded: 2023-09-08T13:55:00
Description: Read this Scientific Article Protocol about Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP at JoVE.com

这项工作由哥伦比亚的Sistema General de Regalías资助，资助号为BPIN 2020000100092，Universidad Icesi - Convocatoria Interna，资助号为CA0413119。MFVT还得到了安第斯大学的助理教授基金的资助。资助实体没有参与手稿的设计、活动执行、数据收集、数据分析和准备。我们感谢 Valle del Lili 大学医院提供来自 SARS -CoV-2 样本的病毒 RNA，并感谢 Alvaro Barrera-Ocampo 博士对手稿的评论。

使用的样本由 Fundación Valle del Lili 大学医院的临床实验室提供，对应于 使用 RT-qPCR 技术检测出 COVID-19 阳性的患者的纯化 RNA。所有患者都为研究提供了知情同意书，该研究得到了 Fundación Valle del Lili 大学医院的人类研究生物伦理委员会的批准。
1. RT-LAMP底漆设计与制备
注：LAMP 引物可用于多种平台，包括 New England BioLabs （NEB） LAMP、Primer Explorer ...

使用比色法和 RT-LAMP 分析检测 SARS-CoV-2 

<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>

尽管RT-LAMP被认为是执行分子诊断的补充方法，但它也有一些局限性和关键步骤，在标准化和实施协议时必须考虑这些限制和关键步骤。
用于检测 SARS-CoV-2 的 LAMP 标准化评估了预混液中的以下参数和成分：（a） 引物的浓度和比对温度;（b） 酶的浓度;（c） 镁浓度;（d） 反应时间;（e） 含有BSA、DMSO、氯化胍等添加剂;（f） 引物的设计;（g） 添加着色剂;（h） 使用内部生产的反?...

严重急性呼吸系统综合症冠状病毒 2 （SARS-CoV-2） 导致 2019 冠状病毒病 （COVID-19）。世界卫生组织于 2020 年 1 月 30 日宣布为国际关注的突发公共卫生事件，并于 2020 年 3 月 11 日宣布为大流行。截至本文撰写之日，大流行已导致超过 7.6 亿例病例和 687 万人死亡1.
这种病毒的影响凸显了需要更好、更准确、更快和更广泛可用的监测工具，以改善传染病的检测和控制2,3。在大流行期间，SARS-CoV-2 诊断测试基于检测核酸、抗体和蛋白质，但核酸的 RT-PCR 检测是金标准4。然而，RT-PCR有一些局限性;它需要专门的设备、基础设施和接受过分子生物学培训的人员，将其应用限制在专业实验室。此外，它很耗时（4-6 小时），不包括将标本运送到实验室的时间，这可能需要第5 天。这些制约因素妨碍了有效的样本处理和获取应急计划和流行病学管理所需的信息。
与RT-PCR相比，逆转录环介导的等温扩增（RT-LAMP）具有多项优势，使其成为设计未来即时诊断检测（POCT）的有吸引力的策略，特别是在资源有限的环境中6。首先，它具有极强的特异性，因为它使用四到六个引物来识别靶序列中的六到八个区域，无论是 DNA 还是 RNA 7,8。其次，由于它在恒定温度下运行，因此不需要复杂的设备（如实时热循环仪）来产生扩增，也不需要训练有素的人员来操作。第三，反应时间很短（~60 min），并且采用了不是很专业的试剂，这使其成为一种具有成本效益的工具6.鉴于上述情况以及 COVID-19 大流行造成的突发卫生事件，该技术可以被视为一种替代诊断方法，该方法快速、廉价且易于在任何研究实验室中实施9.
本文描述了标准化和实施RT-LAMP以使用热循环仪和水浴通过比色法检测SARS-CoV-2的协议（图1）。讨论了关键点、它们的局限性以及推进这些临界点的替代方案。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65662/65662fig01.jpg"> 图 1：使用 RT-LAMP 技术扩增 SARS-CoV-2 的方案。 <a href="https://www.jove.com/files/ftp_upload/65662/65662fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</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

严重急性呼吸系统综合症冠状病毒 2 （SARS-CoV-2） 病毒对人类健康产生了巨大影响。它仍然是对现代社会的威胁，因为许多人因感染而死亡。该疾病通过血清学和分子检测进行诊断，例如金标准实时聚合酶链反应 （RT-PCR）。后者有几个缺点，因为它需要专门的基础设施、昂贵的设备和训练有素的人员。在这里，我们提出了一种协议，概述了在人类样本中使用逆转录环介导的等温扩增 （RT-LAMP） 检测 SARS-CoV-2 病毒所需的步骤。该方案包括 计算机模拟设计引物、制备试剂、扩增和可视化的说明。一旦标准化，这种方法可以在 60 分钟内以低成本和廉价的设备轻松实施并适应任何实验室或护理点。它适用于检测不同的病原体。因此，它有可能在现场和卫生中心使用，以开展及时的流行病学监测。

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>

作者聚焦：使用标准化 LAMP 推进病原体诊断

通过逆转录环介导的等温扩增检测 SARS-CoV-2 病毒

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.

该协议的实施首先要按照上述协议为每个靶基因设计一组引物。2020 年 6 月，从 NextStrain 数据库获得了 5,000 个 SARS-CoV-2 基因组，哥伦比亚基因组的代表性为 10%。对这些序列进行比对，以获得引物设计过程中使用的共识序列。 表 1 显示了为引物 RdRp/Hel 和 RdRp 选择的引物组。基因 N 扩增的引物组来自先前发表的报告14。
协议标准化的第一步是避免N...

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

在这里，我们提供了一个完整的协议，用于标准化和实施通过逆转录环介导的等温扩增 （RT-LAMP） 检测人类样本中 SARS-CoV-2 病毒的方法。这种方法在 60 分钟内完成，可以以低成本和廉价的设备适用于任何实验室或护理点。

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

我们的研究旨在将 LAMP 标准化为诊断前测试，适用于缺乏先进实验室基础设施的现场或初级保健环境。我们寻求促进使用本地可生产的缓冲液和替代染料，减少对进口商业试剂盒的依赖。目前，CRISPR-Cas是一种高效的系统，用于开发快速、特异性和灵敏的病原体诊断方法。现在，该系统具有广泛的补充范围，可检测不同目标样品中的 RNA 或 DNA 靶标。目前的实验挑战涉及防止由扩增子污染和原代细胞引起的非靶标扩增，特别是对于容易形成二聚体的长引物。此外，我们的目标是确定最佳反应条件，以实现染料中视觉上可区分的颜色变化。一旦标准化并得到充分验证，该方法就可以在60分钟内轻松实施并适应任何实验室或护理点，以低成本和廉价的设备，检测几乎任何病原体。因此，它有可能在初级保健中心用于进行及时的流行病学监测。我们的多学科团队专注于从哥伦比亚本土植物中发现分子，试图确定抑制登革热、寨卡病毒或 SARS-COVID-2 等病毒复制的潜力。此外，我们正在进行研究，以开发这些病毒的分子诊断方法。

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.

科研

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.